(1)
Institute of Pathology, Medical University Graz, Graz, Austria
In this chapter, we will provide a short overview on carcinogenesis and epidemiology, as far as it is necessary to understand the resulting morphology. The aim is not to extensively discuss these aspects, because there are many articles, reviews, and books out, which can be used for a more in-depth understanding. Molecular biology and genetics are discussed in the chapters on Molecular Pathology and Metastasis. In these chapters, molecular abnormalities in the different tumors are discussed, and moreover, mechanisms of invasion, migration, and metastasis are described. So here we have only to focus on mechanisms of carcinogenesis to that moment an in situ carcinoma has been formed, and the primary tumor is established. Not much knowledge is available on the genetic mechanisms of benign epithelial tumors, whereas some data are available for mesenchymal tumors. These aspects will be discussed with the respective tumor entity, because it seems to be unique for each entity.
Since the lung is composed of many different epithelial and mesenchymal cell types at different locations (approximately 56 different cell types), from the large bronchi via the different generations of bronchi and bronchioles, down to the alveoli and along the septa to the pleura, it is not surprising that so many different tumors exist. Some tumors are strictly structure related, such as glandular adenomas, which occur only in large bronchi, or alveolar adenoma occurring only peripheral. Other tumors can occur either peripherally or centrally. Some tumors are confined to the pleura, others spread along the interlobular septa into the lung.
17.1 Epithelial Tumors
17.1.1 Benign Epithelial Tumors
Within the group of benign epithelial tumors, there are centrally located bronchial gland adenomas, cystadenoma, pleomorphic adenoma, as well as the peripheral located papillary adenoma and sclerosing hemangioma.
17.1.1.1 Bronchial Mucous Gland Adenoma (Salivary Gland-Type Adenoma)
Definition and Incidence
These are tumors similar to their salivary gland counterparts. The tumors outgrow from and replace mucous glands of the bronchus. They all arise in the central portion of the large bronchi in areas where bronchial glands are normally found. As their salivary cousins, they can have different cell compositions, which will give rise to the different names used. Tumors comprise many cystic spaces or glands filled with mucus. Lining cells are cylindrical or cuboidal. Mitoses and atypia are absent. Sometimes the surface of the tumor is covered by metaplastic squamous epithelium. In the stroma there are inflammatory cells. Myoepithelial cells might occur in specific entities.
Symptoms
These centrally located tumors most often present with symptoms of bronchial obstruction and are often found in the young-aged population; even children might be affected.
Mucous Gland Adenoma
This is the “common” type of these adenomas. It can occur in both sexes and at any age. Cases have been described in peripheral locations but these need to be separated from papillary adenoma [1]. The major symptom is bronchial obstruction causing cough and purulent inflammation.
Gross Appearance
The tumor grows endobronchial and usually induces bronchial obstruction (Fig. 17.1). The size can be from a few millimeters up to few centimeters in diameter. The tumor is grayish red to white; the cut surface is glistening due to mucous production. The tumors most often grow out from the bronchial mucosa with a broad basis.
Fig. 17.1
Bronchial mucus gland adenoma with typical endobronchial growth and obstruction of the bronchial lumen. H&E, bar 500 μm
Microscopy
Histologically mucous gland adenomas are composed of numerous tubules and papillae, often cystic dilated, filled with mucous (PAS positive) and lined by cuboidal or columnar cells, which are either of the goblet or the secretory cell type [2–4] (Fig. 17.2). Rarely oxyphilic cell may be present; exceptionally rare the entire tumor can be composed of oxyphilic cells; some authors have called these variants of bronchial gland adenomas oncocytoma [5–8] (Fig. 17.2a). Clear cells can also be seen, usually admixed with the other mucous-producing cells.
Fig. 17.2
Three different examples of mucus gland adenomas of the bronchus. (a) Is dominated by oxyphilic cells, (b) shows a mixture of serous and goblet cells, (c) is entirely composed of high columnar cells. H&E, bars 50 and 100 μm, and ×260
Immunohistochemistry
The epithelial cells showed strong and diffuse cytoplasmic staining for high molecular weight cytokeratins (cytokeratin 5/cytokeratin 6) and may also be positive for cytokeratin 20 and often lack TTF1 expression [9]. Scattered myoepithelial cells may stain with S100 protein antibodies.
In the differential diagnosis, low-grade mucoepidermoid carcinoma can closely mimic mucous gland adenoma. However, the mixture of squamous and transitional cell complexes in mucoepidermoid carcinoma will immediately guide to the correct diagnosis.
Molecular Biology
There is only one report, which analyzed a bronchial gland adenoma for genetic aberrations. Pfragner et al. reported trisomy for several chromosomes in a tubular adenoma out of cell cultures from the tumor [10].
Serous and Mucinous Cystadenoma Including Borderline Variants
Gross Pathology
These cystic tumors arise most often in central bronchi but may be found peripheral. The tumor is most often asymptomatic [5, 11]. On gross examination the tumor will present as a cystic mass, which easily rupture when prepared or resected surgically. The tumor is usually encapsulated and separated by a thin fibrous band from the adjacent lung. On cut surface the mucus-filled cysts are easily visible (Fig. 17.3).
Fig. 17.3
Mucinous cystadenoma, resection specimen. The tumor is composed of numerous cysts filled with mucus. At the border there is macrophage reaction with many macrophages filled with mucus but also lipids (yellow areas)
Microscopy
They resemble ovarian cystadenoma. However, they present with unilocular or multilocular cysts lined by one layer of tall columnar or cuboidal cells either producing a serous fluid (Fig. 17.4) or mucus (Figs. 17.5 and 17.6). The nuclei are bland, often in a basal orientation; nucleoli if present are small and inconspicuous. Papillary projections may be found. In these areas stratification of cells and nuclei can be seen. A correct diagnosis is only possible on whole resection specimen, although a diagnosis might be suspected on biopsy (Fig. 17.7).
Fig. 17.4
Papillary cystadenoma in a main bronchus, obstructing the lumen. This type of tumor very closely mimics its ovarian cousin. H&E, ×100
Fig. 17.5
Mucinous cystadenoma in a main bronchus. H&E, ×12 (Case kindly provided by Dr. B. Papla, Krakow)
Fig. 17.6
Mucinous cystadenoma of the bronchus. Another case shows predominantly large acini filled with mucus. Overview left, and higher magnification right, showing bland cytology and a mixture of columnar cells including goblet cells. H&E, ×12 and 100
Fig. 17.7
Bronchial biopsy of a serous type of bronchial gland adenoma. On this biopsy the diagnosis can be suspected, because the glands are close together, too large for normal glands, and the cytology of the cells is bland. H&E, bar 200 μm
Differential Diagnosis
Within the differential diagnosis, adenocarcinoma in situ (AIS), colloid or mucinous carcinoma, and congenital pulmonary adenomatoid malformation (CPAM) have to be considered. Well-differentiated mucinous AIS cannot be separated from mucinous cystadenoma on small biopsies, the same applies to colloid carcinoma. The sharp demarcation and the lack of atypia in mucinous cystadenoma will be the main criterion. In contrast to mucinous cystadenoma, colloid carcinoma will present with discontinuous growth along alveolar septa. In cystadenoma, the cells form a continuous line along preexisting structures, either bronchi or alveoli. From CPAM types I and II, the cystadenoma can usually be separated more easily: the CPAM cysts (types I and II) are covered by normal bronchial-type epithelium, show mesenchymal cells within their cyst wall, and if present will exhibit atypical goblet cell hyperplasia, which is a focal goblet cell proliferation within the cyst epithelium [12].
Borderline Variant
In cystadenomas atypia can be present and similar to the tumors of the ovary have been called borderline tumors or variants of serous or mucinous cystadenoma (Fig. 17.8). The borderline variants are still encapsulated, but extensive sampling has to be done to exclude invasion. In these cases there is atypia of the nuclei, enlarged nucleoli, formation of cell papillae, and few mitoses including atypical mitosis [13–15].
Fig. 17.8
Mucinous papillary cystadenoma in a large bronchus. In this case a borderline variant. In (a) overview of the tumor with many cystic spaces filled by mucin. Already areas with higher cellularity are seen. (b) Area with bland cytology but already papillary projections. (c, d) show areas with cellular atypia, crowding, epithelial papillae, and nuclear atypia. H&E, ×12, 100, and 200 (Case kindly provided by Dr. B. Papla)
No data exist in the literature about genetic aberrations or specific markers, associated with the development of these adenomas.
Prognosis and Treatment
Prognosis is usually excellent, if these tumors are completely removed. In mucinous cystadenomas the removal might sometimes be complicated by rupture of the tumor, releasing mucus and within the mucus also tumor cells. This might give rise to recurrence. In case of a borderline variant of cystadenoma, a careful workup of the entire tumor is essential. There might be areas of invasion, which immediately change the diagnosis and also shifts this tumor into the group of mucinous adenocarcinomas.
17.1.1.2 Pleomorphic Adenoma
Pleomorphic adenoma of the lung is a rare tumor but otherwise resembles its counterpart in the large salivary glands [16]. It affects man and woman in almost all ages, except childhood, but most often older patients. There is no gender predominance.
Gross Pathology
Most cases reported so far arise from the large bronchi and may grow with an endobronchial portion into the lumen, causing obstruction. The size may vary from one up to several cm in diameter. On cut surface pleomorphic adenoma are well circumscribed but lack a well-defined capsule. The color is grayish red to myxoid white glistening, depending on the amount of myxoid and glandular areas.
Histology
Pleomorphic adenoma is composed of ducts and glands, resembling bronchial glands. A spindle cell component is usually present, whereas in contrast to the salivary glands chondroid metaplasia is rare; however, myxoid and hyaline changes of the matrix proteins can be seen. Myoepithelial cells are usually present and in some cases may form a major portion [5, 16–18] (Fig. 17.9).
Fig. 17.9
Pleomorphic adenoma of the bronchus. In the upper panel, an overview is shown; in the lower panel, there are ducts and acini embedded in a myxoid stroma with stellate cells. H&E, ×12 and 100
Differential Diagnosis
Pleomorphic adenoma is usually not difficult to diagnose correctly because of its benign cytological features and the biphasic nature. However, in cases with a prominent spindle or myoepithelial component, the differential diagnosis toward myoepithelioma or inflammatory myofibroblastic tumor might be rendered. Important features to correctly diagnose this entity include also positivity for pan-cytokeratin, S100 protein, smooth muscle actin, vimentin, and glial fibrillary acidic protein (GFAP) [1, 17, 19]. In rare instances within a pleomorphic adenoma carcinomas can arise [1, 19].
Immunohistochemistry
Pleomorphic adenoma will stain positively for pan-cytokeratin and high molecular weight cytokeratins (CK 5/CK 6); the myoepithelial component will be positive for S100 protein and smooth muscle actin; some epithelia and myoepithelial cells might coexpress vimentin together with cytokeratin and GFAP.
17.1.1.3 Myoepithelioma
Myoepithelioma is an exceptionally rare benign tumor arising in the central portion of the lung [21].
Macroscopy
Myoepithelioma usually presents as an endobronchial nodule obstructing the lumen. Myoepitheliomas can reach a size of 2 cm before causing symptoms. On gross sections this will be a grayish-white nodule, the cut surface being soft and fleshy.
Microscopy
Microscopically the tumor is composed of small glands and ducts, presenting with an inner cuboidal and an outer spindle or flat cell layer (Fig. 17.10). The inner cells are stained by cytokeratins, the outer layer in addition also by S100 protein antibodies. Mitoses are absent, and the nuclei are small and round, the chromatin is finely distributed, and nucleoli are inconspicuous. In a few cases spindle cell may predominate [22]. The mitotic rate is usually low with approximately 1 in 20 high-power fields.
Fig. 17.10
Myoepithelioma arising from a large bronchus. The tumor is composed of glandular epithelium surrounded by myoepithelial cells, which usually have a spindle cell appearance (upper panel). There are no mitoses and no nuclear atypia. The myoepithelial cells stain positively for S100 protein (lower panel). H&E, immunohistochemistry, ×200
Immunohistochemistry and Molecular Biology
The inner layer of the glands stains for cytokeratin, TTF1, and epithelial membrane antigen and the outer layer for S100 and smooth muscle actin. In one case the spindle cells stained for CD34. P27/kip-1 protein plays a fundamental role in the development of these neoplasms. It shows an aberrant cytoplasmic location [23]. In another investigation p63 expression was demonstrated in this tumor [21]. So TP63 might have an impact on the development of this tumor.
In the differential diagnosis, only myoepithelial carcinoma has to be considered. A proper sampling is essential, especially the basis of the tumor needs to be examined at multiple levels to exclude invasion, which is the only reliable criterion to separate these two entities. Myoepithelial carcinoma will usually show nuclear polymorphism and a few mitoses. Due to the fact that epithelial-myoepithelial tumor is difficult to separate from epithelial-myoepithelial carcinoma, some authors prefer to group these entities together. But a high mitotic rate, tumoral necrosis, and nuclear pleomorphism favor the carcinoma diagnosis [22].
17.1.1.4 Papilloma in Adult and Childhood
Epidemiology and Incidence
Pulmonary papillomas are rare epithelial neoplasms, often an incidental finding on bronchoscopy, for which no incidence is available. The incidence in children is somewhat higher; however, there are also no data on papilloma and papillomatosis in the literature. In our own experience, a papilloma diagnosis was rendered in 210 cases out of 48,676 cases within the lung and pleura collection. In only 25 cases, the diagnosis was confirmed by proving one of the HPV types. Out of these 25 cases, there were 7 cases with a proof of HPV 16 or 18, respectively, whereas in 18 cases, one of the non-oncogenic or facultative oncogenic types was identified. In children bronchial papillomas are often seen together with papillomas in the upper respiratory tract (tracheal, laryngeal, sinonasal papillomas) [24–26]. Papillomas are caused by human papilloma viruses, mainly by types 6b, 11, 16, 18, 31, 33, and 35 [27]. There are data pointing that infection especially in children do occur during delivery via an infected mother [28]. Adult patients are usually younger compared to carcinoma patients. In our experience it occurs always in smokers with a male predominance. There are no specific symptoms. Sometimes papillomas are associated with invasive squamous cell carcinoma [29].
Symptoms
Patients with papillomas present with hoarseness, cough, hemoptysis, wheezing, chest tightness, and non-purulent expectoration. Some patients have no symptoms and therefore papillomas are also found incidentally. Large papillomas might cause obstruction and poststenotic pneumonia. On bronchoscopy papillomas present as small round polyps, usually pedunculated with a broad stalk.
Radiographic Findings
Gross Findings
Papilloma can occur as solitary or multiple nodules or as diffuse proliferation. It projects into the lumen of bronchi like any other endobronchial tumor. Usually it presents as a small tan-white tumor (Fig. 17.11) with a small branching stalk and a smooth or verrucoid surface. However, the gyrated surface with a similar appearance as a colonic adenoma may guide the pathologist into the right direction. In cases of a papillomatosis, the gross appearance is less typical and might simulate features of other tumors (Fig. 17.12). On a closer look, one may appreciate the endobronchial growth and the composition of different nodular structures.
Fig. 17.11
Bronchial papilloma at the resection margin of an upper lobe resected because of squamous cell carcinoma. The papilloma shows the characteristic stromal tree structure and is covered by squamous epithelium. H&E, ×25
Fig. 17.12
Papillomatosis of the lung. Numerous papillary proliferations are seen in the large bronchi. Inset: enlarged view of the lesion (kindly provided by Dr. A. Moresi-Hauff, Gauting)
Histopathology
Papillomas are benign epithelial tumors, characterized by a treelike branching of the stroma, composed of thin-walled capillaries or veins with a hyalinized wall and few mesenchymal cells, covered by an epithelium, which can be either entirely composed of squamous cells or a mixture of cuboidal, cylindrical, and/or transitional cells. On surgical resection specimen, the diagnosis is apparent already on low-power magnification: the treelike structure of the mesenchymal stalk covered by multilayered epithelium, most often of the squamous nonkeratinized type, sometimes with certain degrees of dysplasia (Figs. 17.11, 17.13, and 17.14). Other types of epithelia such as columnar, transitional, and mixed type are found and will be acknowledged in the name [31–33]. Especially in the squamous cell papillomas, koilocytes are seen (Fig. 17.15), and by immunohistochemistry, by in situ hybridization, or by PCR, human papillomavirus can be detected [29, 34]. Koilocytes are characterized by halos close to the nuclei; in addition a bizarre outline of nuclei is usually detected in cases of HPV 16- or HPV 18-positive cases. Also “nuclear rupture” might be seen: nuclear membrane is vanishing and chromatin loses its staining intensity. When dealing with bronchial biopsies, the diagnosis might be not as easy. The papilloma is broken into parts; the treelike structure of the stalk is not seen in its entire length. Therefore one needs clearly defined criteria. The stroma should be branching, usually there is not much inflammatory infiltrate present, the capillaries or veins are thin-walled, the basement membrane is thickened, and there are only a few other mesenchymal cells present. The epithelium is composed of layers of squamous cells, focally showing koilocytic features (Fig. 17.13). In rare cases the epithelium might be of a transitional or even columnar type, in this case with or without goblet cells. In these cases koilocytes are usually not encountered. Dysplasia might be present; some may have carcinoma in situ [35] (Fig. 17.14).
Fig. 17.13
Bronchial biopsy showing fragments of a squamous cell papilloma. H&E, bar 100 μm
Fig. 17.14
Squamous cell papilloma with severe dysplasia. Nuclei are increased, epithelial layering and maturation of cells is lost, focal areas show already dyskeratosis of cells. H&E, ×100
Fig. 17.15
Koilocytes in a squamous cell papilloma. Around nuclei a halo is seen. The more the nuclei show bizarre configuration, the more likely they are infected by an oncogenic type of HPV. H&E, bar 100 μm
Immunohistochemistry
In all cases human papilloma virus (HPV) typing should be done. In the author’s experience, immunohistochemistry for low- and high-risk types is sufficient. HPV typing by in situ hybridization or by PCR might be done instead (Figs. 17.16 and 17.17) [27, 36].
Fig. 17.16
In situ hybridization for human papilloma virus type 6b. There are dense signals in many of the squamous cells. ISH, ×100
Fig. 17.17
PCR for common sequence in human papilloma virus GP5/GP6. A series of cases have been tested
Cytology
The diagnosis of a papilloma cannot be made on cytological specimen, although koilocytes might be correctly diagnosed. The diagnosis requires the structural features. However, by molecular investigation HPV can be detected, which together with koilocytes might be used to make a tentative diagnosis of papilloma.
Molecular Biology
HPV types are oncogenic due to their interference with cell cycle proteins [36, 37]. They can interact with p53 as well as with retinoblastoma gene protein. The most common HPV types in the upper and lower respiratory tract are types 6b, 11, 16, 18, 31, 33, and 35. Whereas types 16 and 18 are regarded as oncogenic types and infer a risk for progression of papillomas to invasive carcinoma, 6 and 11 are associated with recurrent papillomas/papillomatosis but rarely with carcinomas [38]. There is an exception to the rule: within the HPV genome, there are segments designated E1 through E7, which code for different functions. Within E2 there is a regulatory gene controlling the expression of the oncogenic E6 and E7 genes. If E2 is mutated in HPV 11, the virus will show an uncontrolled expression of E6 and/or E7 and thus behaves similar to the oncogenic HPV 16 and 18. This might be an explanation for case reports on HPV 11 in papillomas progressing to invasive squamous cell carcinomas [37, 39–45]. The oncogenic E6 and E7 sequences induce inactivation of p53 and rb1 proteins, respectively, and therefore these control mechanisms for DNA integrity are lost. In addition to an increased expression of p53 and Rb proteins, HPV also reduced the expression of p21 (WAF1) protein [46]. HPV-E6 protein also increases the activity of cyclin A-dependent kinase 2 (CDK2). Malanchi and coworkers also confirmed the downregulation of p21(WAF1/CIP1) and enabled cells to overcome the G1 arrest upregulating oncogenic RAS [36].
Cell of Origin
Detection of HPV by in situ hybridization will show a low amount of viral gene in the basal cells and increased reactivity in the middle and upper part of the epithelium. However, this most probably reflects expansion and multiplication of the viral genome or parts thereof. Most probably reserve cells of the bronchial mucosa are the target of HPV, which then will undergo transformation and expansion, giving rise to the papilloma [47].
Differential Diagnosis
There is no differential diagnosis to be considered in solitary papillomas. In papillomatosis the major differential diagnosis is squamous cell carcinoma. If papillomatosis is seen in children, then squamous cell carcinoma is most unlikely. In adults the differential diagnosis might be difficult or even impossible. Immunohistochemical stains for p63 proteins might be of help, as well as a positive stain for carboanhydrase IX and increased staining for the proliferation marker MiB1(Ki67).
Prognosis and Therapy
Papillomas are cured by surgical resection or laser abrogation. However, recurrence is common. The pathologist should insist that primarily a biopsy is taken, to make the proper diagnosis including HPV typing. Cases with proven oncogenic HPV are at high risk for development of recurrence and progression into squamous cell carcinoma [27]. These patients need to be closely monitored and controlled by bronchoscopy. Since in older age HPV genomic integration is most often accompanied by smoking habits, the risk of developing a squamous cell carcinoma is increased. The proportion of squamous cell carcinomas arising from papillomas is rare. However, a papilloma remnant can sometimes be seen in squamous cell carcinomas, usually in the exophytic part.
If a treatment with antiviral drugs has any effect, it is not clear; HPV vaccination in children might not prevent infection in all cases, since infection can occur during delivery by the infected mother. If vaccination has a protective effect of already infected children will be seen in follow-up observation studies [48].
Variants
Transitional Cell Papilloma
Transitional papillomas are characterized by their transitional epithelium covering an otherwise identical branching stroma stalk. Transitional papilloma often present as a mixed type with squamous cell papilloma (Fig. 17.18).
Fig. 17.18
Variants of HPV-induced papilloma, a mixed squamous and columnar type of papilloma (upper panel), and a columnar cell papilloma (lower panel; this one kindly supplied by B. Papla). H&E, ×150
Columnar Cell Papilloma
Columnar cell papilloma is extremely rare. A few cases have been reported. Columnar cell might be mucin-producing goblet cells or secretory columnar cells (Fig. 17.18). HPV might be detected in this type of papilloma, similar to columnar cell proliferations in the cervix, but might also be negative for HPV. Sometimes cellular atypia can be seen as in the case presented here. So far HPV testing was not reported.
Squamous Cell Intrabronchial Papillomatosis
There exists a rare diffuse form of papillomatosis [25, 49]. A whole lung lobe can be affected. Papillomatosis is characterized by an intrabronchial growth of papillomas, which all together form a multinodular lesion (Figs.17.12, 17.19, 17.20, and 17.21). In papillomatosis the normal columnar epithelium is replaced by metaplastic often dysplastic squamous epithelium, and there is an ingrowth of mesenchymal stalks into the bronchial lumen. Many of these papilloma projections grow and spread intrabronchially, completely filling the lumina. They may grow down into the bronchioles and alveolar ducts and reach the centrilobular portion of the alveoli (Figs. 17.20 and 17.21). This entity is different from what some authors call recurrent papillomatosis, namely, using the term for multiple papillomas spreading within the upper and lower airways [50]. This should instead be called multifocal papilloma but not papillomatosis. Papillomatosis can occur in children as well as in adults. In the newborn the disease is probably a result of infection with papilloma viruses during delivery. Recurrent upper respiratory tract papillomatosis in children younger than 10 years of age will more likely spread to the lower respiratory tract [24, 26, 49]. These tumors undergo spontaneous regression in puberty and usually do not progress to a malignant form. HPV is present in all cases. The oncogenic types 16 or 18 prevail in adults, whereas HPV 6 or 11 in children. In 5 % of the cases it involves lower airways and presents with the symptoms of pneumonia, hemoptysis, and asthma. The dignity of papillomatosis is difficult to assess: due to the fact that the papillomatosis fills the airways completely and may exhibit some nuclear atypia because of the presence of the viral genome, malignant transformation is hard to prove and invasion hard to rule out. The differentiation from microinvasive squamous cell carcinoma developing from papillomatosis can in some cases be impossible (Fig. 17.19c). Radical surgical resection is recommended and the only choice for treatment.
Fig. 17.19
Squamous cell papillomatosis, positive for HPV16. (a) Overview of the papillomatosis, the growth along the bronchial tree is evident; however, squamous cell carcinoma can be suspected. (b) Area of squamous cell metaplasia, the epithelium replaces the original columnar epithelium. (c) Another area suspected as representing invasive squamous cell carcinoma arising in papilloma. H&E, ×12 and 200, bar 100 μm
Fig. 17.20
Squamous cell papillomatosis in a 10-year-old girl, positive for HPV11. H&E, ×150
Fig. 17.21
Same case of papillomatosis, here the koilocytes can be seen. There is some nuclear atypia with polymorphism. H&E, ×200
Glandular papillomatosis has never been described, but in the European rare pulmonary disease collection, a case of intrabronchial papillomatosis has been collected. This proliferation is characterized by an intrabronchial growth of benign-looking papillary structures, covering a delicate mesenchymal stalk. The nuclei of the epithelial proliferation are small and round, chromatin is evenly distributed and fine, and nucleoli are inconspicuous or absent. The lesion is characterized by a bronchiolar columnar and cuboidal cell proliferation forming a single row without mitosis, which extends into the alveolar region replacing normal pneumocytes (Fig. 17.22). This lesion resembles intraductal papillomatosis of the breast. The biological behavior is uncertain, and no follow-up data are available.
Fig. 17.22
Glandular papillomatosis, a lesion so far unknown to the literature. (a) Overview showing an adenoma-like lesion, however, not encapsulated. (b) Higher magnification, some calcifications are within the central area. (c) shows the extension of the papillomatosis into adjacent bronchi and bronchioles. The tumor grows in a similar fashion as intraductal papillomatosis of the breast. (d) High-power view shows a bland tumor epithelium without atypia and organized in a single row of columnar cells, some cells with signs of apocrine secretion. H&E, bars 300, 60, 100, and 30 μm
17.1.1.5 Papillary Adenoma
Definition and Incidence
Radiographic Findings
The tumor presents as a well-circumscribed small peripheral solitary nodule on CT scans.
Gross Pathology
These tumors present as solitary, rarely as multinodular lesion. The nodules are usually small and well circumscribed, measuring from a few mm up to 3 cm in diameter. Cut surface is grayish red and soft and can show a variety of features, such as hemorrhage and sclerosis/scaring. The tumor does not contain a capsule but compresses the adjacent lung parenchyma.
Histopathology
Microscopically the tumor is well circumscribed and shows papillary structures composed of fibrovascular stroma covered by uniform cuboidal to columnar cells, which resemble type II pneumocytes. Clara cells and ciliated cells are present, but also solid areas can be found (Figs. 17.23 and 17.24). Nuclei of cells have typical cytoplasmatic invaginations. Surrounding lung parenchyma can show features of organizing pneumonia, fibrosis, or simple pneumonia due to bronchial obstruction and mucostasis.
Fig. 17.23
Papillary adenoma, a rare tumor arising in the peripheral lung. Upper panel shows the many papillary projections; in the lower panel, details of the papillae are seen. The bland-looking stroma is covered by a single row of columnar or cuboidal cells, some of them resembling Clara cells. Note the bland morphology, without mitosis or nuclear atypia. H&E, ×50 and 200
Fig. 17.24
Papillary adenoma, other case. (a) Low-power overview of the tumor with papillae covered by bland-looking epithelium. In (b) the epithelial cells are cuboidal, some with features of Clara cells. (c) Immunohistochemistry for cytokeratin 7 shows positivity of the single epithelial layer. H&E, ×50, 100, immunohistochemistry ×100
Immunohistochemistry and Molecular Biology
Immunohistochemically, the tumor cells react positively with antibodies for surfactant apoproteins, carcinoembryonic antigen, cytochrome oxidases P-450 1A1/A2 and 2B1/B2, surfactant apoproteins, and Clara cell proteins. Also a positive reaction for TTF1 can be demonstrated. Tumor cells have microvilli but also may develop lamellar bodies. Papillary adenoma is a benign, and morphologically distinctive tumor, which may develop from Clara cells or pneumocyte type II precursors [51–53]. Ultrastructurally many osmiophilic lamellar bodies and electron-dense granules can be demonstrated [51–55]. No mitotic figures, no necrosis, and no mucin can be found.
In experimental mouse model papillary and lepidic pulmonary adenomas could be induced by fibroblast growth factor 10 expression. FGF10 might induce a differentiation of cells to a pneumocyte type II phenotype. Withdrawal from FGF10 expression caused regression of the tumors and loss of the differentiation markers TTF1, SP-B, and proSP-C [56].
Prognosis and Treatment
Radical surgery is curative in almost all cases described so far. However, as stated above atypia and invasion has to be ruled out.
The main differential diagnoses are sclerosing hemangioma/pneumocytoma and carcinomas (primary and metastatic). The absence of atypia, mitosis, and necrosis are features of this tumor. Invasion should be ruled out. However, in two reports the dignity of papillary adenoma have been questioned: Mori et al. [55] described a case, which showed vascular invasion and also dissemination into the surrounding lung. Invasion of the lung and pleura was reported by Dessy and colleagues in their papillary adenoma [53]. The papillary adenoma can be differentiated from intrabronchial glandular papillomatosis because it is a solitary tumor, does not grow intrabronchially, and compresses the adjacent lung.
Biphasic Papillary Adenoma and Myomatous Hamartoma
This tumor was excised with the clinical diagnosis of a benign-looking cystic tumor.
Histologically it was composed of a papillary adenoma with cuboidal and columnar cells, some containing cilia. In addition there were smooth muscle cells in the walls of the cysts, and in the center of the tumor was a myomatous variant of hamartoma embedded. The tumor stained for low molecular weight cytokeratin 7, apoprotein A and B antibodies, and the mesenchymal cells were positive for HHF35, smooth muscle actin; in addition there was an increase of ACTH-positive neuroendocrine cells within the papillary cysts. Similar tumors have been reported. Matsumoto reported on a mixed tumor of the salivary gland type with chondromyxoid stroma. In the glandular wall, myoepithelial components and cartilage formation were embedded. The glandular component was positive for TTF1 and the surface lining cells were positive for surfactant apoprotein A [57]. The five cases reported by Chang showed glandular and spindle cell differentiation. The inner, cuboidal epithelial cell layer (positive for panCK, EMA, and TTF1; some glands also positive for surfactant apoprotein A) was surrounded by an outer layer of myoepithelial cells (high molecular weight CK, S100, SMA, calponin, caldesmon, and p63 positive) [58].
17.1.1.6 Sclerosing Pneumocytoma (Formerly Sclerosing Hemangioma)
Clinical Symptoms and Epidemiology
The tumor is more common in middle-aged women (more than 80 %; mean age 40–45 years) and is rare in Europe and North America but frequent in Asia (Japan and China). The tumor is asymptomatic and usually incidentally detected by chest X-ray. Most often there is a single tumor nodule, but also multiple tumors have been reported [59–61]. The tumor grows slowly and is expansive, but few cases of malignant metastasizing sclerosing pneumocytoma have been reported [62–65].
Misnomers
The name hemangioma is misleading and originally was selected because the first cases presented with massive bleeding and central sclerosis [66]. The epithelial proliferations at the border were erroneously interpreted as reactive [67]. Other terms used for these tumors were pneumocytoma, papillary pneumocytoma, benign sclerosing pneumocytoma, and post-inflammatory pseudotumor. At that time immunohistochemistry was not available and the hemorrhagic part of the tumor was interpreted as hemangioma. In consecutive reports the epithelial nature of the tumor was finally proven [68, 69]. Therefore in the last WHO classification, the name was changed back to sclerosing pneumocytoma [70].
Radiographic Findings
An inhomogenous enhancement is frequently present in sclerosing hemangioma, especially in sclerotic and predominantly papillary types. The presence of a tail sign, intra-tumoral cystic areas, and a prominent artery sign are the most frequent features [71]. In one case also positron emission tomography (PET) was performed, and an increased accumulation of 18F-fluorodeoxyglucose (FDG) was demonstrated [72].
Macroscopy
The tumor can present as an endobronchial mass [73], but more often the tumor is embedded within the lung parenchyma forming a solitary nodule of less than 3 cm [74–76]. There are usually no clinical symptoms except in the rare endobronchial variant, which will cause bronchial obstruction. On cut surface they are grayish red to white in color; the different parts may stick out by their different color composition (Fig. 17.25a). They can be located peripheral as well as central endobronchial [62–64].
Fig. 17.25
Sclerosing pneumocytoma (formerly sclerosing hemangioma). (a) Resected tumor with hemorrhage, one of the reasons for the old name. (b) Tumor with sclerosis in the center. (c) Other case with mild fibrosis. (d) Border between tumor and sclerotic center. (e) Tumor front shows mature-type pneumocytes and immature cells. (f) In rare cases as this one giant lamellar bodies released from pneumocytes can be seen. (g) High power showing the two types of pneumocytes, mature at the surface and immature at the stroma of the tumor. H&E, ×12, 100, and 250
Histopathology
SP can present with a variety of morphological features: there might be central hyalinization, areas of thin-walled veins with extensive hemorrhage (therefore the original name), and areas of papillary epithelial proliferations (Fig. 17.25). Sometimes parts with hyalinization, hemorrhagic areas with hemosiderin-laden macrophages, xanthoma cells, calcification, or chronic inflammation can be quite large [76]. There are two different cell populations: the one at the surface is mature and of a pneumocyte phenotype positive for TTF1, surfactant apoproteins, and low molecular cytokeratins, whereas the cells within the stroma are immature pneumocytes, epithelioid, and round to polygonal with eosinophilic sometimes clear cytoplasm (Fig. 17.25). The tumor cells are large cuboidal epithelial cells forming sheets but also can present as single cells, embedded in a hyalinized stroma. Nuclei are round to oval with inconspicuous nucleoli. Cells lining the papillary structures are cuboidal with prominent nucleoli and cytoplasmatic invaginations. Mitotic figures are very rare, but some tumors will present with atypia and with multinucleated tumor cells. Both types of cells seem to belong to the tumor, based on findings of their monoclonality [77].
Immunohistochemistry and Electron Microscopy
Immunohistochemically the tumor cells at the surface will positively stain for EMA, pan-cytokeratin, surfactant apoproteins, carcinoembryonic antigen, and Clara cell proteins. Epithelioid cells within the alveolar walls are positive for surfactant apoproteins [74], TTF1, and EMA (Fig. 17.26) but negative for cytokeratins and thus may be transformed pneumocytes [68, 78, 79]. Ultrastructural investigations confirmed that the tumor cells are of epithelial origin, most probably pneumocyte precursor cells [80], because they present with whorled bodies and lamellae, which resemble those of type 2 pneumocytes. However, differences were also found, namely, the loss of the apico-lateral and a restricted surface differentiation in the stroma cells, whereas the surface cells form small lumina, microvilli, and multivesicular or multilamellar bodies. It was concluded that sclerosing hemangioma is a proliferation of rather fetal type II pneumocytes [80]. The higher frequency of in women prompted the search for hormonal determinants: these tumors are positively stained by progesterone and estrogen receptor antibodies [81].
Fig. 17.26
Sclerosing pneumocytoma, positivity of many tumor cells for surfactant apoprotein A. This demonstrates that these tumors are derived from primitive pneumocytes. Bar 10 μm
Molecular Biology
Sclerosing hemangioma has been extensively investigated for their cells of origin and for other factors related to their occurrence. In a loss of heterozygosity study, Dacic and coworkers studied sclerosing hemangioma at a few microsatellite markers and found frequent allelic loss on chromosomal arms 5q and 10q. When compared with bronchioloalveolar carcinoma, the microsatellite marker D5S615 was significantly more frequently affected than in BAC. The authors concluded that a putative tumor suppressor gene located on the chromosomal arm 5q might play a role in tumorigenesis of sclerosing hemangioma [82]. In an immunohistochemical investigation, the surface as well as the polygonal stroma cells of sclerosing hemangioma expressed MUC1, thyroid transcription factor 1 (TTF1), and EMA. Thomsen-Friedenreich antigen and cytokeratin were positive only in the surface cells. It was concluded that the two types of cells in sclerosing hemangioma might derive from a common precursor cell through divergent differentiation toward the type II pneumocyte during tumorigenesis [83]. Expression of EGFR but not Her2 was observed in the several cases of sclerosing pneumocytoma. Analysis of gene mutations in EGFR and HER2 and also KRAS sequencing did not reveal molecular alterations, whereas allelic losses at p16 and Rb loci were observed in few cases [84]. Both cell types of SP are positive TTF1, EMA, β-catenin, E-cadherin, and VEGF. In SP no CpG island methylation was seen for p16(INK4a) but low for RASSF1A gene methylation was seen for HOX D9, D11, and D13 in approximately one third of cases, respectively [85]. In several cases of sclerosing pneumocytoma, the tuberous sclerosis complex (TSC) pathways were investigated. The mammalian target of rapamycin (phospho-mTOR), phospho-Akt, hamartin, and tuberin, as well as hyperoxide inducible factor (HIF-) 1alpha and VEGF showed a positive reaction implying that aberrant mTOR signaling may play a role in the development of sclerosing pneumocytoma [86]. The finding of metalloproteinase 9 (MMP-9) in sclerosing pneumocytoma was discussed in relation to the locally invasive growth pattern, whereas the proof of tubulin-alpha was discussed as being responsible for sclerosis [87].
Differential Diagnosis
Inflammatory pseudotumor, clear cell “sugar” tumor (PECOMA), some primary and metastatic carcinomas, and low-grade epithelioid hemangioendothelioma have been considered as probable differentials. Inflammatory pseudotumor usually can easily be separated, because of the mixture of plasma cells, myofibroblasts, smooth muscle cells, as well as histiocytes. Even pure histiocytic variants will morphologically look differently. The clear cell tumor might look similar but will not present with the two cell types mentioned above. Epithelioid hemangioendothelioma might give problems in the differential diagnosis: the sclerotic areas can look similar, hemorrhage is sometimes present, but the pseudo-signet ring cell appearance of the tumor cells within sclerotic areas will usually guide one toward the correct diagnosis. A positive reaction for surfactant apoprotein A and a negative one for endothelial markers will help in making the right diagnosis. Primary and metastatic carcinomas can look similar to sclerosing pneumocytoma – this is especially true for metastatic lobular breast and prostate carcinomas. Since nuclear atypia and mitotic counts are not immediately apparent, immunohistochemical stains might be necessary.
Prognosis and Natural History: Treatment
Unusual associations of sclerosing pneumocytoma were reported for familial adenomatous polyposis [88] and also for Cowden syndrome [89]. If this association is incidental or not cannot be answered yet. SP is a benign tumor. However, malignant variants have been reported. Chan reported lymph node metastasis in one case [62], Iyoda and Wei each recurrences in two other cases [75, 90]. Komatsu and coworkers reported intrapulmonary metastasis [63]; however, this might be questioned, since in another report multifocality was seen with several sclerosing pneumocytomas in both lungs [60]. So sclerosing pneumocytoma might be locally invasive but still has to be regarded as benign – a death due to tumor burden has not been seen in any patient.
17.1.1.7 Alveolar Adenoma (Pneumocytoma)
Epidemiology and Incidence
Alveolar adenoma/pneumocytoma is a rare tumor or tumorlike lesion. It was first described by Yousem and Hochholzer [91]. There are no data about epidemiology, and the incidence has never been assessed. Alveolar adenoma can be regarded as either a developmental disease or a tumor. It is formed by numerous alveoli without any connection to bronchioles or bronchi. Since there is no connection with the bronchial tree other then the channels of Lambert and the pores of Kohn, alveolar adenoma usually present with enlarged and cystic dilated alveoli. These will steadily increase, because lipids and proteins produced by the epithelia are drained insufficiently.
Special Clinical Features
Patients are of older age and equal sex. Alveolar adenoma is incidentally detected, because it will cause compression and thus atelectasis of the adjacent normal lung and consequently causes hypoxia, if large enough. Clinically alveolar adenomas are usually asymptomatic and detected accidentally in routine chest X-ray.
Radiographic Findings
On X-ray only large alveolar adenomas will be detected because of its translucent feature. On HRCT it presents as a cystic translucent well-circumscribed emphysema-like lesion. If the adjacent parenchyma is compressed, the more likely radiologists will be able to correctly diagnose this tumor.
Macroscopy
Alveolar adenoma present as a multicystic structure surrounded by normal lung parenchyma, the latter might be atelectatic. Macroscopically, tumor is solitary tan or grayish white and 1–2 cm in diameter. The mucus-filled cystic structures are easily identified. Secondary bleeding into the cysts can occasionally obscure the tumor and make the diagnosis difficult.
Histopathology
The adenoma is composed of cystic structures lined by flat or cuboidal pneumocytes (Fig. 17.27). Within the lumen PAS-positive material (surfactant proteins) is usually found. Foamy macrophages can be seen with ingested PAS-positive material. There are no bronchi or bronchioles and consequently also no medium-sized blood vessels. The entire tumor is composed of enlarged alveoli. Ultrastructurally the epithelial cells contain lamellar bodies and blunt surface microvilli and other features of type 2 pneumocytes. The interstitium between the cysts varies in thickness and contains myxoid collagenous matrix and myofibroblasts.
Fig. 17.27
Alveolar adenoma, (a) a case with large cystic spaces without a connection to bronchioles. (b) Alveolar adenoma with bleeding and compressed adjacent lung. Again no bronchioles are seen. Higher magnification of second case, showing flat pneumocytes covering the surface of the cystic dilated alveoli. H&E, ×12, 25, 100
Immunohistochemistry
In case of uncertainties, immunohistochemical stains using surfactant apoprotein antibodies will highlight the alveolar lining cells. These cells are also positive for TTF1.
Differential Diagnosis
Other cysts within the lung are the main differentials: in emphysema bronchioles can be seen, and thus this will be not a complicated differential diagnosis. Bronchogenic cyst will present with a bronchial epithelium, a wall with smooth muscle cells, and rarely also cartilage. Congenital pulmonary adenomatoid malformation (CPAM) of type I and II can be separated, because the epithelium again is of bronchial type and within the cyst wall elements of normal bronchi can be found. In addition CPAM is a disease most often found in children, whereas alveolar adenoma is most often detected in older patients of both sexes. Atypical adenomatous hyperplasia (AAH) might be occasionally difficult to differentiate from alveolar adenoma if there are a lot of inflammatory changes in the adenoma. In these adenomas the pneumocytes are enlarged and show reactive changes. However, atypia is not as prominent as in AAH. In addition in AAH, respiratory bronchioles and alveolar ducts can be found in the vicinity, which would be not seen in the adenoma. Lymphangiomatosis and lymphangioleiomyomatosis might be other problematic lesions to differentiate. Lymphangiomatosis presents with cystic spaces, but the endothelial cells are usually flat. The cyst walls are usually fibrosed. Immunohistochemical stains for cytokeratins or podoplanin (D2-40) will unequivocally enable the correct diagnosis. Lymphangioleiomyomatosis also can present with multiple cysts. However, the cyst epithelium is flat, and usually at some foci areas of myoblast, proliferations will be seen, which enable the correct diagnosis.
Prognosis and Natural History
The etiology of alveolar adenoma is unknown. The process represents most probably a neoplastic epithelial proliferation predominantly of type II pneumocytes. Alveolar adenoma grows slowly, there are no mitoses, and only isolated nuclear staining for Ki-67 can be found in pneumocytes at the peripheral part of the tumor. These tumors do not infiltrate, recur if resected, and do not metastasize.
Molecular Biology
Roque et al. showed a clonal chromosomal aberration of der(16)t(10;16)(q23;q24) in 19 % of the cases. This may serve as an argument of a neoplastic character [92].
17.1.1.8 Multifocal Nodular Pneumocyte Hyperplasia (MNPH)
Epidemiology and Incidence
Micronodular pneumocyte hyperplasia is a tumorlike lesion associated with tuberous sclerosis and lymphangioleiomyomatosis [93]. Its incidence is unknown, but it is more rare than lymphangioleiomyomatosis (LAM). In contrast to LAM, MNPH is not restricted to females but can also occur in men. No clinical symptoms are recorded.
Radiology
MNPH might present with subtle cystic changes, if many nodules are present. However, cystic changes are not as pronounced as in LAM.
Macroscopy
This lesion is composed of multiple small nodules within the peripheral lung. The nodules are clinically silent and are therefore detected incidentally on HRCT. Their size can be between 2 and 8 mm. The nodules are tan white well demarcated from the lung. Small cysts are always present but macroscopically are inconspicuous.
Microscopy
Histologically the nodules are composed of tumor cells, which form nests and groups on the surface of alveoli but also within alveolar septa (Fig. 17.28). The cells resemble immature and mature pneumocytes without atypia, which will stain for low molecular weight cytokeratins, EMA, and surfactant apoproteins [93, 94]. The cells proliferate within alveolar septa. There is no progression into malignant tumors known and even no progression of these lesions into larger tumors.
Fig. 17.28
Multifocal nodular pneumocyte hyperplasia (MNPH). (a) A case with several nodules without a capsule. (b) High magnification of the cells, which clearly resemble pneumocytes type II. (c) Many of the cells are positive for glycogen. (d) A small MNPH focus with predominantly immature pneumocytes. (e) Immunohistochemistry for surfactant apoprotein A. H&E, ×50, 250, 100, PAS, ×100, immunohistochemistry, bar 10 μm
Molecular Biology
A germline mutation of TSC2 and an immunohistochemical staining for tuberin have been demonstrated in LAM as well as in MNPH [94–96]. In another investigation mutations have been found in TSC1 and TSC2 [97]; therefore, MNPH is associated with tuberous sclerosis complex. Mutation of the TSC1 and TSC2 genes induces genetic instability, a reason for the occurrence of several benign tumors [98, 99].
Differential Diagnosis
The differential diagnosis is meningothelial nodules and chemodectomas (negative for cytokeratin and surfactant ApoA) and AAH (no growth within alveolar septa). Morphologically MNPH resembles cells of sclerosing pneumocytomas (both are based on a proliferation of cells differentiated into pneumocytes). So it was speculated that MNPH might be a precursor lesion of sclerosing pneumocytoma. However, this has never been proven. Even more, no case of sclerosing pneumocytoma was reported with adjacent MNPH lesions, and in addition sclerosing pneumocytoma does no exhibit mutations in the TSC genes but alterations of the mTOR pathway [86]. In other investigations some similarities were found between sclerosing pneumocytoma and pulmonary in situ adenocarcinomas [84, 85]. Based on these studies, MNPH and sclerosing pneumocytoma are most probably initiated by two different pathway activations but resulting in tumors of pneumocyte lineage, the former without, the latter with malignant potential.
Prognosis and Therapy
This proliferation is benign; all cases so far reported never developed into larger tumors or malignant neoplasms. No therapy other than resection has ever been applied. Resection, however, in all instances was done on the basis of a suspicious clinico-radiological diagnosis of either early malignancy or LAM.
17.1.1.9 Endometriosis
Although endometriosis is not a tumor, it most often presents as a tumor. In cases with pleural location, it causes pain and effusion, usually hemorrhagic. In addition it presents with multiple small nodules and is therefore mistaken as metastasis on X-ray and CT scan examinations. Intrapulmonary endometriosis is mistaken as a tumor for two reasons: by CT scan, it appears either as a single nodule or with multiple small nodular densities, again suspected as malignant tumor. Histologically misdiagnosis is also not uncommon; the proliferation is regarded as mixed epithelial-mesenchymal tumor.
Endometriosis can occur in the lung as well as in the pleura. Endometrial glands are surrounded by stroma cells. Fresh and old hemorrhage are present within this lesion. Cytokeratin antibodies can stain the epithelium, whereas the stroma cells will express estrogen and progesterone receptors (Fig. 17.29). The most important aspect is to not overdiagnose this lesion for a metastatic carcinoma or worse a carcinosarcoma.
Fig. 17.29
(a) Intrapulmonary and pleural endometriosis. The pulmonary lesion is composed of bland endometrial glands, some of them with Arias-Stella phenomenon. Between the glands typical endometrium-type stroma cells as seen, many of them transformed. Note fresh and remnants of old hemorrhage. B-E another case of endometriosis in the pleura. (b) Overview with thickened stroma of the pleura and lymphocytic infiltrations (right lower corner). (c, d) Shows two endometriosis foci, in c the epithelial nature is obvious, whereas the focus in d might be taken as mesenchymal proliferation. By immunohistochemistry using cytokeratin 14 antibodies, the epithelial nature of this focus is confirmed, but also negative stroma cells are seen. H&E, ×150, bars 100, 50, and 20 μm
17.1.1.10 Intrapulmonary Thymoma
Epidemiology and Incidence
Intrapulmonary thymoma is an extremely rare primary tumor within the lung. There are no data about the incidence of these tumors, only single case reports. More common are metastatic thymomas or thymomas infiltrating the lung directly from the mediastinum. The pathogenesis of these tumors is subject of speculation; an embryonal displacement of thymic “anlagen” is suspected or its development from germ cells [100].
Clinical Presentation
Some intrapulmonary thymomas present with myasthenia gravis, similar to their mediastinal counterparts [72], but most of them will not cause symptoms, other than due to enlargement and obstruction.
Radiographic Findings
Radiologically thymomas will present as solitary nodules up to 10 cm in diameter, usually well circumscribed.
Macroscopic Pathology
Macroscopically tumors are up to 10 cm and are located in the hilum, in the peripheral lung, or subpleuraly [101]. These will show a thin fibrous capsule and are clearly demarcated from the surrounding lung parenchyma.
Histopathology
Histologically primary pulmonary thymomas present most often as thymomas type A or AB, rarely other types are seen (Fig. 17.30). Malignant variants, such as type B3, are exceedingly rare among these rare tumors. Most often the spindle cell component (type A) will predominate; type B1 areas are usually restricted to small foci within the tumor [102, 103]. Consequently the lymphocytic compartment is not as prominent as it is seen in the mediastinal AB thymomas. The nuclei are small, chromatin is finely dispersed, and nucleoli are either absent or inconspicuous. Fibrous septa usually separate the spindle cell clusters. The diagnosis might be achieved by fine needle aspiration cytology, but enough material is necessary, to perform the necessary immunocytochemical stains. A rare type A3 malignant variant of the A-type thymomas has also been seen, but in these cases a metastasis from the mediastinum has to be excluded.
Fig. 17.30
Intrapulmonary thymoma, (a) shows an area of B1 type, in (b) there are infiltrating tumor cells with dense desmoplastic reaction, in (c) immunohistochemistry for pan-cytokeratin, staining the epithelial tumor cells. H&E and immunohistochemistry, bars 20 μm
Immunohistochemistry
The tumor cells are positive for cytokeratin (cytokeratin 19) and EMA; the lymphocytes stain for CD45RO, CD4, CD8, CD99, and Tdt.
Prognosis and Treatment
Tumors are benign, and they are best removed by complete excision. Cases which present with myasthenia gravis will behave worse than cases without clinical syndromes [102]. There are rarely malignant variants of thymomas reported [101]; however, when dealing with a thymoma of types B2 or B3, one should primarily exclude metastasis from a mediastinal primary.
Differential Diagnosis
There are many different tumors to be considered in the differential diagnosis of intrapulmonary thymomas: spindle cell carcinoid, metastasizing leiomyoma, inflammatory pseudotumor (myofibroblastic tumor), pleomorphic carcinoma, and malignant lymphoma.
Spindle cell carcinoids will show a rich vascular network and stain for neuroendocrine general markers (chromogranin A, synaptophysin); metastasizing leiomyoma on high-power magnification will clearly show myofilaments in their cytoplasm, which can be highlighted by smooth muscle markers (e.g., smooth muscle actin). In both tumors there is no lymphocytic focus, where spindle cells are intermingled with immature lymphocytes. Inflammatory pseudotumor, especially the mixed variant, can be problematic to differentiate on H&E stains alone. The infiltrative growth of IPT will be one clue to the correct diagnosis, and a positive cytokeratin or EMA stain will definitely exclude IPT.
Pleomorphic carcinomas of the lung, especially the pure spindle cell carcinoma, might be problematic too. But, nuclear polymorphism, frequent mitosis, and again an invasive growth into the surrounding lung will easily separate these two spindle cell neoplasms. Malignant lymphoma is not a problematic differential diagnosis in this setting. High-grade non-Hodgkin lymphomas are easily separated due to their nuclear polymorphism, large nuclear and nucleoli size, and coarse chromatin. This differential diagnosis might only be considered, if dealing with a B3 thymoma. In this case an immunohistochemical stain for cytokeratin 19 will clearly rule out the lymphoma. Low-grade lymphomas, such as marginal zone lymphomas of MALT/BALT type on the other end of the spectrum, are rarely a differential diagnosis, because of their rich lymphoid cell infiltration and the absence of spindle cells or thymoma cells of the type B1. Again cytokeratin 19 will highlight the thymoma cells, whereas the lymphoma will be negative.
A rare differential diagnosis, which might create considerable problems, is a metastasis from a biphasic endometrial stroma tumor/sarcoma. Morphologically the epithelial components will be clearly separated from the spindle cell component, the lymphocytic background infiltration will be more scarce, and these lymphocytes will express mature lymphocyte markers, such as CD20 and CD3. The tumor cells will express cytokeratins and also CD5 and CD10.
Molecular Biology
There are no genetic data for intrapulmonary thymomas and also only scarce data for those arising in the mediastinum. Comparative genomic hybridization has been performed on mediastinal thymomas and also some markers have been evaluated, but if these data can be transferred, also intrapulmonary thymomas has to be confirmed [104–106].
17.2 In Situ Carcinoma and Precursor Lesions
17.2.1 Preneoplastic Lesions – Squamous Cell Dysplasia
Actually our knowledge on preneoplastic lung lesions is limited: for a few carcinomas, the preneoplastic lesion is known, for most carcinomas not. In addition there is no good knowledge about driving genes, which induce progression into carcinoma. We still do not know the factors responsible for progression, invasion, etc. Molecular biology of these preneoplasias is mentioned where necessary; otherwise, these will be discussed in the next subchapter on malignant epithelial tumors.
For squamous cell carcinomas, the preneoplastic lesion is known for a while [107]. In the large bronchi, there exists a protection program for toxin/carcinogen exposure, which goes from goblet cell hyperplasia to squamous cell metaplasia and on to squamous cell dysplasia or intraepithelial neoplasia [108]. So far only a few factors influencing this progress have been identified, such as TP53 mutations, allelic loss and/or inactivating mutations of FHIT on 3p14, and inactivation of genes on 5p like AP1. VEGF seems to play a role in progression, since it is upregulated in rapid progressive squamous cell dysplasia with prominent vascular intraepithelial growth pattern. However, the interrelation of genes upregulated and downregulated has not been elucidated, so we are presently limited to single gene abnormalities, which do not allow understanding the process fully.
The grading of squamous cell dysplasia is quite subjective. In analogy to the cervix, also in the bronchus grading of dysplasia is done by the presence of cytological atypia, mitosis, and pattern. Whereas grade 3 does not cause a problem for most pathologists, the differentiation of grades 1 and 2 from reactive or regenerative proliferation in squamous cell metaplasia will cause disagreement between different experienced observers. Below are given the official definitions of grading of squamous dysplasia. It should be noted that there are discussions to reduce grading to low- and high-grade dysplasia, where high grade corresponds to grade 3 whereas low grade includes grade 1 and 2. We will follow this two-tiered graduation.
Low grade (G1+G2): expanded basal cell layer into the lower two thirds of the epithelium, mild to moderate atypia (anisocytosis, pleomorphism), maturation in the upper third, and rare mitosis.
High grade (G3): expansion of the basal cell layer up to the surface, complete loss of orientation, atypia present in all layers, which results in an epithelium looking the same in the basal and apical site, mitosis in all layers (Figs. 17.31 and 17.32).
Fig. 17.31
Squamous cell dysplasia (intraepithelial neoplasia). (a) Low-grade dysplasia with expansion of basal cells into the middle of the epithelium; a few atypical cells are seen, one with dyskeratosis. (b, c) High-grade dysplasia, the atypical basal cells have replaced the normal epithelium throughout the whole thickness; atypia and mitosis is seen until the surface. (d) High-grade dysplasia with transition into in situ squamous cell carcinoma within a bronchial gland; there is dyskeratosis, apoptosis of single cells, and high-grade nuclear atypia. H&E, bars, 50 μm
Fig. 17.32
Squamous cell dysplasia (low grade) with sharp transition from the adjacent goblet cell hyperplasia. This sharp demarcation is helpful when deciding on dysplasia. H&E, 100 μm
The vascular variant of dysplasia is characterized by an ingrowth of capillaries into the squamous epithelium (Fig. 17.33). Atypia and mitosis might be mild or even absent; however these are rapid progressive lesions due to the expression and release of VEGF [109].
Fig. 17.33
Vascular variant of squamous cell dysplasia. (a, b) Show two cases of this type of dysplasia. The cellular features as well as nuclear atypia are mild. (c) Shows the ingrowth of ill-formed capillaries into the epithelium. H&E, bar 50 μm, ×100, immunohistochemistry for CD31, bar 50 μm
Squamous cell in situ carcinoma cannot always be separated from high-grade dysplasia especially in nonkeratinizing carcinoma; however, it can be done in cases with focal “maturation,” which means keratinization/dyskeratosis of single cells or small groups or formation of squamous pearls. The separation of dysplasia from regeneration and squamous metaplasia is another sometimes difficult decision. Nuclei in regeneration can be large and polymorphic, intranuclear vacuoles might suggest atypia and inflammation, and bleeding might add in a suspicious morphology (Fig. 17.34). In difficult cases immunohistochemistry for p63 and p53 proteins can help. P63 protein stains basal cells of the bronchial epithelium, and in case of metaplasia and dysplasia, these cells expand into the upper layers of the epithelium. P53 can also be used, as squamous dysplasia usually occurs in cigarette smokers and most of them carry a TP53 mutation in their epithelia (Fig. 17.35).
Fig. 17.34
Atypia in regenerating epithelium. There was a previous biopsy resulting in bleeding and necrosis with fibrin on top. At the border a squamous metaplasia has developed, which shows polymorphism of nuclei, some with dense chromatin. Inflammation and fibrin should prevent here to render a diagnosis of dysplasia. H&E, bar 50 μm
Fig. 17.35
Immunohistochemistry for p63 (upper panel) and p53 (lower panel). In the upper panel, there is squamous metaplasia; the basal cells still form a single or double row stained by p63. Staining with p53 antibodies in the lower panel shows a high-grade dysplasia, where the cells with TP53 mutations have expanded into the surface of the epithelium. Immunohistochemistry, bars 50 μm
17.2.2 Atypical Adenomatous Hyperplasia
Atypical adenomatous hyperplasia and bronchiolar columnar cell dysplasia are two preneoplastic lesions confined to the alveolar and bronchiolar periphery, respectively. Atypical alveolar hyperplasia (AAH) [110–112] is visible already at low-power magnification (Fig. 17.36a).
Fig. 17.36
Atypical adenomatous hyperplasia (AAH). Several cases are shown. In cases from Southeast Asia, most often there is no inflammation in AAH (a; case provided by Y. Shimosato), whereas in European cases most often there is considerable inflammation and in some cases also fibrosis of the septa present (b, c). The major criterion and difference to in situ adenocarcinoma is less nuclear atypia and the presence of intercellular gaps between the tumor cells (d, e). Multiple AHH can be seen in a lung but rarely close by as in (f). Here several AAH lesions are present, but one of them already associated with an invasive adenocarcinoma (f, g, arrow shows the lesion). For comparison in (h), an in situ adenocarcinoma is shown. Here the tumor cells form a tight layer of cells, the gaps are no longer present, and nuclear polymorphism and atypia is increased. H&E, ×25, 100, 12, and 250, bars 50 and 20 μm
If the resected lung is fixed in an expanded way and thin tissue slices expanded under water are examined under light, the tiny spots with grayish thickened septa can be seen.
Histologically the normal epithelium is replaced by either pneumocyte type II-like atypical cuboidal cells or by columnar cells. The nuclear and nucleolar size is increased; nuclei are usually round to oval, nucleoli are polygonal, and intranuclear inclusion bodies are frequently seen, representing entrapped surfactant apoprotein. Between the cells, lined up as a single row, there are gaps (Fig. 17.36). When these gaps are lost, usually also higher grades of atypia are seen, mitotic rate increases, and focally epithelial papillae are formed; the diagnosis changes to in situ adenocarcinoma (AIS, Fig. 17.36h). The WHO classification [70] draws a line of 5 mm size between AIS and AAH, which, however, does not make sense. The problem of distinction of AIS and AAH can only be solved by cytomorphology and structure: in AAH there is no alveolar collapse, no atelectasis, and no desmoplasia, and the alveolar structure is rigid. The cytomorphology of the cells is uniform, most often pneumocyte-like, nuclei are slightly enlarged, nucleoli are either invisible or inconspicuous, and mitosis is absent. In AIS there can be alveolar collapse and atelectasis and no desmoplasia, the nuclei are larger compared to AAH, and nucleoli are increased and can be polymorphic [113]. A few mitotic figures can be seen, usually 0–2/HPF. Size is not a reliable criterion for distinguishing AAH and AIS: cases of AIS as small as 2 mm have been seen by the author and AAH cases with up to 8 mm too (Fig. 17.36). As in other organ systems, size is not reliable. In addition as we usually examine tissues after formalin fixation, shrinkage is another problem changing the real size.
17.2.3 Bronchiolar Columnar Cell Dysplasia
In contrast to AAH, bronchiolar columnar cell dysplasia (BCCD) can only be seen at the microscope under high magnification [114]. In BCCD atypical cells, gradually replace normal bronchiolar/bronchial epithelium. Normally bronchial/bronchiolar epithelium is composed of different cell types, as ciliated, secretory, goblet, Clara, and reserve cells. All these cells look different and have differently sized and shaped nuclei. In early stages of BCCD, a monomorphous proliferation of cells replace these differentiated cells and gradually expand within the epithelium (Fig. 17.37). Atypia can be low as well as high grade. In contrast to regeneration, BCCD appears monomorphic. As in AAH the important molecular events driving this preneoplasia into adenocarcinoma is unknown. However, BCCD can give rise to adenocarcinomas arising in small bronchi and bronchioli, in contrast to AAH, which is the precursor for non-mucinous adenocarcinomas arising from the bronchiolar-alveolar junction zone.
Fig. 17.37
Bronchiolar columnar cell dysplasia (BCCD). In A, B, and C, BCCD is shown at three different levels of bronchioles. In (a) BCCD is seen in small bronchioles. The bronchiolar layer of differentiated cells is replaced by uniform cells with enlarged nuclei with increased nucleoli. Also chromatin abnormalities are seen. In (b) BCCD is within a terminal respiratory bronchiole. The layer of Clara cells (right) is replaced by atypical cells, a few multinucleated. The nuclei of these cells are enlarged, nucleoli are increased, and nuclear membrane is accentuated. In (c) BCCD is seen in a terminal bronchiole at the opening into an alveolar duct. Note the abrupt transition from the normal cuboidal to an atypical epithelium. In (d) in the upper side of this bronchiole, scattered atypical cells are seen; on the lower side, the epithelium is replaced by squamous cell metaplasia. In (e) regeneration of the epithelium is shown. In regeneration the different types of cells are retained, and although there is some atypia, this is not the uniform proliferation of dysplastic cells. In (f) another example of BCCD is shown, and the difference of dysplasia to regeneration is evident. H&E, ×100, 200, bar 50 μm
17.2.4 Atypical Goblet Cell Hyperplasia
Atypical goblet cell hyperplasia (AGCH) is difficult to recognize: the nuclei are compressed at the basal cell border, and the chromatin structure is invisible. As in BCCD the growth pattern of the cells is more important: atypical, goblet, or signet ring cells are replacing the normal epithelium, resulting in a monotonous pattern. In contrast to goblet cell hyperplasia in dysplasia, the atypical cells replace the normal epithelium completely (Fig. 17.38). Atypical goblet cell hyperplasia might give rise to the different mucinous adenocarcinomas of the lung [115]. AGCH is often found in CPAM types I and II. From our study it seems obvious that this represents the preneoplastic lesion for the rare adenocarcinomas of childhood [12].
Fig. 17.38
Goblet cell dysplasia. (a, b) Shows two examples of goblet cell dysplasia. These might be precursor lesions for mucinous adenocarcinomas. In (c, d) atypical goblet cell hyperplasia within congenital pulmonary adenomatoid malformation type I and II is shown. In C the atypical proliferation is in the cyst epithelium, whereas in (d) the proliferation is growing into adjacent lung tissue. In contrast (e–g) show already lesions, which are regarded as in situ adenocarcinoma. (e) Is an intraepithelial growth of adenocarcinoma forming bridges similar what is seen in breast carcinomas. In (f) the proliferation forms roman type of bridges, again similar to what is seen in breast carcinoma. In (g) finally there is papillary growth pattern, which might give rise of a papillary adenocarcinoma. H&E, ×150, 100, 60, 200
17.2.5 Neuroendocrine Cell Hyperplasia
Neuroendocrine cell hyperplasia (NEH) is divided into NEH associated with fibrosis, bronchiectasis, carcinoid, and diffuse NEH of unknown cause; in addition there is tumorlet and nodular NEH. At present it is unclear, to what extent NEH is associated with neoplasia, and the factors influencing neoplastic progression are unknown. However, NEH is most probably a preneoplasia for carcinoids but not for the high-grade neuroendocrine carcinomas!
Neuroendocrine cells in the lung can be found in two places: as single dispersed neuroendocrine cells within the bronchial tree and as neuroepithelial bodies in the peripheral lung. Neuroendocrine markers, such as chromogranin A (CGA), Bombesin the frog analog of gastrin-releasing peptide (GRP), synaptophysin, PGP 9.5, and γγ-enolase (neuron-specific enolase (NSE)) can identify the cells [116–118]. They are part of Feyerter’s diffuse neuroendocrine system of the body [119, 120] and play a role in fetal lung development. Genes regulate growth and differentiation of the bronchial buds and the joining with the coelomic structure, but neuroendocrine cells act locally in fine tuning of the development in response to a variety of stimuli, as in hypoxia [121]. Two of the growth hormones expressed during fetal lung development act later on in neuroendocrine carcinomas as autocrine growth factor loop: GRP and adrenocorticotropin (ACTH) [122, 123].
NEH can be a reactive process, such as in bronchiectasis. The proliferation of the NE cells is the answer for obstruction. By this proliferation repair mechanisms are stimulated to restore normal lung structure as it did work in fetal lung. This question was addressed in publications by J. Polak’s group: in children dying from respiratory distress syndrome, bombesin content was lowered [121]. In an experimental model, CGRP was increased after 7 days of hypoxia [124].
A similar process might be the basis of NEH in UIP and other idiopathic interstitial pneumonias, this time starting from peripheral NE cells and neuroepithelial bodies. NE hyperplasia in the vicinity of carcinoids might be stimulated by growth factors released from the tumor, but this has not been proven so far.
Whereas single neuroendocrine cells are usually not identifiable on H&E-stained sections, NEH can be diagnosed when clear cell clusters are seen within the mucosa (Fig. 17.39). Nodular NEH can be characterized by an increase of these cells into clusters. A tumorlet is an aggregation of several nodular clusters of neuroendocrine cells separated by bundles of stroma but all together appearing as a small tumorlike lesion (Fig. 17.40). The major feature differentiating tumorlet from carcinoid is the presence of these dissecting fibrous bands between the neuroendocrine nodules. Tumorlets are usually less than 5 mm in diameter. They most likely represent early carcinoids. They are incidentally found in the same setting as NEH, i.e., close to carcinoids, and in fibrosis or obstructive lung disease.
Fig. 17.39
Neuroendocrine hyperplasia (NEH). In (a) a neuroepithelial body is shown, a normal but rare finding in an adult lung. (b) Shows NEH within a bronchus, the cells have clear- or pale-stained cytoplasm. (c) Shows small nodular proliferations of neuroendocrine cells in the wall of an already obstructed bronchus. (d) Is from another case with obstructive bronchitis and consecutive NEH. (e) Is NEH in the wall of small bronchi in a case of diffuse idiopathic neuroendocrine hyperplasia (DIPNEC). (g) Shows NEH in small bronchi and bronchioles highlighted by an immunostain for chromogranin A. (h) Is a resection specimen resected because of purulent bronchiectasis and pneumonia. In this case many small grayish-white nodules are seen, representing NE hyperplasia and tumorlets. H&E, bars 20, 50, 100 μm, ×50
Fig. 17.40
Tumorlet and carcinoid. In (a) there is a carcinoid and scattered foci of NEH, demonstrated by chromogranin A immunohistochemistry. (b) Shows a tumorlet in the vicinity of an adenocarcinomas, the transition zone between the two tumors is seen magnified in (d). (c) Shows another tumorlet. Here the nodules are joined together by fibrous bands. (e–g) Shows tumorlets, the neuroendocrine cells highlighted by chromogranin A immunohistochemistry, and a large tumorlet is seen in (h) here demonstrated by immunohistochemistry for PGP9.5. H&E, ×50, bars 100, 20 μm, immunohistochemistry, bars 0.5 mm, 100 or 50 μm
If there is no known cause found for a diffuse NE hyperplasia, these are regarded as idiopathic.
17.3 Malignant Epithelial Tumors
17.3.1 Epidemiology
In contrast to many other organs in the lung, the vast majority of tumors are belonging to the malignant epithelial category. In the early 1900s, lung cancer was a rare tumor, but around the 1920s an increase of lung cancer was recorded in central European Pathology Institutes [125–127]. This has mainly to do with exposure to cigarette smoke. Whereas cigar smoking was common in the upper classes in the nineteenth century, but too expensive for the lower class population, this changed completely when cheap manufactured cigarettes appeared. Approximately some 15–20 years after introducing the cheap cigarette at the turn of the nineteenth century, lung cancer appeared on the scene and since then has not stopped rising until recently reaching the number one tumor worldwide [128–130]. The following decades saw an uprise of squamous and small-cell carcinomas until the 1950s. These were called the smoking-related carcinomas, whereas adenocarcinomas were regarded as not cigarette smoking associated [128, 131]. This view was also “confirmed” by experimental cigarette smoke inhalation studies in mice, rats, and hamsters, which resulted in adenomas and adenocarcinomas. The tobacco industry used these findings as arguments that cigarette smoking at all is not carcinogenic.
Within tobacco smoke, side- as well as mainstream, approximately 600 different chemical compounds are identified, from which roughly 10 % are carcinogenic on their own; however, many others act together and are carcinogenic too, for example, by chemical interaction [132–141]. Also within the respiratory tract, several enzymes act as modifiers, which can transform precarcinogens into carcinogens, for example, by oxidation at specific CH3 or NH sites. In addition specific polymorphisms within the P450 cytochrome oxidases might also be important [142]. Most carcinogens belong to carbohydrates and nitrosocomponents, amines, and more complex chemicals. Several chemical side chains have been identified as being responsible for their carcinogenic action, such as instable C=O, HN=NH, and C=NH binding sites. Of note are especially HN=NH and C=NH binding sites as these can directly interact with DNA nucleotides and result in point mutations.
Many tobacco carcinogens induce strand breaks on the double-stranded DNA. It has become clear that this follows exact rules: normally DNA is in a coiled or supercoiled state, and oxygen radicals, for example, cannot act on the coding nucleotides. So strand breaks can occur only when the DNA is uncoiled and opened for transcription. This is the reason why cells (especially different kinds of stem cells) undergoing mitosis are more prone to acquire genetic hits, whereas differentiated cells divide rarely and therefore are protected from injury. DNA repair mechanisms have been less studied; however, the few reports showed a contribution to the development of lung cancer. In the study by Park et al., a polymorphism at the 8-oxoguanine DNA N-glycosylase 1 gene involved in base excision repair showed increased dose-dependent risk for lung cancer in smokers [143]. The DNA damage repair genes, mutY and mutM, prevent G to T mutations caused by reactive oxygen species. In mice deficiencies of both genes resulted in lung and ovarian tumors. G to T mutations were identified in 75 % of the lung tumors at codon 12 of KRAS [144] and it seems that this is gene specific. ERCC1 if highly expressed in tumors was able to counteract chemotherapy-induced DNA strand breaks resulting in shorter survival of patients with NSCLC, whereas patients with tumors negative for ERCC1 showed a prolonged survival [145]. So more in-depth studies on the different repair mechanisms are necessary to see how much these enzyme systems are involved in carcinogenesis. Another enigma is that mutations often occur on specific genes. One explanation might be that first of all mutations can occur only at sites of an uncoiled DNA, i.e., a DNA area which is to access, which is common for cell cyle associated genes. Oxygen radicals cannot approach coiled and supercoiled DNA.
Besides tobacco smoke as a collection of major carcinogens, some other causes are emerging especially from China. There is indoor cooking, where many carcinogens are released and due to ill ventilation are inhaled in high concentrations [146, 147]. Other possible causes are toxic and carcinogenic waste in industrial areas (this will be discussed in detail in the pneumoconiosis chapter).
However, the story is not complete, if we do not also see the action of cancer prevention systems. As the lung is exposed to outside air, it is also exposed to many natural carcinogens present in the environment. Therefore during evolution the organ has developed several defense mechanisms to protect it from injury. These mechanisms have evolved over millions of years starting with old mucociliary and macrophage clearance, followed by enzymatic defense, and finally also by the action of the immune system.
The mucociliary escalator system is composed of the mucus-producing cells and the ciliated cells. Mucus is produced by the goblet cells along the bronchial surface epithelium and the bronchial glands. Mucus forms a fine liquid surface layer, into which particles as well as chemical substances are impacted and diluted. The ciliated cells move the mucus constantly toward the larynx, and therefore many inhaled substances either do not contact the surface epithelium or only for a short time period. Therefore the action of toxic substances is limited. In the alveolar periphery including the small bronchioles, macrophages constantly patrol through the airspaces and remove any harmful substances by phagocytosis, again preventing toxic injury.
The anatomy of human airways is characterized by asymmetric branching of bronchi/bronchioli. One bronchus divides into a main branch with approximately two thirds of the diameter, whereas the minor bronchus has a diameter of one third. This branching results in a disturbed airflow with turbulences at the bifurcations. Large particles >10 μm impact at the bifurcations of the larger bronchi, and only small particles of <2 μm can reach the alveolar periphery. Thus the inhalation of larger particles is prevented and the air is cleared.
Less well known is the protective enzymatic system of the airways. There are two major systems: class one and two; class one consists of the cytochrome P450 oxidases, microsomal epoxide hydrolase, and myeloperoxidase and the antioxidases superoxide dismutase, catalase, and glutathione peroxidase [142, 148]. In class two there are several enzymes such as the glutathione S-transferases family enzymes capable of protecting from oxygen radical injury by a redox mechanism [148, 149].
In addition to enzymes, also pro- and anti-inflammatory proteins/lipoproteins are involved in maintaining homeostasis of the airway system. How these interact with inhaled toxicants/carcinogens is not known. Immunoglobulins especially from the A class protect against inhaled bacteria by opsonization. Surfactant apoproteins and other substances such as Clara cell proteins also are involved in a protective manner but can even be produced and secreted by adenocarcinomas – so their function in health and disease is still not understood.
17.3.2 Carcinogenesis: Our Current Sight on the Development of Cancer
In these paragraphs we will focus on the development of carcinomas from normal epithelium to dysplasia to in situ carcinoma. Invasion, migration, and metastasis will be discussed in the metastasis chapter.
Several conditions need to be functioning for carcinoma development from the early changes in the epithelium to preneoplastic lesions and finally to in situ carcinoma. Some of these factors are general, working in different carcinoma types, whereas other factors are subtype specific, and even individual settings do occur. Generally each preneoplastic cell needs appropriate nutrition, especially supply of amino acids for the synthesis of nucleotides to be used for each round of DNA replication, and oxygen as well as glucose as a provider of energy since each cell division consumes energy in the form of ATP. These early events of carcinogenesis are less explored than the development of genetic abnormalities. Preneoplastic cells also acquire genetic aberrations, which will result in cell cycle stop at one or both cell cycle checkpoints where DNA integrity is proven, repaired in case of failure, or cells are sent into apoptosis, if the gene defect is irreparable. So these checkpoints as well as apoptosis need to be inactivated. In addition genes harboring DNA defects (deletion, mutation, amplification, rearrangements/translocations) will translate into proteins, which are detected by immune cells. This might be recognized by immune cells, which will immediately attack the neoplastic cells. So preneoplastic cells need to develop mechanisms to avoid immune cell attack. Stem cells are another factor, which can play a role in some tumors, but this is not a general rule. Finally once a tumor has formed an in situ lesion, tumor cells need to transverse the basal lamina, interact with the stroma, and migrate. Within the primary tumor due to accelerated growth, hypoxia develops, as new blood vessel formation do not keep with tumor growth. So tumor cells have to change their metabolism into anaerobic glycolysis.
17.3.2.1 Changes in Metabolism: Access to Nutrition and Oxygen
It has been shown that many enzymes as ATP synthase subunit D, beta1,4-galactosyltransferase, cytosolic inorganic pyrophosphatase, glutathione-S-transferase M4, prolyl 4-hydroxylase β-triosephosphate isomerase, and ubiquitin thiolesterase (UCHL1) are overexpressed in adenocarcinomas and most likely also in preneoplastic lesions such as AAH [150]. Tumor cells express exclusively the embryonic M2 isoform of pyruvate kinase, which is necessary for aerobic glycolysis and thus provides a selective growth advantage [151]. Glycolytic ATP supports growth under hypoxic conditions. Glutamine conversion into the tricarboxylic acid cycle by glutaminase and alanine aminotransferase is essential for KRAS-induced anchorage-independent growth. Changes in the mitochondrial complex III metabolism generate reactive oxygen species, which is also required for KRAS-induced anchorage-independent growth through ERK-MAPK signaling [152]. This finding was further explored in the study of Beuster, who showed that L-alanine aminotransferase (ALAT) promote mitochondrial metabolism by L-alanine production and enhanced D-glucose uptake. ALAT enhanced anchorage-dependent and anchorage-independent growth and enhanced ATP production via the Warburg effect [153]. The Warburg effect is characterized by glucose uptake and breakdown through the process of aerobic glycolysis. The importance of the glutamine (Gln) in cancer development and progression has been studied by van den Heuvel and coworkers. A variety of essential products to sustain cell proliferation, such as ATP and macromolecules, are provided. GLS1 is the key enzyme. NSCLC cell lines depend on Gln for glutaminolysis to a varying degree, in which the GLS1 splice variant GAC plays an essential role [154]. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) overexpression induces progression in NSCLC, again pointing to the importance of the glucose metabolism in cancer development [155]. GLUT1 another enzyme in the glucose metabolic cycle is overexpressed in smoking-associated carcinomas preferentially in squamous cell type. GLUT1 overexpression was also correlated with EGFR-negative and KRAS mutation-positive carcinomas [156].
The second most important factor is oxygen supply by increase in vascular density. In experimental carcinogenesis induced by N-nitrosobis(2-hydroxypropyl)amine in rats, VEGF, VEGFR-1, and VEGFR-2 were upregulated in malignant and premalignant lung lesions. In addition, VEGF mRNA and VEGFR mRNAs were overexpressed in adenocarcinomas and squamous cell carcinomas [157]. In the study by Fontanini and coworkers, increased microvascular density (MVD), accompanied by overexpression of VEGF and p53, was seen in normal bronchial epithelium, moderate dysplasia, in situ carcinoma, and invasive cancer. The association between MVD, VEGF expression, p53 mutations, and preinvasive lesions of the bronchial tree suggests that neoangiogenesis is an early event in NSCLC [158].
17.3.2.2 Mutations in Mitochondrial Genes
Mitochondria are the energy-producing factories of the cell. Therefore it seems logical that cancer cells also use enzymes to suit their higher energy consumption. Increase in the rate of glycolysis is one of the metabolic alterations in most cancer cells. In the study by Pelosi et al., mutations in the D-loop of mitochondrial DNA (mtDNA) were found in 22 % of lung cancers and mutations in the mtDNA in 57 %; there were changes in the mononucleotide or dinucleotide repeats, deletions, or multiple insertions. Instability in the D-loop region of mtDNA and decrease in mtDNA copy number seems to be involved in carcinogenesis [159]. MtDNA copy number changes are a biologic response to mtDNA damage and dysfunction. Polycyclic aromatic hydrocarbons (PAHs) may cause mitochondrial toxicity. Workers with high PAH exposure showed higher mtDNA copy number changes, as well as higher levels of genetic and chromosomal DNA adducts, micronuclei, telomere length, and epigenetic p53 gene-specific promoter methylation alterations [152]. In the study by Kadota, missense mtDNA mutations were found in 9 of 13 mtDNA coding genes. They found a variety of mtDNA mutations and mtDNA polymorphisms in human lung cancer, some of them involved in human lung carcinogenesis [160].
Mutations of TP53 are frequently in lung cancers. Synthesis of cytochrome c oxidase 2 (SCO2) is one of the genes’ downstream of TP53 and regulated by it. In p53-deficient cells SCO2 gene is disrupted and this leads to a metabolic switch toward glycolysis. So via SCO2 p53 is coupled to mitochondrial respiration and shows another function of mutated p53 explaining the Warburg effect in cancer cells [161]. Two other genes coding for the enzymes ATPase6 and NADH dehydrogenase 3 are mutated in 86 % of lung cancer cases. It seems both are cooperating during cancer development [162].
17.3.2.3 Proliferation, Cell Cycle, and Chromosomal Strand Breaks
Once the supply of oxygen and nutrition is solved, the preneoplastic cells need to high check the cell cycle machinery. Here a variety of mechanisms can be used to overcome the cell cycle controls. Stimulatory factors USF-1 and USF-2 dimerize to regulate transcription through E-box motifs in target genes. In phenotypically normal bronchial tissues, USF-2 was highly expressed at 1 cm distance from the tumor. USF-2 was restricted to ciliated cells in normal bronchial epithelium but more strongly expressed in dysplastic epithelium as well as in small- and large-cell neuroendocrine carcinoma and squamous cell carcinoma. USF-2 represents an early molecular marker for the development of bronchial dysplasia and non-adenocarcinoma lung cancer and may also play a role in bronchial carcinogenesis by promoting cell proliferation [163].
Oxygen radicals (reactive oxygen species (ROS)) play a major role in carcinogenesis, as many tobacco carcinogens act via this mechanism. ROS induce DNA strand breaks and point mutations but also interfere with cell cycle checkpoint function [164]. Also the inactivation of protective enzymes plays a role in the initial process of cell transformation. MTH1, MUTYH, and OGG1 play important roles in mammalian cells to avoid an accumulation of oxidative DNA damage, both in the nuclear and mitochondrial genomes, thereby suppressing carcinogenesis and cell death. But in carcinogenesis one or several of these enzymes can be inactivated by mutation, which will ultimately lead to carcinoma formation [165]. The tumor suppressor gene RASSF1A regulates the stability of mitotic cyclins and the timing of mitotic progression. RASSF1A localizes to microtubules during interphase and to centrosomes and the spindle during mitosis. The overexpression of RASSF1A induced stabilization of mitotic cyclins and mitotic arrest at prometaphase. RASSF1A is frequently silenced by hypermethylation of its promoter. Depletion of RASSF1A by RNA interference accelerated the mitotic cyclin degradation and mitotic progression. It also caused a cell division defect characterized by centrosome abnormalities and multipolar spindles [166]. Thus silencing might be another cause of increase of mutations, deletions, and other kind of chromosomal aberrations during early carcinogenesis.
MicroRNAs (miR) are involved in many cell cycle processes. How much they are involved in early carcinogenesis has not been extensively explored. Mascaux and colleagues showed that miR-32 and miR-34c expression decreased from normal bronchial tissues of nonsmokers to squamous cell carcinomas. Others behaved differently at successive stages, such as miR-142-3p or miR-9, or are only altered from a specific stage, such as miR-199a or miR-139. miR follows a two-step evolution, first decreasing during the earliest morphological modifications of bronchial epithelium and thereafter increasing at later stages of lung carcinogenesis [167]. In this respect the regulation of the tumor progression locus 2 (TPL2) by miR fits well into this concept: TPL2 acts as a suppressor of lung carcinogenesis and is genetically altered in lung cancer patients. Upregulation of miR-370 downregulate TPL2 and activates RAS signaling. Low TPL2 levels correlate with accelerated onset and multiplicity of urethane-induced lung tumors in mice. TPL2 antagonize oncogene-induced cell transformation and survival through a pathway involving p53 downstream of JNK and be required for optimal p53 response to genotoxic stress [168].
17.3.2.4 Apoptosis
The more tumor cells acquire genetic aberrations, the more important is control of apoptosis checkpoints for these cells. Otherwise tumor cells would be sent to apoptosis due to irreparability of their genome. To overcome apoptosis tumor cells can inhibit the checkpoint control by several mechanisms: mutation of TP53 results in its inability to recognize DNA damage. Since p53 protein cooperates with p16 in regulating apoptosis, and as p16 is also often mutated/deleted, the overall effect is apoptosis escape of the tumor cell [169–171]. Another mechanism to escape apoptosis is facilitated via the PTEN NFkB pathway. The promoter of PTEN can be downregulated by NFkB. Restoration of PTEN expression inhibited NFkB transcriptional activity and augmented TNF-induced apoptosis, indicating a negative regulatory loop involving PTEN and NFkB. PTEN is, thus, a novel target whose suppression is critical for antiapoptosis by NFkB [172]. In small-cell carcinoma, another mechanism of antiapoptosis is used: suppression of aurora kinase A (AURKA) increased apoptosis of SCLC cells. An activation of AURKA resulted in upregulation of Bcl-2 and downregulation of Bax, thus preventing apoptosis [173].
17.3.2.5 Stem Cell Theory
The debate on lung cancer stem cells is still ongoing. One of the problems in research is the misunderstanding of researchers about the organogenesis of the lung. From fetal lung development, it is well known that the bronchial buds are proliferating toward the coelom structure and finally merge with it. Lung stem cells are not a homogenous cell population, as there are stems cells within the central portion with a capability to give rise to many different cell types, stem cells along the bronchial tree, and finally stem cells in the bronchioloalveolar junction zone with much less differentiation capabilities. Since peripheral adenocarcinomas arise from this junction zone, stem cells in these carcinomas are quite different from stem cells in the central area where SCLC arises. SCLC seems to be close to central stem cells with a capability to move within the epithelium, easily invade the stroma early on, and differentiate into SCC upon chemo- or radiation therapy (transdifferentiation).
Inhibition of EGFR, Src, and Akt signaling suppressed SOX2 and the self-renewal growth and expansion of stem cells in NSCLC. In contrast other stem cell factors like Oct4 and Nanog played a lesser role in regulating self-renewal [174]. In a subsequent study, Li and colleagues studied the stem cell markers Bmi1, CD133, CD44, SOX2, Nanog, OCT4, and Msi2 in lung cancer. Bmi1, CD133, CD44, SOX2, Nanog, and Msi2 mRNA and protein were abundantly expressed in lung cancer. Nanog expression was exclusively upregulated in lung cancer tissues [175].
In a study focusing on brain metastases, CD15 and CD133 were identified as possible markers of a stemlike cell population [176]. Furthermore CD133 expression correlated with chemoresistance and increased tumorigenicity in vitro and in vivo accompanied by increased expression of Akt and Bcl-2. CD133 expressing small-cell neuroendocrine cancer cells had also an increased expression of the mitogenic neuropeptide receptors for gastrin-releasing peptide and arginine vasopressin [177]. However, to make the story a bit more complicated, in another study, cells surviving radiation therapy showed upregulation of CD44, SNAIL, and PDGFRβ and showed features of EMT pointing to the possibility that there are more than one factor related to stem cell morphology [178]. This leads one to the fact that stem cells but probably also other cancer cells can move liberally within the epithelial layer. Most of us have seen or will see SCLC cases, where the primary tumor is 5 mm in diameter, whereas the patient present with brain metastasis as large as 3 cm. There is only one explanation: carcinoma cells start to invade the stroma early on, long before a radiologically visible tumor is formed. In the case of multiple AAH, we found an almost identical genotype in all the lesions and in only one focus an invasive adenocarcinoma [179]. The only logical explanation is that cells out of the initial precursor stem cells move out and create other foci from where similar precursor lesions start to evolve. Multifocal carcinomas seen in different organs might be explained by this phenomenon. There are no studies in pulmonary carcinogenesis, which had this in focus. However, in urinary bladder intraepithelial neoplasia, it has been shown that a reduction in integrin β4 plays a role in migration of carcinoma in situ cells along laminin [180]. Similarly another adhesion molecule E-cadherin, when downregulated, was associated with circumferential non-muscle-type βactin organization, increased motility, and random cell migration [181]. Again in another organ preneoplasia PanIN, it was shown that MUC5AC, upregulated by GLI1, interfered with the membrane localization of E-cadherin, leading to decreased E-cadherin-dependent cell-cell adhesion and promoted migration and invasion. GLI1 induced the nuclear accumulation of βcatenin in a MUC5AC-dependent manner [182].
17.3.2.6 Driver Genes and Bystander Genes: Better to Be Called Cooperators
With all these many genes involved in carcinogenesis, one might be totally confused, as it seems that there is no general rule, which gene induces what. However, we have learned through the findings of EGFR- and KRAS-mutated lung adenocarcinomas that we have to discern driver genes from bystander genes. In mouse models KRAS-mutated gene constructs induced a proliferation of stemlike cells at the bronchioloalveolar junction zone, which via hyperplasia and AAH-like lesions progressed into adenocarcinoma in situ [183]. However, KRAS needs a cooperation of mutated TP53 and deletion of PTEN to progress further into an invasive adenocarcinoma. So KRAS functions as a driver gene. Similar findings are seen in mutated EGFR adenocarcinomas. Cooperator genes are either cooperation partners of the driver or have other important functions for the maintenance of the developing carcinoma. This is nicely addressed by the work of Chitale: in KRAS-mutated adenocarcinomas, growth is upregulated by the mutated driver KRAS itself, no other passenger mutation is necessary for this function. In EGFR-mutated adenocarcinomas a passenger mutation is required: DUSP4 is involved in negative feedback control of EGFR signaling. DUSP4 is underexpressed due to a single-copy loss on 8p. DUSP4 functions as a growth suppressor in EGFR-mutant lung adenocarcinoma, and furthermore DUSP4 loss is associated with p16/CDKN2A deletion, which acts as a G2-S checkpoint control and might send cells to apoptosis, in case of ROS-induced DNA alteration [184]. Another gene TWIST1 collaborates with the EGF pathway in promoting EMT in EGFR-mutated lung adenocarcinoma [185]. So these genes are not bystanders but cooperating genes fulfilling specific functions, which the driver cannot do. Similarly in KRAS-mutated adenocarcinoma, exclusive gene fusions were found for CD74-NRG1, SLC3A2-NRG1, EZR-ERBB4, TRIM24-BRAF, and KIAA1468-RET. The CD74-NRG1 fusion activated HER2-HER3 signaling, whereas the EZR-ERBB4 and TRIM24-BRAF fusions constitutively activated the ERBB4 and BRAF kinases, respectively [186].
As of the end of 2015, these driver genes were identified in pulmonary carcinomas, with some variations among different races: mutations in EGFR, KRAS, NRAS, BRAF, PIK3CA, MET, and CTNNB1. Novel driver mutations were identified in LMTK2, ARID1A, NOTCH2, and SMARCA4. Furthermore there are rearrangements in ALK, RET, ROS1, FGFR2, AXL, and PDGFRA. Among recurrent alternative splicing events, exon 14 skipping in the proto-oncogene MET identified it as another cancer driver. For several genes there was a strong association with smoking history of patients [187]. Similar finding were reported for Asian patients; however, the frequency for each gene alteration was different [182]. In African Americans again a similar genetic profile was seen for NSCLC [188]. In a Chinese study involving never-smokers 75 % harbored EGFR mutations, 6 % had HER2 mutations, 5 % had ALK fusions all involving EML4 as the 5′ partner, 2 % had KRAS mutations, and 1 % harbored ROS1 fusions. BRAF mutation was absent [189]. Independent from race EGFR mutations were highly associated with female sex, Asian race, and never-smoking status; ALK rearrangements were strongly associated with never-smoking status and more weakly associated with the presence of liver metastases. ERBB2 mutations were strongly associated with Asian race and never-smoking status; single mutations were seen for PIK3CA, ALK, or MET [190]. When looking up adenocarcinomas in smokers, EGFR, STK11, and KRAS were most common; in squamous cell carcinomas, the following driver genes were identified: PTEN (16.1 %), STK11 (8.3 %), and PIK3CA (7.2 %) were the three most frequently enriched genes in smokers with SCC. DDR2 and FGFR2 were exclusively present in smokers with SCC, whereas EGFR, c-Met, and PIK3CA alterations were found in the nonsmoker population with SCC [191].
17.3.3 Common Carcinomas
Adenocarcinoma has replaced squamous cell carcinoma as the leading carcinoma, comprising 40–60 % of all pulmonary carcinomas. The percentage of small-cell carcinoma is decreasing to the range of 8–12 % in Austria, Germany, and other Western and Central European countries; however it is still high in eastern parts of Europe. Squamous cell carcinoma in Austria decreased from 35 % to 18 % but increased again within the last 5 years to 27 %, probably due to immigrants from southeastern Europe. Squamous cell carcinoma is still high in Eastern European countries. The number of large-cell carcinomas for a long time remained stable at 12 % but since 2005 decreased to 4 % due to the use of immunohistochemical markers for the differentiation of NSCLC, which shifted almost two thirds of them into the adenocarcinoma and a smaller fraction into squamous cell carcinoma.
By X-ray and CT scan, carcinomas present as a tumor mass with ill-defined border. Spicule radiating from the tumor center represents outgrowth of the tumor into the periphery. In situ carcinoma, especially adenocarcinoma, is characterized by ground-glass densities, whereas invasive portions will show higher densities.
Due to the invention of ultrasound-guided biopsy in bronchology, the size of biopsies decreased considerably during the last few years. For that reason the WHO classification now also included a new classification for small biopsies and cytology specimen [70].
The previous editions of the WHO classification of lung tumors were mainly based on the experience from resected tumors. This is especially true for lung carcinomas. This means the classification is based on the experience from less than 30 %, whereas more than 70 % of lung carcinomas are diagnosed on small tissue samples or on cell preparations. In the past this was not important, because there were not much therapy options available for patients with non-resectable lung carcinomas. In these periods oncologists asked for the differentiation of small-cell (SCLC) versus non-small-cell carcinomas (NSCLC); everything else was unimportant.
This has changed dramatically! New chemotherapeutics have been introduced, which are efficient under certain circumstances and in defined tumor entities; targeted therapy has finally also found its way into lung cancer treatment.
Several of these therapies were especially suitable for adenocarcinomas, or even squamous histology had to be excluded before this therapy was applied. Cisplatin-based chemotherapy showed better outcome in adenocarcinomas; nucleotide excision repair enzyme (ERCC1) is more prevalent in squamous cell carcinomas, which result in repair of DNA damage induced by platinum compounds, thus explaining why adenocarcinomas responded better. The thymidylate synthase blocker pemetrexed is efficient in most adenocarcinomas and not in squamous cell carcinomas.
Finally activating mutations of the epidermal growth factor receptor (EGFR) are most often found in never-smokers, in females, and in patients with adenocarcinoma histology. Other molecular targets such as ALK kinase gene fusion with the EML4 gene (and others) has been recently shown in patients with adenocarcinomas. ROS1 rearrangement followed again in adenocarcinomas.
Angiogenesis is essential for the primary tumor as well as for metastasis. The secretion of VEGFs facilitates most often neoangiogenesis. Tumor blood vessels are fragile and are prone to rupture. Using antibodies against VEGF (bevacizumab) and more recently by FGF receptor inhibitors, the vascularization can be inhibited and regression of the tumor is induced.
17.3.3.1 Squamous Cell Carcinoma (SCC)
Clinical Symptoms
SCC usually presents with weight loss, hemoptysis, hoarseness, cough, mucopurulent expectations, and due to frequent bronchial obstruction by an endobronchial component also purulent pneumonia. On X-ray as well as on CT scan, it presents as a central mass, which often also involves regional lymph nodes.
Gross Morphology
SCC is most often a tumor mass located in the central bronchial tree. Regional lymph nodes are often directly involved by an ingrowth of the carcinoma (Fig. 17.41). On cut surface the tumor can look coarse granular in keratinized forms – the granules correspond to sheets of keratinized cells. Necrosis is common and can be widespread. There is a rare form of SCC occurring in the peripheral lung (Fig. 17.42). In most of these cases, a previous inflammatory lesion is reported, most often previous treatment with cytotoxic drugs.
Fig. 17.41
Gross morphology of a squamous cell carcinoma (SCC), located in an upper lobe bronchus. The endobronchial growth is seen as well as some lymph node metastasis in N1 nodes
Fig. 17.42
Unusual variant of a SCC arising in peripheral location. In addition to the carcinoma (white nodules), there is also bronchiectasis and emphysema
Histology
SCC is defined by a platelike layering of cells, keratinization of groups or single cells, intercellular spaces or gaps, and bridges (represented by desmosomes and hemidesmosomes) (Figs. 17.43, 17.44, and 17.45). Squamous cell carcinomas can be keratinizing or nonkeratinizing; however, they are not different with respect to prognosis. Similar to the cervix, there exists a rare small-cell variant of SCC, which might cause diagnostic problems because of simulating small-cell neuroendocrine carcinoma. However, the former has visible nucleoli, and also intercellular gaps are focally seen (Figs. 17.46 and 17.47). In addition this variant also expresses high molecular weight cytokeratins.
Fig. 17.43
Bronchial biopsy of a SCC. This case shows sheets of keratinized tumor cells. The cell borders are clearly visible by the intercellular gaps. There is a lot of apoptotic cells and infiltration by neutrophils, which point to autophagy. H&E, bar 50 μm
Fig. 17.44
Another case of SCC. The intercellular gaps/bridges are clearly seen; in addition there is a row of palisading cells on the left side, a feature not uncommon in SCC. There is single cell keratinization and considerable nuclear atypia, placing this case more into the high-grade type. H&E, bar 10 μm
Fig. 17.45
Electron microscopy of an SCC. The intercellular gap between two tumor cells is shown. The cell connection in the middle is closed by a desmosome. There are other desmosomes and hemidesmosomes present (densely stained structures). Different filaments such as cytokeratin filaments insert in these structures. ×6,000
Fig. 17.46
Small-cell variant of SCC. In the upper panel, the infiltrating tumor is seen in a transcutaneous needle biopsy. The nuclei are dense and dark stained; however, there is ample cytoplasm. In some foci a peripheral palisading was seen. In the lower panel, an immunohistochemical stain for p63 protein confirmed the diagnosis of SCC. H&E, bar 10 μm, immunohistochemistry, bar 20 μm
Fig. 17.47
Other case of small-cell variant of SCC, which in addition showed positivity for NCAM in about 10 % of tumor cells but also stained for p40. H&E, bar 20 μm
There exist no general accepted grading system for squamous cell carcinoma. In the AFIP fascicles, keratinization was proposed for grading: a SCC with more than 20 % keratinized tumor cells was graded as G1, less as G2, and if there are only single keratinized cells or no keratinization at all, this was graded as G3.
In my opinion this grading is not accurate, as it does not reflect biological behavior. In some SCC keratinized carcinoma cells are exclusively found in lymph node metastasis, whereas the nonkeratinized component is seen in the primary tumor. In addition as the classification changed into keratinizing and nonkeratinizing SCC, keratinization cannot be used anymore. In many malignancies mitosis is used for grading, and I adapted this for SCC and other lung carcinomas. It proved to be very useful. These are the recommended features:
G1 = mitotic counts 0–3/HPF (×400), minor nuclear polymorphism, small nucleoli (Fig. 17.48).
Fig. 17.48
SCC well differentiated with some nuclear polymorphism but only two mitoses (arrow)
G2 = mitotic counts 4–8/HPF, nuclear polymorphism clearly visible, medium-sized nucleoli (Fig. 17.49).
Fig. 17.49
SCC, G2 type. Keratinization of single cells is present; five mitotic counts are present. There are still good visible intercellular gaps. H&E, bar 20 μm
G3 = mitotic counts >8/HPF, nuclear polymorphism clearly visible, a few scattered multinucleated cells might be encountered; nucleoli are enlarged and irregularly shaped (Fig. 17.50).
Fig. 17.50
High-grade SCC with more than eight mitoses per HPF. Here intercellular gaps are hardly seen; the carcinoma looks almost undifferentiated. However, there are areas with single keratinized cells (arrows), and also this carcinoma expressed SCC markers. H&E, bar 20 μm
In looking for markers predictive for survival, Kadota and coworkers compared keratinizing, nonkeratinizing, basaloid, and clear cell subtypes, as well as single cell invasion, nuclear diameter, and tumor budding, and found that only these later factors were independent prognostic factors [192].
The etiology of squamous cell carcinomas is to almost 100 % linked to cigarette smoking, especially to filterless cigarettes. Some other agents inducing SCC are metals such as cadmium and arsenic, but also radon and uranium exposure has been linked to SCC [193–197]. Additional to these chemicals also HPV similarly to the cervix might be involved in SCC development. HPV-induced papillomas exist in the airways, from the upper respiratory tract to the bronchi. In most cases non-oncogenic HPV types have been demonstrated in these papillomas. However, in rare instances oncogenic types have been proven, which subsequently developed into SCC [27, 29, 43]. While HPV 16 and 18 directly interfere with the mitosis checkpoint controls RB1 and TP53, HPV11 by itself is not oncogenic, unless there is a mutation in the E2 sequence, which controls the oncogenic E6 and E7 sequences [37, 38]. All patients reported so far had HPV gene sequences in their tumors but also were heavy smokers. So the final clue if HPV alone is able to induce SCC is still missing. It is more likely that HPV infection together with smoking accelerate the development of the carcinoma, as most of these patients are of much younger age.
Immunohistochemistry
SCC expresses several differentiation markers, which can be used for diagnostic purpose. High molecular weight cytokeratins such as acidic CK3, CK5, and CK6 and basic CK13 and CK14 stain SCC, and also desmocollin3 and the basal cell marker p63 or its splice variant p40 are useful, especially in small biopsies or cytologic specimen [198–201]. Helpful is also a cell membrane-accentuated staining with cytokeratin antibodies (Figs. 17.51 and 17.52). To differentiate primary pulmonary squamous cell carcinomas from those of other locations within the upper respiratory tract, this is not always possible. SCC from the esophagus can be differentiated by the positivity for CK4, which is not expressed by pulmonary SCC. Laryngeal SCC cannot be differentiated, because this shares the same immunoprofile, whereas SCC from the oral cavity might express CK1 and CK2, which is not expressed by the pulmonary SCC [202].
Fig. 17.51
Immunohistochemical markers in SCC. (a) Histology of an SCC, (b) shows reactivity for p40 with stained almost all tumor cells. (c) A staining for p53 can sometimes be used, as almost all patients with SCC are smokers, and therefore have mutations of TP53 gene. (d) Staining for cytokeratin 5/cytokeratin 6, a high molecular cytokeratin present in SCC. Bars 20 and 50 μm
Fig. 17.52
Immunohistochemistry for cytokeratin 5/cytokeratin 6, showing the cell membrane-accentuated staining pattern. Bar 50 μm
Genetic Abnormalities in SCC and Targets for Therapy
Gene aberrations are common in SCC. Gains are found on chromosomes 2, 3q, 5p, and 8q, whereas deletions are common on 3p, 5q, and 8p. The most specific aberrations are gain of 7p and 8q, whereas the most specific deletions are on 13q and 19p when compared to adenocarcinomas and small-cell neuroendocrine carcinomas [203–206] (and unpublished data by CGH and array CGH). Several targetable genes have been identified in SCC so far: amplifications and activating mutations of FGFR1 [207]; inactivating mutations or deletions of PTEN [208]; amplifications of PDGFRα [209], MCV1, SOX2 [210], EGFR [211, 212], and HER2NEU [213]; and mutations of CDKN2A [214], NOTCH1 [215], FGFR2 [191], and DDR2 [191, 216]. TP53 is frequently either mutated, deleted, or has a truncation mutation [217, 218], whereas PI3K and AKT1 are mutated or amplified in many cases [216, 219–222]. For some of these genes, therapeutic drugs are available as dasatinib for DDR2 mutation and FGFR kinase inhibitors for FGFR1 amplifications (Fig. 17.53). However, as SCC carries concomitant genetic aberrations, inhibition as in adenocarcinomas might not work: a good example is FGFR1 amplification, which can be accompanied by PI3KCA activating mutations – FGFR1 TKI inhibition therefore will not work.
Fig. 17.53
FISH analysis for amplification of FGFR1. The FGFR1 probe is labeled in red; the centromere probe in green. There are many cells of this SCC, which show clusters of FGFR1 gene signals. Such a case would need further analysis for concomitant genetic abnormalities before applying FGFR1 inhibitor therapy. ×630
A basal cell variant of SCC does exist, and in the previous WHO edition, basaloid cell carcinoma was listed as a variant of large-cell carcinoma [223]. In the new WHO classification [70], both are now unified into the variant basaloid squamous cell carcinoma (Figs. 17.54 and 17.55). In basaloid squamous cell carcinoma (BSCC), there might be either regular SCC elements even with keratinization or cases which are entirely basaloid without any differentiation. Immunohistochemical markers such as p40, p63, cytokeratins 5/cytokeratins 6, and desmocollin-3 will help in confirming the diagnosis [224]. This marker expression was the main reason for reclassification. p40 was also proven in BSCC in another study [225]. In basaloid carcinoma there is a uniform population of large cells with vesicular large nuclei, nucleoli are not prominent, but good visible. The cells form sheets and nests. On low-power magnification, the basaloid pattern is easily seen. It resembles basalioma of the skin: there is an outer layer of cells forming a palisading ring and an inner portion, where the cells are totally disoriented, i.e., cells lie in any direction. On higher magnification numerous mitoses are seen. Sometimes the organoid pattern may resemble a neuroendocrine morphology, but this vanishes on closer examination. Basaloid carcinoma is a highly aggressive carcinoma with a poor prognosis despite aggressive chemotherapy. This might be due to a specific mRNA expression profile, with upregulated factors for cell cycle progression, and some genes related to maintenance of stem cell-like features, while genes related to squamous differentiation are repressed. Among the genes specific for BSCC, SOX4 and IVL discriminate it from regular SCC [226].
Fig. 17.54
Basaloid variant of SCC, here the classical type with features reminiscent of basalioma of the skin. There is an outer row of tumor cells with palisading, whereas the other cells are totally disoriented. H&E, ×200
Fig. 17.55
Basaloid variant of SCC, here a case in the previous WHO classification placed in the large-cell carcinoma group, now regrouped in SCC because of expression of SCC markers. There is still some kind of palisading of tumor cells, but otherwise no clear differentiation is seen. Cell borders are visible, in few cells intercellular gaps are seen. H&E, bar 20 μm
Cytology and small biopsy classification for SCC: nuclei usually with coarse chromatin, nucleoli middle sized, keratinization of single cells or groups, and intercellular gaps visible on small-cell groups (Fig. 17.56), layering of cells if there are large sheets of cells (in well-differentiated SCC); in addition in biopsies – layering of cells and basal cell layer. Keratinization is highlighted in PAP stain or similar (Fig. 17.57).
Fig. 17.56
Biopsies of SCC, where only surface parts of the carcinoma has been taken. Invasion is not present. The morphology however confirms SCC. H&E, bar 200 μm
Fig. 17.57
Cytology of SCC, clockwise from upper left: the tumor cells show intercellular gaps, a keratinized cell is also present; in this photograph a keratinized tumor cell is surrounded by other carcinoma cells; in the third graph, emperipolesis of red blood cells by a carcinoma cell. In addition another SCC cell has also been phagocytosed (cannibalism); in the last graph, several keratinized carcinoma cells are seen (tadlepol cells). PAP stain, ×400 and 630
17.3.3.2 Adenocarcinoma
Clinical Findings
The clinical symptoms are usually very unspecific including weight loss, fatigue, and less often cough. Hemoptysis is usually not a feature, but blood-tinged mucus expectorations might be seen. On X-ray and CT scan, this is usually a peripheral lesion, sometimes close to the pleura. Some small carcinomas present entirely as ground-glass opacities – these correspond most often to adenocarcinoma in situ.
Gross Morphology
Non-mucinous adenocarcinomas present as grayish-white solitary nodule or mass. Mucinous adenocarcinomas appear with a grayish-white cut surface and abundant gelatinous material. Colloid adenocarcinomas also look gelatinous; however, whitish small foci are scattered within these mucin lakes, like speckles. AIS appears grayish with a finely cystic structure, representing rigid extended alveoli. There are rare adenocarcinomas in central portions, most often histologically of the bronchial gland type again with some mucin seen on cut surface, and adenocarcinomas arising from small bronchi and bronchioli, which do not present with specific features, just solid whitish-grayish nodule (Fig. 17.58).
Fig. 17.58
Examples of adenocarcinomas of the lung, (a) large adenocarcinoma of central type (bronchial gland type), (b) mucinous adenocarcinoma, (c) small peripheral adenocarcinoma arising in lung fibrosis, (d) diffuse mucinous adenocarcinoma (pneumonia type), (e) adenocarcinoma with extensive pleura involvement (pseudomesothelioma type), (f) adenocarcinoma with massive intrapulmonary metastasis
Histology
Adenocarcinomas can present with different morphological pattern, such as lepidic, acinar, papillary, micropapillary, solid, and cribriform. In most ACs different patterns are mixed; acinar and papillary are the most common combinations. The pure forms are quite rare. Lepidic AC is characterized by a tumor cell growth along preexisting alveolar septa (Fig. 17.59a). In contrast to adenocarcinoma in situ, lepidic AC will always have an invasive focus, which should be >5 mm. In acinar AC the tumor forms well-defined gland-like acini, surrounded by a small rim of stroma, but sometimes the stroma might be thin (Fig. 17.59b, c). In papillary AC the tumor forms papillae projecting into a widened lumen. The papillae have stoma stalks, which are formed by newly formed blood vessels and some myofibroblasts (Fig. 17.59e). The tumor cells grow along these papillae. In micropapillary AC the tumor cells form micropapillae, which in contrast to the papillary form have no stroma, but consist of epithelial proliferations, projecting into the lumen (Fig. 17.59d). This type of AC is characterized by downregulated cellular adherence, the reason why these tumor cells easily disconnect from the septa and form small-cell clusters. This structure is also seen in lymph node metastasis, where the tumor cells lie within some liquid secretions. Solid AC is defined by a solid growth pattern (Fig. 17.59f) and can present with a small amount of mucin-producing cells: a minimum is two times five cells in two different fields. By the use of immunohistochemistry, another form of solid AC has arrived, characterized by solid growth pattern and TTF1 positivity.
Fig. 17.59
Patterns in adenocarcinomas: (a) lepidic, the tumor cells grow along preexisting alveolar septa; (b) acinar, the tumor cells form an acinar glandular structure; (c) acinar with morula formation, in this cases within acini solid structures called morules are formed, similar to what is seen in some endometrial adenocarcinomas; (d) acinar mixed with micropapillary, the micropapillary component is composed of groups of tumor cells without a stroma stalk; (e) papillary, the tumor cells cover a stroma stalk, which is a newly formed mesenchymal structure with mesenchymal cells and new blood vessels; (f) solid, the tumor cells form solid cell complexes, the basal orientation of the nucleus is seen, and the cytoplasm shows fine vacuolation; (g) cribriform, the tumor cells form primary, secondary, and tertiary acini; (h) bronchial gland type, the tumor cells simulate serous cells of the bronchial glands. H&E, bars, 20 and 50 μm
Cribriform AC has not been included in the new WHO classification, but this subtype does exist. It resembles metastasis of colon carcinoma. The tumor presents with complex acinar structures, which have formed secondary and tertiary lumina out of a primary acinus (Fig. 17.59g).
Adenocarcinomas usually have large vesicular nuclei and prominent nucleoli; the chromatin is most often lightly stained or unstained (euchromatin more abundant than heterochromatin). Nucleoli tend to be larger and more bizarre, the less the AC is differentiated. Nuclear membrane is accentuated by chromatin; the cytoplasm can be finely vacuolated or present with larger vacuoles. The content of these vacuoles is not always mucin but may also contain some proteins, lipo-, and glycoproteins, if the cells are differentiating toward secretory columnar cells.
As the lung is a 3D structure, where alveoli fill up all spaces, and our sections just confront us with a 2D picture, some uncertainties remain: Are acini and papillary structures real different? Or are these only different views and section planes of the same acinar structure. This might be resolved by applying new techniques producing 3D views of the acini using step sections and 3D reconstruction.
Invasion in adenocarcinomas can be difficult to assess, because in contrast to SCC adenocarcinomas show often less prominent desmoplastic stroma formation, especially in the well-differentiated forms. Invasion in AC can be diagnosed, if a desmoplastic stroma is present, if lymphatic or blood vessel invasion is seen, and if pleural invasion is present. Another help in the assessment of invasion is alveolar collapse (atelectasis). This is the area where one should look for desmoplastic stroma cells (Fig. 17.60). Invasion also implicates a change in morphology: When adenocarcinoma cells invade, the nice arrangement along alveolar surface structures as in lepidic type is impossible. Instead the tumor cells usually arrange themselves into small acinar or tubular, papillary, or solid structures or invade as single cells (Figs. 17.60 and 17.61).
Fig. 17.60
Assessment of invasion in adenocarcinomas; in the upper panel, solid adenocarcinomas invade the stroma causing proliferation of myofibroblastic stroma cells and a granulocytic infiltration as part of desmoplastic stroma formation. In the lower panel, there is only mild desmoplastic reaction with few stroma cells, but single cells and small groups are within the septum and reactive endothelia and few myofibroblasts are seen. H&E, bars 20 and 50 μm
Fig. 17.61
Central scar in an adenocarcinoma. Tumor cells are within the scar and also in dilated lymphatics, a sign of worse prognosis. H&E, bar 100 μm
In the WHO classification, airspace spreading is mentioned. In this condition tumor cells are free floating or moving within the alveoli and are separated from the primary tumor. This can be difficult to assess: small complexes of carcinoma cells lying within airspaces might be well attached at an alveolar septum, which will be seen on serial sections. So a freely floating tumor cell complex especially if these are close to the tumor will require step sections and/or 3D reconstruction to prove. In addition airspace spreading might be an extension or outgrowth of tumor cells or just reflect tumor cells moving along alveolar septa, as precursor cells already do. Another aspect is artifact: sectioning of the tissue block might transfer tumor cells. Airspace spreading has no impact on metastasis; however, it is important as a resection margin, because from these cells recurrence can occur. Airspace spreading is not invasion and not intrapulmonary metastasis: invasive tumor cells have access to vessels, move within the stroma, and interact with it, and metastasis means establishment of tumor cells at a different area of the lung clearly separated from the primary tumor. In intrapulmonary metastasis usually there will be areas of tumor cells within lymphatics.
In situ AC (AIS) is a rare form of AC. AIS is defined as a proliferation of carcinoma cells along alveolar septa, completely covering the surface (Fig. 17.62). They can produce epithelial papillae; invasion or desmoplasia should be excluded. Different cell types are involved: Clara-like cells, pneumocyte II-like cells, columnar cells, and goblet cells (Fig. 17.63). Most often AIS presents with a mixture of these cellular differentiations; however, pure Clara cell- or pneumocyte-like AIS does occur. AIS is a precursor of peripheral adenocarcinomas arising at the bronchioloalveolar junction zone. AIS can be non-mucinous or mucinous; however, in cases of multiple nodules of mucinous AIS, a careful examination and step sections are required to rule out invasion. In mid- and central portions of the lung, AIS has not been identified so far.
Fig. 17.62
In situ adenocarcinoma (AIS), the tumor has been incidentally detected and removed. On the left side, the tumor is shown, consisting of lepidic growth pattern without invasion. In the right side, two different differentiation grades are seen: above a single cell row with hardly any mitosis, a grade 1, and below cells with larger nuclei, some epithelial papillae, and a few mitotic counts, graded as 2. Also surfactant nuclear pseudoinclusions are seen. H&E, ×12, 60, and 100
Fig. 17.63
Adenocarcinoma entirely composed of Clara cell-like tumor cells, a rare finding as most adenocarcinomas are composed of a mixture of cells of the bronchioloalveolar junction zone. H&E, ×400
Microinvasive adenocarcinoma (MIA) is another entity based on histology and CT scan. On CT this type of AC is characterized by ground-glass opacity as in AIS. On histology a small invasive focus is seen, whereas the AC is lepidic in the majority. The invasive focus should be ≤5 mm in diameter (Fig. 17.64). The reason for creating a separate entity, different from AIS and lepidic AC, is that MIA confers the same good prognosis as AIS, i.e., a 100 % survival after surgical removal [227, 228]. The term microinvasive or minimally invasive AC was already proposed for the 1999 WHO classification by Y. Shimosato, based on his experiences with small size adenocarcinomas [229, 230] (and personal communication). In his proposal invasion should be less than 10 % of the whole tumor diameter. However, this proposal was rejected by the majority of the WHO panel members.
Fig. 17.64
Microinvasive adenocarcinoma (MIA); two examples are shown. At the top adenocarcinoma shows a small focus of invasion into the bronchial wall – the only focus in this case. In the middle another small adenocarcinoma is shown, differentiated in a mucinous type, again with a small focus of invasion into a bronchial wall, shown in higher magnification at the bottom. H&E, bars 200 and 50 μm
Adenocarcinoma Variants
Invasive Mucinous AC (IMAC)
This is a newly created entity, defined by invasion, abundant mucin production, and a columnar or goblet cell morphology [70]. There is an additional sentence, which will create confusion: “This entity should replace mucinous bronchioloalveolar carcinoma.” Mucinous bronchioloalveolar carcinoma was defined in the 1999 and 2004 WHO classification as noninvasive adenocarcinoma, so it is the same entity, which we now call either mucinous or non-mucinous AIS. So a carcinoma, which already was defined as AIS now, is placed into invasive mucinous AC. Abundant mucin is another imprecise term: how much is abundant? This will open individualized IMAC diagnoses according to what each pathologist regards as abundant.
In two recent investigations, large series of invasive mucinous AC have been presented. In both outcome was not different from non-mucinous AC, pointing that TNM staging is important, but differentiation into mucinous AC has no impact on prognosis [147, 231]. However, in both studies KRAS mutations are the most frequent driver mutations (over 50 % of cases); some other cooperating genes were identified such as deletion of p16, mutations of BRAF and PI3KCA, as well as gene fusions of CD74-NRG1, VAMP2-NRG1, TRIM4-BRAF, and TPM3-NTRK1. ALK1 rearrangements were seen in a similar frequency as in non-mucinous AC; surprisingly mutations of TP53 were rare, although in this type of AC, smoking is common.
So how IMAC can be more precisely classified: mucin production is seen in more than 70 % of tumor cells. Tumor cells can present as columnar cells where mucin is stored in small vacuoles and secreted toward the apical cell portion. Secretion can be simple release of the mucin or can be facilitated by holocrine secretion, i.e., a portion of the apical cytoplasm is extruded together with the mucin. In other cases mucin is stored in large vacuoles apical of the nucleus, which results in a goblet cell morphology. Mucin secretion is also apical. Since mucin production is not synchronized in AC, the cells are usually in different stages of synthesis. This can result that some cells do not show mucin, others show small amounts, and others show signs of release. In case of uncertainty, a stain for any of the MUC proteins (MUC1, MUC2, MUC5AC) will help to solve this problem. Mucins synthesized and secreted by IMACs are all acidic, so they will stain by Alcian blue stain at pH <2.5.
As in non-mucinous AC, also the mucinous AC can present with a lepidic (rare), acinar (frequent), papillary (frequent), micropapillary (less common), and solid pattern (Fig. 17.65). As in non-mucinous AC also in mucinous AC, solid and micropapillary patterns confer a worse prognosis [147, 232].
Fig. 17.65
Invasive mucinous adenocarcinomas. (a) Colloid mucinous adenocarcinoma, (b) mucinous cystadenocarcinoma with a pseudocapsule, (c, d) are mucinous adenocarcinomas with acinar and papillary pattern, and goblet cell differentiation; (e) mucinous adenocarcinoma with signet ring cell component (arrow), (f) is a mucinous adenocarcinoma with predominant signet ring cell pattern. (a–f), H&E, bars 50 μm, (f) ×150
There are two rare variants of invasive mucinous adenocarcinoma, the multifocal and the pneumonia type (Fig. 17.58b, d). The multifocal mucinous invasive adenocarcinoma is usually composed of more than five nodules separated from each other but usually within the same lobe, more rarely within different lobes but on the same side. Some of the nodules are in situ AC, others are clearly invasive. There is no lymphangiosis carcinomatosa, so these nodules seem to arise from a precursor clone, which spread early on and created several independent tumors, but if genetically analyzed they will show a similar genetic profile. The other variant is the pneumonia type. This was long ago recognized as pneumonia carcinomatosa in the old German literature. Here the tumor cells float within the mucus as small clusters of cells, usually mixed with mucophages. There is no visible nodule, often both lungs can be involved, and airspace spreading is common. Very often this type of carcinoma cannot be surgically removed, despite lymph node metastasis is uncommon. On small biopsies this type cannot be differentiated from colloid carcinoma. The patients are often of young age. If there is any relationship to CPAM has never been explored.
Colloid Adenocarcinoma
Colloid AC has been lumped with mucinous cystadenocarcinoma, although there is some difference: mucinous cystadenocarcinoma is usually a centrally located carcinoma, whereas colloid adenocarcinoma most often is a peripherally located carcinoma. In colloid AC, there is no capsule, the tumor grows diffusely into adjacent lung tissue, whereas in mucinous cystadenocarcinoma, there is a fibrous pseudocapsule (Fig. 17.65a, b). In addition for mucinous cystadenocarcinoma, there is an cystadenoma-carcinoma sequence, with mucinous cystadenoma, borderline mucinous cystadenoma, and mucinous cystadenocarcinoma.
Colloid AC shows huge amount of mucin secretion, which is already visible at gross inspection: the cut surface is glistening with gelatinous material. The tumor cells are focally at alveolar septa, forming strands of cells firmly attached to the wall. Some cells may float within the mucus. It seems they have developed a special metabolism, as these floating cells look well and viable, so their nutrition might involve also secreted material. In some cases one needs to make several sections until tumor cells are encountered. The cells can present with goblet or columnar cell morphology or a mixture of both. Also some cells with Clara cell morphology might be encountered between the columnar cells. Nuclei are usually round, smaller than the average AC cell nucleus, chromatin is finely dispersed, and nucleoli are small.
In mucinous cystadenocarcinoma the cells are tall columnar, goblet cells can be seen, but they are usually not numerous. In contrast to colloid, AC lymph node metastasis is most often present. The nuclei are much more pleomorphic, chromatin is coarse granular, and nucleoli are enlarged and sometimes bizarre. Mitosis is more frequent compared to colloid AC. At the invasion a desmoplastic stroma reaction is seen.
Enteric Adenocarcinoma
Enteric adenocarcinoma is a rare form of AC resembling a colonic AC. The structure is acinar, sometimes cribriform, otherwise the cytomorphologic features are similar to the other non-mucinous AC (Fig. 17.66). The most important and helpful finding is the brush-border formation seen at the top of the cells. This carcinoma does not produce mucin. Enteric adenocarcinomas are usually positive for cytokeratin 20 but usually express TTF1.
Fig. 17.66
Enteric adenocarcinoma, a rare entity; in the upper panel, an overview is shown, the carcinoma is acinar, and an extensive desmoplastic stroma reaction is present. In the lower panel, the characteristic features are shown: enteric brush border with microvilli as in colonic adenocarcinomas. This case was positive for CK20; some cells expressed CDX2, other TTF1. H&E, bars 100 and 20 μm
Fetal Adenocarcinoma
Fetal type of adenocarcinoma was described in 1995. It resembles a fetal developmental stage of around the 12th week of gestation. At that time the bronchial buds are branching, the cells are full of glycogen, and the nuclei are at an apical position within the cytoplasm. In this type of carcinoma, again the cells might look like clear cells, because glycogen is removed by tissue processing. The nuclei are also at the apical top of the cell. In contrast to pulmonary blastoma, which we discuss later, morules are either not present or exceptionally rare (Fig. 17.67). When considering the apical nuclear position, one has to consider what also happens during fetal maturation: initially all nuclei are apical, but with every week passing, more and more nuclei shift down to the middle of the cells and finally to the basal position. This can be seen also in fetal AC: so this carcinoma reflects the maturation of the bronchial bud in every respect. Nuclei at mid position and even same basal-oriented nuclei do not abolish the other diagnostic criteria. Most fetal ACs are well differentiated with round nuclei, chromatin is often finely distributed within the nucleus, and nucleoli are small. Mitosis is rare, less than 3/HPF. However, there is a rare high-grade form; the architecture is the same, but the cytomorphological features are different: nuclei are enlarged and pleomorphic, nucleoli are enlarged, and mitosis can be up to 8/HPF (Fig. 17.67c, d).
Fig. 17.67
Fetal adenocarcinoma, two cases are shown. (a, b) Represent a well-differentiated form, which is the most common one. The tumor presents with an acinar pattern, the tumor cell cytoplasm is clear, and the nuclei are in part apical but also in mid portion and in other cells already at the final bottom side of the cell. The clear cytoplasm is a result of dissolving glycogen by tissue processing. The lower case (c, d) is a high-grade fetal adenocarcinoma with cell crowding, more atypia, and frequent mitosis. H&E. bars 100 and 50 μm
Signet Ring Cell Adenocarcinoma (SRC-AC)
Signet ring cells are most often scattered within mucinous AC; however, they rarely can occur as a carcinoma entirely composed of these cells, which has to be called SRC-AC. In a series of mucinous AC, few cases have been identified (Fig. 17.65e, f). The pulmonary SRC-ACs in contrast to the SRC-ACs of the gastrointestinal tract behave not like high-grade carcinomas but instead behave as other mucinous AC. So biologically they are different from their GI tract cousins [147].
Immunohistochemistry of AC
Non-mucinous ACs are predominantly arising from the bronchioloalveolar junction zone, where precursor cells for Clara cells and pneumocyte II are located in niches (niches where several alveoli and their alveolar duct join). These cells express TTF1, are positive for napsin A, and most often also for one or several of the surfactant apoproteins (Fig. 17.68). With respect to cytokeratins, these carcinomas express CK7 and CK18/CK19. They are usually negative for basal cell markers such as p63. In adenocarcinomas arising in small bronchioles and bronchi, there is a tendency to lose TTF1 staining and also to express focally p63. If the AC arises further proximal, TTF1 is negative, CK7 might be only focal positive, and AC can express CK20. This is especially true in AC of bronchial gland type (Fig. 17.59h). They are usually also positive for CEA and secretory component but negative for CDX2 (Fig. 17.69).
Fig. 17.68
Immunohistochemical markers in adenocarcinoma. (a, b) Staining for Napsin A, the papillary adenocarcinoma is positive with granular reaction in the cytoplasm, whereas in the solid adenocarcinoma only few cells are positive. Staining for cytokeratins are helpful in establishing the diagnosis of an adenocarcinoma, but there might be some exceptions: in (c) most of the cells from mucinous adenocarcinoma stain for CK7, however, a minority also for CK20 (d). An aid in the diagnosis of primary pulmonary adenocarcinoma is antibodies for surfactant apoprotein B (e) and TTF1 (f). However, TTF1 might be negative in some colloid adenocarcinomas (g). Not a diagnostic marker but an aid in separating primary lung versus gastric metastasis is b-catenin (h). Bars
Fig. 17.69
Immunohistochemistry for laminin (a) and fibronectin (b) can be used in those cases where invasion cannot be decided on H&E morphology. Staining for CEA and secretory component (c) can be used for adenocarcinomas arising in central bronchi. Staining for S100 protein will highlight dendritic cells, which are preferentially seen in papillary adenocarcinomas (d). ×200 and 100
Mucinous AC are most often positive for TTF1 and CK7, but a minority will show positivity for CK20 and few also for CDX2, a marker for enteric differentiation. Even two cell populations might be encountered, one staining for TTF1 and the other for CDX2 [147]. CDX2 as well as CK20 is positive in enteric AC, although in different case series, also positivity for TTF1 and CK7 was reported [233–235]. MUC1, MUC2, and 5 AC proteins are positive in all types of mucinous AC. Colloid AC is usually positive for CK7, sometimes CK20; however they are all positive for TTF1. In addition all colloid AC harbor KRAS mutation. Fetal AC is positive for TTF1, surfactant apoprotein antibodies, especially SApoB, and CK7. Signet ring cells are again positive for CK7and TTF1.
Another remarkable variant does exist, which is psammomatous adenocarcinoma. Usually this is an acinar or papillary structured AC with many psammoma bodies, at least 3–5/HPF. This type of AC is not different from acinar or papillary AC with respect of biological behavior, but since this type of AC is most frequent in AC from the ovary and thyroid, it needs to be mentioned. In these cases an immunohistochemical investigation is necessary, and TTF1 is not helpful in differentiating this from thyroid AC (Fig. 17.70). Here immunohistochemistry for surfactant apoproteins is the only way of sorting metastasis out. Clear cell changes occur in almost every non-small-cell carcinoma type, so this should be described but is of no diagnostic or prognostic value (Fig. 17.71).
Fig. 17.70
Psammomatous variant of adenocarcinoma. The tumor presents with clear cell pattern; some cells contain eosinophilic material, which is also found outside the cells. In addition numerous psammomatous bodies are encountered. The differentiation from thyroid carcinomas will need additional immunohistochemical investigation. H&E, ×200
Fig. 17.71
Clear cell pattern in adenocarcinoma; this is nonspecific; the adenocarcinomas will behave as any other type. H&E, bar 20 μm
Prognostic Factors
TNM staging is the most important factor for behavior of AC. The higher the stage, the worse the prognosis. Within AC there are some cases, which are so far not investigated: AC with low T but high N, and AC with high T and low N. Some of these cases can be explained, for example, by a micropapillary component, but not all of them. Architecture has been included now into grading; however, I still prefer cytomorphology over architecture. I also do not accept AIS or MIA as low grade, because this reflects staging.
Grading in Adenocarcinomas
G1 = mitotic counts 0–2/HPF, minor nuclear polymorphism, well-formed acinar or papillar structures, and important a single row of carcinoma cells
G2 = mitotic counts 3–7/HPF, nuclear polymorphism clearly visible, nucleoli enlarged, acinar, papillary pattern, but cells form double layers and also few cell papillae
G3 = mitotic counts >8/HPF, nuclear polymorphism clearly visible, prominent irregular formed nucleoli, solid, micropapillary, and also cribriform patterns
There are exceptions within the variants: fetal adenocarcinoma with regular nuclei, few mitoses, and no polymorphism is a slowly progressing AC G1, whereas there exists also a high-grade form, G3.
Another prognostic factor is invasion of the adenocarcinoma cells into lymphatics within a central scar predicts worse behavior. In most cases lymphatics can be easily identified by their thin wall and their endothelial cells; in a few cases, staining using antibodies against podoplanin might be necessary. In addition invasion into blood vessels and into the pleura is a sign of worse prognosis, whereas lymphatic invasion outside scar does not change prognosis (Fig. 17.72) [236, 237].
Fig. 17.72
Invasion of adenocarcinomas into blood vessels (upper panel), lymphatic vessels (middle), and into the pleura (lower panel). H&E, bars 100 and 50 μm
Cytology and Small Biopsies in AC Diagnosis
Almost 80 % of ACs are in stage IV when diagnosed. This means that most often, the diagnosis is established on small biopsies (bronchial, transbronchial, transthoracic/transcutaneous) or cytological material (EBUS-/EUS-guided fine needle aspiration, transthoracic needle aspiration, bronchial brush cytology, bronchial washings, and BAL). Due to the possibility of specific treatable driver gene mutations in the tumors, a significant portion of this already tiny material has to be preserved for molecular analysis. Therefore less is available for immunohistochemistry. This has led to a restrictive use of differentiation markers.
There are classical features which enable the diagnosis of adenocarcinomas in cytological specimen and small biopsies: polar orientation of nuclei, vesicular chromatin, large nuclei in high-grade adenocarcinoma, intranuclear inclusions (surfactant proteins), papillary complexes often in 3D spheres, mucin secretion, and goblet cell differentiation in mucinous adenocarcinomas (Fig. 17.73), and in addition in biopsies, acinar, papillary, micropapillary structures, solid with intracellular mucin, and solid with cytomorphological features suggestive of AC. In any case of uncertainty, immunohistochemistry is applied. However, only the two-marker approach is recommended: TTF1 for AC and p40/p63 for SCC. With classical cytomorphology and these two markers, almost 95 % of AC (and also SCC) can be correctly diagnosed (Fig. 17.74). There remains a small portion of so-called not otherwise specified carcinomas.
Fig. 17.73
Cytology of adenocarcinomas; the tumor cells show basal orientation of nuclei (a), clustering of cells into papillae (c, d, f), cytoplasmic vacuoles (a, b), ill-defined cell borders (d). Some tiny microvilli can be seen (c), multinuclear cells (b), and coarse chromatin pattern (d) best seen on H&E or PAP stains. Some tumor cells can mimic tadlepool cells characteristic for squamous cell carcinomas, but the basal located nucleus will help (e). Giemsa, H&E, PAP stains, bars 10, 20, 50 μm
Fig. 17.74
Small biopsies in adenocarcinomas: top a bronchial biopsy showing nicely arranged acini; a papillary pattern is seen in the transthoracic needle biopsy (middle). No invasion was encountered, however in a biopsy one can only state that invasion is not present. A tiny little transbronchial biopsy shows a cluster of cells from an adenocarcinoma. Not much information can be retrieved from such a biopsy, and even molecular analysis is not possible. H&E, bars 10, 50, 100 μm
This tendency of submitting less tissues and requesting more tests has led to the invention of the cellblock technique for cytology. Aspirated cells are transferred into liquids and submitted to pathology. Cells are centrifuged directly into warm liquid agarose forming a cell pellet at the bottom, or aspirates are transferred into a clotting substance. The so formed cell pellet is fixed in formalin and can be embedded in paraffin as a tissue biopsy. With this technology serial sections can be performed and immunohistochemistry is possible for different markers, if necessary.
Classification and Classification Problems
A new 2015 WHO adenocarcinoma classification has been published and should replace the 2004 WHO classification. This new classification also includes statements on the diagnosis of carcinomas in biopsies, which is becoming a major issue due to new treatment options (targeted therapy, see above).
What Are the Major Changes? (Table 17.1)
Table 17.1
Comparison of changes in the WHO classification of adenocarcinomas
WHO classification 2004 | WHO classification 2015 |
|---|---|
BAC with variants as mucinous, non-mucinous, and mixed mucinous-non-mucinous BAC | Replaced by in situ adenocarcinoma (this makes sense, because BAC was already classified as an noninvasive, i.e., in situ adenocarcinoma with variants as mucinous, non-mucinous, mixed mucinous-non-mucinous AIS) |
Minimal invasive adenocarcinoma was proposed by Y. Shimosato in 1999; the invasive focus should be less than 10 % of the tumor diameter, i.e., in a carcinoma of 15 mm diameter, invasion should not exceed 1.5 mm (but this was rejected by the majority of pathologists from the USA) | Minimal invasive adenocarcinoma (invasive portion less 5 mma) |
Mucinous, non-mucinous, mixed mucinous-non-mucinous MIA | |
Mixed adenocarcinoma | Predominant acinar Predominant papillary Predominant micropapillary Predominant solidb Adenocarcinomac Invasive mucinous adenocarcinomad |
Mucinous (colloid) adenocarcinoma | Mucinous (colloid) adenocarcinoma |
Mucinous cystadenocarcinoma | Now merged with colloid adenocarcinoma |
Fetal adenocarcinoma | Fetal adenocarcinoma |
Signet ring adenocarcinoma | This entity was skipped, but signet ring cells should be mentioned in the descriptione |
Clear cell adenocarcinoma | This entity was skipped; clear cells might be mentioned in the description Enteric adenocarcinoma; this is a newly accepted variant, which is characterized by a morphology mimicking colonic adenocarcinoma and by the expression of markers of colonic adenocarcinomas |
We have tried to solve these problems based on our own experience and literature data with invasive mucinous AC type. This results in a modification of the classification of invasive mucinous AC (Table 17.2):
Table 17.2
Classification of mucinous adenocarcinomas in comparison to non-mucinous types – the way I classify these tumors
Invasive non-mucinous AC | Invasive mucinous AC |
Predominant acinar | Predominant mucinous acinar |
Predominant papillary | Predominant mucinous papillary |
Predominant micropapillary | Predominant mucinous micropapillary |
Predominant solid (mucin producing and/or TTF1+) | Predominant mucinous solid |
Predominant cribriform | Predominant mucinous cribriform |
With predominant or focal signet ring cell component |
More problems in the present classification: AAH as the precursor lesion is not well separated from AIS. AAH is defined as an atypical proliferation of alveolar cells along the alveolar septa, without invasion. The lower degree of atypia and a size less than 5 mm are regarded as the main difference from AIS. However, grading of nuclear and cellular atypia is very subjective and thus not really helpful – no practicing pathologist would do morphometry. The most important feature of gaps between the neoplastic cells in AAH and the close of AIS cells should be more clearly stated. This feature points to the biology of tumor growth: a slow-growing lesion as AAH will leave a space between the tumor cells, whereas in the rapid-growing carcinoma, the cells use all spaces for their developing daughter cells. This has been proposed by the group of Shimosato [230]. In this classification AAH is characterized by a single row of atypical pneumocyte-like cells, proliferating along the alveolar surface, with intercellular gaps. As a caveat atypical cells must completely replace the alveolar epithelium; otherwise this is regeneration or reactive hyperplasia! The former high-grade AAH was transferred to AIS. AIS was characterized as an atypical proliferation along the alveolar surface, without invasion, and without alveolar collapse. The involved part of the lung is rigid and entirely covered by this proliferation. Epithelial papillae might be present. There are no longer gaps between the atypical cells.
Genes and Targets for Treatment in Adenocarcinoma
Although this will be more in deep discussed in the molecular pathology chapter, here a list of targetable driver genes are listed:
EGFR mutations in exon 18, 19, 20, 21, and few rare ones in exons 22–24. The best responders are with deletions within exon 19, followed by point mutations in exon 21. These also account for approximately 90 % of all mutations. The frequency of mutations is highest in Southeastern Asian patients (up to 65 %), less in Caucasians (12 %), and low in African Americans (6–8 %).
AKL1 gene rearrangement: The most common fusion partner is EML4, which also resides on chromosome 2 (inversion). This is seen in approximately 4–8 % of patients. Immunohistochemistry can serve to sort out negative cases; those with 3+ intensity staining by immunohistochemistry will almost always be positive by FISH analysis (Fig. 17.75).
Fig. 17.75
Immunohistochemistry for ALK1, all tumor cells are strongly stained (3+). These will be positive by FISH in almost every case, and even in FISH-negative cases, treatment by ALK inhibitor will improve the patient’s condition. ×100
ROS1 translocation is another gene fusion type of genetic aberrations found in AC. It accounts for approximately 2–4 % of patients. Also in these cases, immunohistochemistry should be used to sort out the negative cases.
KIF5B is one of the fusion partners for either ALK1 or RET. The KIF5B-RET fusion gene is caused by a pericentric inversion of 10p11.22-q11.21. This fusion gene overexpresses chimeric RET receptor tyrosine kinase, which can spontaneously induce cellular transformation. Besides KIF5B, CCDC6 and NCOA4 can form fusion genes with RET. Patients with lung adenocarcinomas with RET fusion gene had more poorly differentiated tumors, are younger, and more often never-smokers.
MET is another receptor tyrosine kinase bound to cell membranes in NSCLC. The ligand for MET is HGF, originally found in hepatic carcinomas. This receptor came into consideration in lung carcinomas, because amplification of MET or alternatively upregulation of HGF was identified as a mechanism of the resistance in EGFR-mutated adenocarcinomas. MET amplification was rare in NSCLC but upregulation of MET is approximately 20 % in NSCLC including adenocarcinomas and squamous cell carcinomas (Fig. 17.76).
Fig. 17.76
FISH for cMET, left a negative case with two signals for MET and centromere probes; right a positive case with clusters of MET amplicons. CISH, bars 10 μm
KRAS mutations are found in 25 % of all adenocarcinomas but in >50 % of mucinous AC. At this time there are only phase I and II trials targeting the downstream proteins ERK and mTOR.
Some rare genetic aberrations are in amplifications and mutations in ERBB2 (HER2Neu) and BRAF, which can be targeted by drugs available for other malignancies.
A marker for response to chemotherapy of platinum compounds has been reported. ERCC1 is a member of the DNA repair enzyme machinery. In those cases, where ERCC1 is highly expressed, this type of chemotherapy is ineffective (Fig. 17.77) [145].
Fig. 17.77
Immunohistochemistry for ERCC1, left a case with strong nuclear expression (SCC); right a case with almost negative staining (adenocarcinoma). Bars 50 μm
17.3.3.3 Large-Cell Carcinoma (LC)
Gross Morphology and Clinical Picture
LC is usually a large tumor, which will present with unspecific clinical findings such as weight loss, cough, and sometimes hemoptysis. Since LC is most often peripheral in location, symptoms due to bronchial obstruction are rare (Fig. 17.78). On X-ray and CT scan, the tumor presents as a mass lesion, which on PET-CT will also show tracer uptake.
Fig. 17.78
Macroscopic picture of a large-cell carcinoma with typical peripheral location
Histology
LC is defined by large cells devoid of any cytoplasmic differentiation and large vesicular nuclei (>26 mμ). Nucleoli are sometimes as prominent as in AC. They have a well-ordered solid structure but no palisading, no rosettes, or any other characteristics (Fig. 17.79). By electron microscopy differentiation structures can be seen such as hemidesmosomes, tight junctions, intracytoplasmic vacuoles with microvilli, and ill-formed cilia. This fits clearly into the concept of a carcinoma, at the doorstep of adenocarcinoma and squamous cell carcinoma differentiation. Mitotic counts can be numerous or scarce; despite this carcinoma is a grade 3. LC numbers have dramatically decreased due to the use of immunohistochemistry, because many of them are either solid undifferentiated AC or SCC. Those cases expressing TTF1 and CK7 are now regarded as undifferentiated AC; those with positivity for p63/p40 and CK5/CK6 are now undifferentiated SCC. Therefore only few cases remain in LC. In addition as this is a diagnosis of exclusion, this diagnosis can only be made after careful analysis of a resected tumor specimen (Fig. 17.80). In biopsies this carcinoma will fall under NSCLC NOS.
Fig. 17.79
(a–f) Examples of large-cell carcinomas. In most of them, nuclei show coarse chromatin, enlarged middle-sized nucleoli, and accentuated nuclear membrane. The cytoplasm can be vacuolated or clear, nuclear size is >26 μm, and cell borders are most often vague. (e, f) Represent a case, which at a first glance resemble large-cell neuroendocrine carcinoma with rosette-like structures, but as the other cases was negative for neuroendocrine markers, TTF1, and p40. H&E, bars 10 and 20 μm
Fig. 17.80
Immunohistochemistry in large-cell carcinomas: (a) pan-cytokeratin staining; (b) vimentin can be expressed in some cases, but usually a coexpression with cytokeratins; (c, d) CK7 with different intensities; (e) focal and only weak staining for p63; (f) absence of TTF1; and (g) in rare cases a few cells can stain for neuroendocrine markers; here chromogranin A (h) and in rare instances LC might be positive for CEA but is negative for markers of germ cell tumors. Bars 10, 20, 50 μm
By cytology the cells looks like any undifferentiated carcinoma. Nuclei are large centrally positioned, diameter >26 μm, chromatin is coarse, nucleoli are middle sized, cytoplasm basophilic without any differentiation, and cells form small and large clusters.
17.3.3.4 Neuroendocrine Carcinomas
Within this group, typical, atypical carcinoid, and small and large-cell neuroendocrine carcinoma is placed. Each of these tumors show infiltrative growth as any other carcinoma; each can set metastasis and might kill the patient, if not treated properly. However, there are also differences. Typical carcinoid is a slow-growing tumor, which rarely set metastasis. Atypical carcinoid is of intermediate malignancy, with a higher frequency of metastasis. Both carcinoids behave biologically different from the two high-grade carcinomas: metastasis after surgical removal does not occur before 7 years, and the risk of dying from recurrence and metastasis peaks around 12 years after surgery [238]. This was also a reason for Masson-Hamperl (Virchows Arch [Pathol Anat] 266:509–548, 1927) to name these tumors carcinoids, i.e., carcinoma-like but not identical. In addition by the name carcinoid, it was also implicated that this is an epithelial tumor. In the last decade, several attempts were made by non-pulmonary pathologists to change the classification according to their classification in the gastrointestinal tract, using neuroendocrine tumor, well-differentiated neuroendocrine carcinoma, and high-grade carcinomas [239]. However, in contrast to GI tract tumors, the lung tumors are different in several aspects: there is no common genetic alterations among them, they do not evolve from each other, and the low grade arise from neuroendocrine cells and share precursor lesions such as tumorlets, whereas the high grades are from undifferentiated probably stem cell-like precursor cells. Three of them are associated with cigarette smoking, whereas typical carcinoid is not [240, 241]. So a change of the name without having new definitions at hand would be like changing the emperor’s clothes (he is naked: in the fairy tale of The Emperor’s New Clothes).
Small-Cell Neuroendocrine Carcinoma (SCLC)
Epidemiology
SCLC together with SCC formed the major part of pulmonary carcinomas from the early twentieth century until the early 1990s in Austria. During the 1990s this changed, adenocarcinoma became the number one, SCC dropped dramatically, whereas SCLC remained with about 25 % of lung carcinomas stable. In the early 2000, SCLC started to drop also and is seen today in about 8 % of pulmonary carcinomas. One of the reasons are the changes of smoking behavior 20 years back: filter cigarette has completely replaced the filterless one; due to lowered nicotine content, smokers more frequently and more deeply inhaled tobacco smoke to reach the desired nicotine level. Due to the latency period of 15–30 years after starting with smoking (for females the latency period is shorter, for males longer), this fits well with our observation.
Gross Morphology and Clinical Symptoms
SCLC will show symptoms such as hemoptysis, cough, and rapid weight loss; SCLC can present as a small tumor with large metastasis at detection. Hormonal symptoms can be found in some cases, most often due to production and release of corticotropin, serotonin, calcitonin, and parathyroid hormone [242].
Small-cell carcinoma is defined by nuclear size of 16–23 mμ (not so small!), dark-stained nuclei (mainly composed of heterochromatin), inconspicuous or lacking nucleoli, small cytoplasmic rim, often invisible in light microscopy, and fragile nuclei. On frozen sections the cytoplasm can be quite broad, and so these carcinomas can be misdiagnosed as non-SCLC. In frozen sections carefully investigate the nuclear details! To assess the nuclear size, just look for adjacent lymphocytes or granulocytes: those have diameters of 7 and 14–16 mμ, respectively. The fragility of the nuclei gives rise to chromatin encrustation of veins, where the chromatin gets trapped at the basal lamina (Fig. 17.81).
Fig. 17.81
(a–c) Different examples of SCLC. In (a) a well-preserved bronchial biopsy, an organoid structure is visible, even some ill-formed rosettes. The nuclear characteristics are most important: nuclear size 18–23 μm, dense chromatin, invisible nucleoli, and nuclear crowding. The amount of cytoplasm can vary, depending on fixation time. In frozen section SCLC will look essentially as here. (b) Surface area of a bronchial biopsy. The tumor cells are spreading within the epithelium. It is very likely that SCLC arise from stem cell-like precursors in this area. The carcinoma cells from the very first beginning can move within the epithelium and across the basement membrane like stem cells do. (c) Another case with less well-preserved tumor cells. However the nuclear features are visible, and lymphocytes (7 μm) or granulocytes (14–16 μm) will serve as measurement standards for the size of the tumor cell nuclei. (d) Ill-formed rosette in a SCLC, not very common in biopsies. (e) Carcinoma cells invading the squamous metaplasia. (f) Transthoracic biopsy, not properly fixed, which causes this dense nuclear picture. In such a case, one might be forced to search for better preserved areas. (g, h) Crush artifacts in bronchial biopsies. In (g) there is a characteristic finding of encrustation of small veins by tumor DNA. This is common in SCLC due to the high rate of apoptosis and the rapid growth. Such a finding is suggestive but not diagnostic. (h) A biopsy where SCLC might be suspected. Immunohistochemistry can help in some cases. H&E, bars 50, 20, 10 μm
SCLC is regularly positive for the neuroendocrine marker NCAM, often for synaptophysin and NSE but most often negative for chromogranin A. The best marker is NCAM with a strong membranous staining. SCLC is usually positive for low molecular weight cytokeratins (CK18) and will show a capping-like reaction, i.e., the positive staining is like a cap on one side of the cell (this is the area where intermediate filaments are concentrated together with neurosecretory granules). SCLC produces hormones, such as adrenocorticotropin (ACTH), but also substances interfering with the blood coagulation system (Fig. 17.82). In contrast to carcinoids, SCLC more often are positive for heterotopic hormones (i.e., hormones usually not found in adult lung). If SCLC is combined with any other type of carcinoma, it is defined as SCLC, combined form. There is one exception: carcinosarcoma that can have an SCLC component. In these cases the diagnosis is carcinosarcoma. In these cases I personally list all the components present in the tumor.
Fig. 17.82
Immunohistochemistry and electron microscopy of SCLC. (a) Staining with cytokeratin shows a cuplike pattern, due to an uneven distribution of intermediate filaments (see g). (b) This case of SCLC was detected because of Cushing syndrome. Immunohistochemistry with antibodies for corticotropin (yellow) showed the reason for high cortisol levels in the blood. Upon chemotherapy the hormone levels dropped down to normal. (c–e) A case of SCLC where in (d) the typical cytokeratin staining pattern is nicely seen and contrasts well with the staining of normal epithelial remnants. The membranous staining for NCAM (e) is one of the most helpful aids in SCLC diagnosis. (f) SCLC in contrast to carcinoid has usually few neurosecretory granules (small dark dots in the cytoplasm), which explains why staining for chromogranin A is often negative (below threshold). The cytoplasmic rim in the tumor cells points to the shrinkage normally seen in formalin-fixed specimen. (g) Two neighboring tumor cells. The cell border is not well delineated but the concentration of intermediate filaments is evident. Among these proteins are cytokeratins, explaining the cuplike reaction. (a, b), ×600, 100, (f, g), ×3,000, 7,000, bars 20 μm
SCLC is defined by high mitotic counts; rarely visible organoid pattern; round, ovoid, or spindle-shaped nuclei between 16 and 23 mμ in diameter; dense heterochromatin; invisible nucleoli; and small or even invisible cytoplasm. By electron microscopy usually neurosecretory granules can be found. By immunohistochemistry SCLC are positive for low molecular weight cytokeratins (CK 7/CK 8, CK 18/CK 19), neuroendocrine markers (NCAM, synaptophysin, NSE, rarely CGA), and also for some hormones (Figs. 17.81 and 17.82). TTF1 is positive in most SCLC with a high percentage of stained nuclei – the function of TTF1 in SCLC is not known. In our experience a positive reaction for gastrin-releasing hormone and ACTH is most often seen. The secretion of ACTH can cause Cushing’s syndrome.
High copy number gains are detected in SCLC encoding JAK2, FGFR1, and MYC family members. Most common losses are seen in RB1 and 59 microRNAs of which 51 locate in the DLK1-DIO3 domain. Alterations of the TP53 gene and the MYC family members were predominantly observed in SCLC. Potential drug targets might be the AKT-mTOR and apoptosis pathways in SCLC [243]. In array CGH unbalanced aberrations are in almost every chromosome; a specific gain on chromosome 3q was however seen in two thirds of SCLC discerning it from LCNEC [244] (Fig. 17.83). A further analysis of the area might disclose some markers suitable for this differential diagnosis.
Fig. 17.83
Comparative genomic hybridization of small-cell and large-cell neuroendocrine carcinomas. SCLC in blue, LCNEC in violet, overlaps of both are in orange. There are some characteristic numeric aberrations: in chromosome 3q SCLC have gains, whereas LCNEC is normal, in chr.10q: SCLC has losses toward the telomeric end, LCNEC no; chr.9q LCNEC has gains, SCLC not; chro.16q SCLC has losses, LCNEC no, but LCNEC has gains in chr.16p; and finally SCLC has losses on chr.17p, LCNEC not. All these aberrations will need further investigation for specific genes within these regions
SCLC can occur combined with other non-endocrine carcinomas, which is then acknowledged as combined SCLC. SCLC was previously staged as either limited or extensive disease. A change to TNM is now mandatory.
SCLC is sensitive for chemotherapy and radiotherapy in almost 100 %. However, the prognosis is still pure. Recurrence does occur in most cases, and metastasis is most often present, even when the primary tumor is small. Less than 20 % of patients survive more than 5 years. New therapy might be available within the next years, interfering with the regulation of cell proliferation (inhibitors of tyrosine kinases such as the Src kinase family members, etc.). Another feature of SCLC is a change of the phenotype in recurrent disease: there might be a predominant squamous cell component and even no SCLC. There are two explanations for this phenomenon, and both have been proven: within the SCLC there are hidden non-SCLC tumor cells, which have a growth advantage, when the SCLC tumor is destroyed by chemotherapy and, second, SCLC cells themselves might react to chemotherapy by differentiating into non-SCLC variants, which are more resistant to chemotherapy (transdifferentiation; Fig. 17.84).
Fig. 17.84
SCLC resection after primary chemotherapy with clinical and radiological response. Within the scars there were small remnants of the carcinoma, focally showing transdifferentiation into squamous cell carcinoma. H&E, ×150
In small biopsies and cytology, SCLC is characterized by a nuclear size of 18–23 μm in diameter (3× lymphocyte, 1.5× granulocyte), dense chromatin, invisible or tiny nucleoli, and small rim of cytoplasm; the comparison with internal size markers is useful, as shrinkage due to formalin fixation affects also lymphocytes and granulocytes. Typically the carcinoma forms minimal cohesive cell groups but rarely rosettes (Fig. 17.85). By immunohistochemistry positivity for NCAM (CD56) and for low molecular weight cytokeratin is helpful. In cytokeratin immunohistochemistry the important feature is a focal cuplike staining, which corresponds to a concentration of intermediate filaments on one side of the cells, usually where also neurosecretory granules are found. By NCAM the staining is membrane based and even retained in necrotic areas. Chromogranin A is most often less helpful, because the small numbers of neurosecretory granules present in SCLC very often results in low protein concentration below the detection rate of the CGA antibody. NSE and synaptophysin are the two other markers, which can be used; however, it should be noted that these are less sensitive and can stain tumors within the differential diagnosis of SCLC such as PNET.
Fig. 17.85
Cytology of SCLC, (a–d), and comparison to carcinoid (e). Clustering of the tumor cells (crowding) is common in this tumor (a, d); the nuclear features are best seen in (b, c): dense chromatin, no visible nucleoli, small cytoplasmic rim, and cell cannibalism. In contrast carcinoids (e) present with epithelial clusters, well-organized nuclei have coarse chromatin, nucleoli are visible and enlarged, and abundant cytoplasm is present. Mitosis is rarely seen in cytological preparations of carcinoids. Giemsa (a, b, e), PAP (d), and modified Giemsa-azure blue (c); bars 10 and 20 μm, in (c) ×630, in (e) ×400
Large-Cell Neuroendocrine Carcinoma (LCNEC)
On gross examination the only feature that might point to LCNEC are large areas of necrosis, which by themselves are not specific. Clinically LCNEC present as a tumor mass on CT scan and X-ray. There are no specific clinical symptoms.
Large–cell neuroendocrine carcinoma is defined by a neuroendocrine pattern, i.e., rosettes, trabeculas, and solid cell nests. On low-power LCNEC looks organoid, similar to a carcinoid, but on higher magnification abundant mitoses are obvious. By counting the number of mitoses, one can easily reach 20 per high-power field, making a total of up to 200 per 2 mm2, which is never reached by atypical carcinoid. LCNEC is defined by large polymorphic nuclei 25–35 mμ, a coarse granular chromatin, and large, landscape-like necrosis (Fig. 17.86) [245, 246]. To confirm the diagnosis, a staining for neuroendocrine markers is recommended, such as NCAM, synaptophysin, chromogranin A, and also NSE. LCNEC can produce hormones as SCLC. LCNEC is also positive for low molecular weight cytokeratin. LCNEC can occur combined with other pulmonary carcinomas; if combined with non-SCLC, the diagnosis is combined LCNEC; if combined with SCLC, the diagnosis is combined SCLC.
Fig. 17.86
Examples of large-cell neuroendocrine carcinomas (LCNEC) in (a–d), and a mixed LCNEC with adenocarcinoma as well as a spindle cell carcinoma in (e, f). In (a) large necrosis is seen, rosettes are ill formed. Rosettes are better seen in (b–d); in addition the nuclear features show enlarged nuclei, coarse chromatin, enlarged nucleoli, and frequent mitosis (can be up to 25/HPF). (e) A mixed LCNEC (left) with adenocarcinoma (right) and spindle cell carcinoma (f) is presented. Immunohistochemistry for chromogranin A (g), synaptophysin (h), and NCAM (i). In contrast to SCLC, this high-grade neuroendocrine carcinoma is less often intensely stained for NCAM but more common for CGA and synaptophysin. H&E, bars 20 and 50 μm
The prognosis in LCNEC is similar to SCLC. Surgery is recommended for LCNEC in stages I–IIIA. In recent time also a similar chemotherapy regimen is favored, similar to SCLC. A majority of patients respond to these treatments; however, recurrence and metastasis are as high as in SCLC. Recent investigations have found some genes specifically altered in LCNEC: FGFR2 mutation was detected exclusively in LCNEC [247], and in another study, mutations in TP53, and STK11, were seen frequently, whereas mutations of PTEN rarely in LCNECs [248]. As tyrosine kinase inhibitors do exist for FGFRs, this finding might open potentially a new treatment strategy. Another finding useful for the differentiation of SCLC and LCNEC is the finding that CDX2 and VIL1 in combination showed sensitivity and specificity of 81 % for LCNEC, while BAI3 showed 89 % sensitivity and 75 % specificity for SCLC [249].
In small biopsies LCNEC can be diagnosed, if rosettes and trabeculas are present, and the nuclei are large (diameter >26 μm), the chromatin is coarse, and the nucleoli are middle sized. High mitotic counts might be encountered, whereas the large necrotic areas might not be seen (Fig. 17.87). Immunohistochemistry will be an aid. In cytological preparations the diagnosis is more difficult. If the nuclear features are present and numerous mitoses are seen, an immunocytochemistry for neuroendocrine markers should be performed.
Fig. 17.87
LCNEC can sometimes easily be diagnosed on transthoracic core needle biopsies if the rosette pattern is clearly visible. A stain for one of the neuroendocrine markers will confirm this diagnosis. H&E, bars 50 and 10 μm
Carcinoid, Typical, and Atypical
Clinically carcinoids present by symptoms of obstruction due to the endobronchial part of the tumor. This results in productive cough and recurrent infections in the tumor-bearing lobe. On X-ray and CT scan, a most often centrally located tumor is seen (Figs. 17.88 and 17.89). On bronchoscopy an almost characteristic bleeding is reported, whenever the tumor is touched by the bronchoscope. Symptoms by the release of hormones are rare; Cushing’s syndrome can be seen due to the release of corticotropin.
Fig. 17.88
CT scan of a carcinoid. The tumor is visible at the lower left side, located within a bronchus with obstruction of the peripheral branches
Fig. 17.89
Typical carcinoid (upper and middle panel and atypical carcinoid (lower panel). The resection specimen is shown, from the resection margin a polypoid tumor is visible, obstructing both upper and lower left bronchus (17-years old boy). In the middle the tumor is seen (after frozen section margin analysis), and the mucus accumulation is visible behind the tumor. In the atypical carcinoid, an intrapulmonary metastasis was already present
Typical carcinoid is defined by neuroendocrine structures, such as rosettes, trabecules, and solid nests, 0 or 1 mitosis per 2 mm2, and absence of necrosis. You will usually find central capillaries or veins in the rosettes. In general carcinoids are well vascularized, which is the cause why they tend to bleed when touched by the endoscope during bronchoscopy. The rosette is the functional structure where carcinoid cells release their hormones and biogenic amines into the local circulation. The nuclei of typical carcinoids are uniform, round, with finely dispersed chromatin, and inconspicuous nucleoli (Fig. 17.90). There are some variants, which can create problems in diagnosis, such as spindle cell carcinoid and oncocytic carcinoid. The spindle cell carcinoid cannot be diagnosed without immunohistochemistry. The entire tumor or large parts of it is composed by spindle cells arranged in whorls without stroma in between them. A few capillaries might be seen. This rare variant behave the same as any other typical carcinoid. Also carcinoids, which synthesize and secrete some hormones such as parathyroid hormone and calcitonin, can present with bone or amyloid formation.
Fig. 17.90
Typical carcinoid and variants. In (a, c) a more solid and nesting pattern dominates in this case; the typical rich capillary network is nicely demonstrated. (b) This carcinoid shows rosettes and trabecules. In (d) rosettes dominate the pattern. (e) This is an unusual oxyphilic carcinoid with giant mitochondria on electron microscopy. (f) shows bone formation and (g) amyloid deposition in carcinoids. In carcinoids with bone formation, usually parathyroid hormone and/or calcitonin secretion is found in the tumor cells, whereas amyloid is based on calcitonin production and secretion. (h) A spindle cell carcinoid is a rare variant where the diagnosis can only be made with the aid of immunohistochemistry. H&E, bars 10, 20, 50 μm, ×200, 100, 150
Atypical carcinoid is defined by two to ten mitoses per 2 mm2, and/or presence of necrosis, and again neuroendocrine structures. The nuclei of ATC are usually larger; enlarged nucleoli are seen more frequently (Fig. 17.91a, b). In both carcinoids there is an invasive/infiltrative growth into the lung, and lymphatic and blood vessel invasion can be found in some cases. Some carcinoids can metastasize, but so far there are uniform predictive markers for the biological behavior. In general atypical carcinoids with mitotic counts >5/2 mm2 or carcinoids with lymphatic or blood vessel invasion will behave more aggressively, will metastasize, and will ultimately kill the patient (Fig. 17.91c). This group comprises 25 % of atypical carcinoids and single cases of typical ones.
Fig. 17.91
(a) Small necrotic foci are one of the hallmarks of atypical carcinoid; another feature is increased mitosis (b); (c) lymphatic invasion can be found in both carcinoids, but more frequently in atypical ones. Lymphatic invasion should be investigated in the border zone of carcinoids especially around blood vessels. They cannot be seen in the tumor center. H&E, ×250, 150, 100
In addition those carcinoids, which have more than two losses on distal chromosome 11q (LOH), and those with multiple chromosomal losses (<5), also show a pure outcome [240, 241, 250]. There is an overlap of chromosomal losses and higher mitotic counts, however, not a 100 % concordance. On the other hand, typical carcinoids with 0–1 mitotic counts per 2 mm2 and without lymphatic or blood vessel invasion have a good prognosis, when completely resected – in our experience a 100 % survival, no recurrence, and no metastasis (L0, V0, N0) [238]. But there remain a group of carcinoids, most atypical ones, for which the prognosis cannot be predicted.
Diagnosis of carcinoids can be made on biopsies and cytology. However, it is recommended to produce Cytoblock from cytological material for additional immunocytochemistry (Fig. 17.92). However, a differentiation into typical or atypical carcinoid is often impossible, unless there are two mitoses within the specimen.
Fig. 17.92
Cells from a carcinoid derived from fine needle aspiration. A Cytoblock was prepared, which enables to cut serial sections for H&E and immunocytochemistry, here chromogranin A. Small rosettes are visible in these clusters of cells. Bars 50 μm
Carcinoids can produce multiple hormones and neurotransmitters. The synthesis and secretion are not coordinated as in normal neuroendocrine cells. So the production can be seen by immunohistochemistry, but there might be no secretion. The production of hormones has no correlation with the biological behavior. In rare cases there is no detectable hormone production but instead an increase of mitochondria (oncocytic carcinoid); this is easily visible on H&E stain, because the mitochondria stick out as tiny eosinophilic granules (Fig. 17.93).
Fig. 17.93
Immunohistochemistry and electron microscopy of carcinoids. (a) Staining for chromogranin (b) staining for NCAM, (c) synthesis of vasointestinal peptide in a typical carcinoid, (d) synthesis of parathyroid hormone in a carcinoid. (e) Electron microscopy of a typical carcinoid shows multiple neurosecretory granules, and also the intimate association of the tumor cells with the capillary in the center. This structure corresponds to the rosette, seen on light microscopy. (f) Oncocytic carcinoid, here only single neurosecretory granules are found, but multiple mitochondria, some giant forms. ×100; ×2,500 and 3,000
Carcinoids can be found as central tumors or peripheral. Central carcinoids usually show an iceberg phenomenon: a small part of the tumor produces bronchial stenosis by an endobronchial component (tip of the iceberg), whereas the major part lies within the lung parenchyma. Therefore carcinoids should never be locally excised like hamartomas. There is no preferred location; all lobes can be affected equally.
Based on genetic studies, it can be speculated that NCAM, its 140 kDa isoform precursor (NCAM 140), Zinc finger protein-like 1, and sorting Nexin 15 might be involved in the genesis of carcinoids [250].
Staging: staging of large-cell neuroendocrine carcinomas is done as usual. The IASCL and UICC new staging manual recommends staging of all neuroendocrine tumors including SCLC and carcinoids. We have staged carcinoids since the 1980s and have found this to be a very useful prognostic marker.
There is an ongoing discussion since decades that neuroendocrine tumors share a common precursor cell and even that the highly aggressive ones might develop from the low malignant ones. This hypothesis is based on the behavior of all four tumors to express general neuroendocrine markers, to synthesize hormones, and to form neuroendocrine structures. So these tumors share some phenotypic features. When looking at the genotype, it is obvious: these tumors have not much in common. Whereas SCLC and LCNEC have typical chromosomal aberrations at chromosomes 3, carcinoids have a few aberrations, and at chromosomal locations, uncommon in the high-grade forms – except chromosome 11q where LCNEC and atypical carcinoids share similar losses. If a high-grade tumor could develop out of a low-grade one, then chromosomal aberrations, such as gains and losses, should be retained in the high-grade carcinoma, and other aberrations should be seen on top of those [241, 244]. In the development of SCLC and LCNEC, tobacco carcinogens are the main factors, whereas in carcinoids not much is known about the inducing factors. In atypical carcinoids approximately 50 % are smokers and the other half never-smokers, and only in smokers also TP53 gene mutations do occur [251]. It has become clear that carcinoids can develop from neuroendocrine cell hyperplasia and also from tumorlets, whereas SCLC and LCNEC do not. Some new information point to the fact that primitive stem cells of the lung posses’ neuroendocrine features, and in organogenesis of the lung, neuroendocrine cells are probably the first differentiated cells within the epithelium, which we have proven in a study using fetal lung from different gestational ages. SCLC in our present understanding develops from primitive stem cells of the central lung, which exhibit a neuroendocrine phenotype. Therefore the primary expansion of carcinoma cells within the epithelium is not visible, because the cells look like undifferentiated stem cells (Fig. 17.94) [252]. And most probably these tumor cells are capable of immediately invading the stroma. In surgical resection specimen carcinoma cells can be recognized within the epithelium as sheets but also as single cells interspersed between normal bronchial epithelium, probably representing the precursor lesion.
Fig. 17.94
Genetically engineered mouse model with SCLC in an early stage. In this stage the carcinoma cells can be seen replacing the normal epithelium, and the cells are indistinguishable from the basal cells. A few cells have started to move into the stroma. H&E, bar 20 μm (Courtesy of Adi Gazdar)
There are different signaling cascades, driving neuroendocrine differentiation in high-grade carcinomas. Human achaete-scute homolog-1 (hASH1) was identified as responsible for inducing a neuroendocrine phenotype in SCLC, also proven in mouse models [252–255]. However, ASH1 does not induce small-cell carcinoma by itself but can induce a neuroendocrine phenotype in other cell types as well, for example, in Clara cells in a mouse model of undifferentiated lung carcinoma with expression of neuroendocrine features, but not reproducing the SCLC morphology [256]. Loss of RB1 and p130, mutation of TP53, and probably alterations of other genes are responsible for the induction of SCLC [257]. ASH1 is an important regulator of neuroendocrine differentiation in normal and fetal lung but also plays an important role in neuroendocrine differentiation of high-grade carcinomas, whereas seems less important in carcinoids [258, 259]. Some other genes within the Achaete-scute complex-like1 such as ASCL1, hASH1, and Mash1; atonal homolog 1 genes as ATOH1, hATH1, MATH1, and NEUROD4 genes (ATH-3, Atoh3, MATH-3); and neurogenic differentiation factor 1 (NEUROD1, NEUROD, BETA2) might be candidates as well, because they also show differential expression among lung tumors. Tumors with high levels of ASCL1 also express neuroendocrine markers, and this is accompanied by increased levels of NEUROD1. ATOH1 expression was found in adenocarcinomas with neuroendocrine features. Aberrant activation of ATOH1 leads to a neuroendocrine phenotype similar to what is observed for ASCL1 and might be another mechanism for NSCLC with neuroendocrine phenotype [260]. Interestingly hASH1 is not only responsible for the neuroendocrine phenotype in SCLC and LCNEC but has also other much broader function. Knockdown of hASH1 gene in human lung cancer cells in vitro suppressed growth by increasing apoptosis, whereas forced expression of hASH1 in human bronchial epithelial cells decreases apoptosis. This can be interpreted that hASH1 promotes remodeling in lung epithelium through multiple pathways [261].
ASH1 is placed within the Notch signaling pathway and seems to be antagonized by HES1. By overexpression of NOTCH-1, NOTCH-2, or the Notch effector protein, human hairy enhancer of split-1 (HES1) in SCLC cell cultures and Notch proteins but not HES1 caused a profound growth arrest. Active Notch proteins led to marked reduction in hASH1 expression and activation of phosphorylated ERK1 and ERK2. So in contrary to the oncogenic function of Notch in other tumors, Notch in the setting of highly proliferative hASH1-dependent neuroendocrine carcinomas causes growth arrest and thus acts like a suppressor [262]. This findings fit well with the observation on Notch-inactivating mutations in EGFR-mutated adenocarcinomas under tyrosine kinase inhibitor therapy. In EGFR-mutated and tyrosine kinase inhibitor-resistant adenocarcinomas, mutations in the Notch genes seem to regulate the transdifferentiation into a carcinoma of small-cell neuroendocrine phenotype [263].
Differential Diagnosis of Neuroendocrine Tumors
High grade can be easily differentiated from the low-grade ones by their number of mitoses. The differentiation of LCNEC from SCLC is not always easy: there are rare overlapping cases, which do not fit well into one of these categories. Helpful features are nuclear size >25 μm favors LCNEC, almost 100 % positivity for TTF1 favors SCLC. By cytokeratin stain SCLC shows a cuplike reaction, whereas in LCNEC the cell membrane and cytoplasm is circumferentially stained. LCNEC can be differentiated from other undifferentiated carcinomas by the staining for neuroendocrine markers (>30 % of cells for either NCAM, CGA, synaptophysin). In SCLC almost 100 % of tumor cells stain for NCAM; the staining for the other neuroendocrine markers is variable.
SCLC cannot be differentiated from small-cell carcinomas of other location, because the staining pattern is identical. Also LCNEC can occur outside the lung and a metastasis from the upper respiratory tract within the lung can be hard to differentiate from a lung primary. Within the differential diagnosis of SCLC, other blue small round cell tumors need to be discussed. A cytokeratin positivity differentiates SCLC from PNET and the tumors of the Ewing sarcoma group. Synovial sarcoma presents with larger cells if epithelioid, and in case of the sarcomatoid variant, the cell nuclei are all spindle type, whereas in SCLC there are always two types of nuclei present, a polygonal one and a plump spindle type.
In our current understanding of carcinogenesis, we have learned that different phenotypic features in carcinoma cells are not always related to each other. Capability of invasion, metastatic potential, lymphatic invasion, are capabilities, which some carcinoma cells acquire, others not. The ability to produce hormones can be an advantage for some carcinoma cells, because they can produce their own growth hormone, synthesize receptors for these factors, and so get independent for growth stimuli (autocrine growth stimulatory loop). But this by no means can be taken as a proof of a common ancestry. In SCLC such loops exist: gastrin-releasing peptide or corticotropin is synthesized by the tumor cells; they express receptors for these hormones. The receptor-hormone ligand interaction activates the RAS signaling system either directly or indirectly by stimulating synthesis of ligands for tyrosine receptor kinases; this results in proliferation. SCLC in cell cultures can double their cells within 30 days [264–267].
NSCLC with NE features is defined as a non-small-cell carcinoma (SQCC, AC, LC) with positivity for at least one neuroendocrine marker, such as NCAM, CGA, and synaptophysin in up to 25 % of tumor cells. Since there is no prognostic difference among non-SCLC with and without neuroendocrine features, this diagnosis is clinically of no importance. The diagnosis can only be made by immunohistochemical stains, because these carcinomas do not show neuroendocrine structures (Fig. 17.95) [268].
Fig. 17.95
Comparison of non-small-cell carcinoma with neuroendocrine features and SCLC. Left a small-cell variant of SCC, the inset shows scattered tumor cells which express NCAM, whereas to the right a SCLC is shown and in the inset more than 60 % of tumor cells expressing NCAM. H&E, bars 20 μm
17.3.4 Carcinomas with Clear Cells
Carcinomas with clear cells: This was a separate entity, but as clear cells can occur in almost every carcinoma, this is now mentioned in the description. Take care on frozen sections: these cytoplasms are by no means clear but are well stained. There is another caveat: clear cell carcinomas in the lung are most often metastases of renal clear cell carcinomas and rarely lung primaries. Primary pulmonary carcinomas entirely composed of clear cells are vimentin negative and cytokeratin positive (renal will often show coexpression of cytokeratins and vimentin; Fig. 17.96a, b). In addition renal carcinoma metastases are usually centered along pulmonary arteries and show large infarct-like necrosis.
Fig. 17.96
(a, b) Undifferentiated primary pulmonary carcinoma entirely composed of clear cells. This would have corresponded to the former clear cell variant of large-cell carcinoma. (c, d) Hepatoid carcinoma showing strands of tumor cells, which on higher magnification resemble hepatocytic carcinomas. There are some inclusions, which look like Mallory bodies (d). (e–g) Carcinoma with rhabdoid morphology; especially in F the growth pattern is interesting, as the tumor cells grow underneath the pneumocytes and thus might simulate an epithelioid angiosarcoma or any other epithelioid sarcoma. The eosinophilic inclusion bodies are quite good seen in (f); the cytokeratin stain in (g) highlights the less intense staining of the tumor cells compared to the normal epithelium. The inclusion bodies are even better seen as negative corpuscles in the cytokeratin stain. H&E, ×25, 150, and 200
17.3.5 Rhabdoid Carcinoma
Rhabdoid carcinoma is characterized by a solid growth pattern, often overlaid by a reactive proliferation of pneumocytes, which can give these tumors a pseudoalveolar pattern and a pseudo-composition of two cell populations. Within the cytoplasm of the tumor cells, eosinophilic inclusion bodies can be found, similar to those seen in rhabdomyosarcomas. These inclusion bodies are stained by eosin and are negative for striated muscle markers but positive for vimentin. The nuclei are large with a diameter >26 μm, chromatin is coarse, and nucleoli are middle sized. Rhabdoid carcinoma can be diagnosed on small biopsies or cytology (Fig. 17.96e–g).
17.3.6 LC of Hepatoid Phenotype
LC of hepatoid phenotype is characterized by large cells which resemble hepatocytic carcinoma. The cells form sheets of cells, the nuclei are large, chromatin is coarse, and nucleoli are enlarged. The cytoplasm is eosinophilic; some inclusions can be seen in a few cells, which resemble Mallory corpuscles (Fig. 17.96c, d).
17.3.7 Lymphoepithelioma-like Carcinoma
Sheets of undifferentiated tumor cells embedded in a lymphocyte-rich stroma characterize lymphoepithelioma–like carcinoma. On frozen sections it might be difficult to encounter the tumor cells. The carcinoma cells are positive for cytokeratin 7 and cytokeratins 13/14; the lymphocytes in most cases are B cells (Figs. 17.97 and 17.98). A diagnostic feature is the intense intermingle of tumor cells and lymphocytes, i.e., lymphocytes are everywhere in between the tumor cells, and seem to be associated with the carcinoma, similar to what is seen in thymomas. In cases from Southeast Asia, most lymphoepithelial-like LCs are positive for EBV, and EBV seems to play a role in carcinogenesis, whereas in Caucasians these carcinomas are negative for EBV.
Fig. 17.97
Lymphoepithelioma-like carcinoma. In (a, b) two cases are shown, where the tumor cells are hardly to discern in the dense lymphocytic background infiltration. By pan-cytokeratin stain in (c), the infiltrating tumor cells are highlighted. The tumor cells are large; the nuclei are enlarged as well as the nucleoli. Chromatin is coarse granular and the nuclear membrane often are dark stained to the high traffic of nucleic acids between nucleus and cytoplasm. The cytoplasm is usually pale stained by H&E. In (d–f) another case is shown. Here the initial decision of primary tumor and lymph node metastasis was hard, because the clinical information was scarce. The tumor cells formed large strands in a dense lymphocytic stroma. The extent of tumor infiltration is best seen on pan-cytokeratin stain (f); however of diagnostic help is the staining for cytokeratin 14 (e), which is characteristic in many of the tumor cells. H&E, ×200, immunohistochemistry bars 50 μm
Fig. 17.98
Two cases of adenosquamous carcinomas, both are of mixed type; in the upper panel, there are cells with keratinization as well as cells forming acinar structures (H&E). In the lower panel, another case is shown, again some single cells with keratinization but a majority of cells producing mucin (PAS stain). ×400
17.3.8 Adenosquamous Carcinoma
Adenosquamous carcinoma, although not regarded as a major type of pulmonary carcinomas, will be discussed here. A mixture of squamous and adenocarcinoma cells characterize it; each component should be represented by at least 10 %. Adenosquamous cell carcinoma can present as a collision tumor, i.e., an adenocarcinoma and a squamous cell carcinoma merges. But there are also true mixed adenosquamous carcinomas. In these cases within cell clusters, both differentiations are seen. In contrast to high-grade mucoepidermoid carcinoma, keratinization does occur in adenosquamous ones. In addition mucoepidermoid carcinoma is a centrally located carcinoma with an endobronchial component, whereas adenosquamous carcinoma is usually peripherally located. Studies on these carcinomas have shown that despite the two phenotypes, these carcinomas represent a clonal proliferation [269], which has also an impact for molecular testing: these carcinomas can harbor a mutation for EGFR and also for EML4-ALK and ROS1 [270–272] (Table 17.3).
Table 17.3
Useful immunohistochemical markers for differentiation; ApoA: surfactant apoprotein A
LMW CK | HMW CK | P40 | TTF1 | ApoA | CK20 | S100 | NE markers | |
|---|---|---|---|---|---|---|---|---|
Squamous cell | + | + | + | ± | − | − | − | − |
Small cell | + | − | ± | + | − | − | − | + |
Adenocarcinoma | + | − | − | +a | + | ±c | +d | − |
Large cell | + | ± | ± | ± | − | − | − | |
LCNEC | + | − | ± | − | − | − | − | + |
Adenosquamous | + | Focal + | + | +b | ± | − | − | |
Mucoepidermoid | + | Focal + | − | +b | − | Focal+ | Focal+ | − |
17.3.9 Diagnosis on Small Biopsies and Cytology Preparations
Most pulmonary carcinomas are detected at a late stage when metastasis already had occurred. Therefore in approximately 75 % of NSCLC and 90 % of SCLC, no resection is possible and instead small biopsy samples or fine needle aspirates have to be used for diagnosis. Therefore most of the time, pulmonary carcinomas are diagnosed with these small samples. In the 2015 WHO classification, this has been taken into account, and diagnostic criteria were adapted also to cytology preparations and small biopsies. The criteria for diagnosis have been included in the respective entity, but some general remarks are discussed here.
- (a)
Cytology
Cytological material comes as smears from fine needle aspirations and from brush. The cell preparations can be of various qualities depending on the experience of the clinician performing the smears. Tumor cell may be well preserved and easy to diagnose. Sometimes the accurate diagnosis might be impossible, especially in high-grade and undifferentiated carcinomas.
Immunocytochemistry is possible on smears but requires distaining of the smears followed by immunocytochemical procedures. This is time consuming and usually allows not more than two antibodies to be evaluated.
Cellblock technique is a good alternative. Cells from aspiration or brushes are not smeared on glass slides but dissolved in physiological solutions. The cells are centrifuged and coated in agarose or fibrinogen. After fixation in formalin, the cell pellet can be embedded in paraffin and sectioned. With this preparation immunohistochemical investigations can be done as usual. Molecular and genetic investigations can be done on cytological material; however, it is limited to a few investigations (sample size limitations). EGFR mutation testing can be done, if enough tumor cells are present (at least 200 cells).
- (b)
Biopsies
Biopsies are obtained from bronchial mucosa (usually in centrally located carcinomas), from the peripheral lung via bronchi (transbronchial biopsies), and transthoracic with a core needle from peripheral tumors. Three to five biopsies and two to three needle biopsies from different sites are usually sufficient and will allow immunohistochemical investigations, mutation analysis, FISH, or CISH/SISH. Up to 30 sections can be done with good biopsies. It is advised to perform up to 15 unstained sections together with the H&E-stained sections for any additional investigations.
- (c)
Diagnostic criteria in cytological preparations and biopsies
Tumors can be diagnosed most often with the same accuracy as in resected specimen. Immunohistochemistry will assist in the diagnosis of undifferentiated carcinomas. In the few remaining cases, which do not show any differentiation either histologically nor expressing any differentiation, marker should be diagnosed as carcinoma not otherwise specified (NOS). These cases should be transferred to molecular investigation for driver gene mutations as adenocarcinomas or squamous cell carcinomas.
- (d)
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Ancillary techniques for the subtyping of lung carcinomas
Some special stains can still be used for subtyping of carcinomas. PAS and other mucin stains can be used to diagnose mucinous adenocarcinomas. Immunohistochemical stains can be used to help differentiate squamous cell, large cell, and adenocarcinomas. Squamous cell carcinomas express high molecular cytokeratins, most useful is CK5/CK6, and almost all cells are also positive for the basal cell marker p63 or better for the truncated version p40. Adenocarcinomas are negative for CK5/CK6 and positive for CK7; a few single cells can be stained for p63 rarely for p40. In addition non-mucinous adenocarcinomas will express NapsinA and surfactant apoproteins A and B. Mucinous adenocarcinomas will express CK7 a few also CK20, but most are negative for CDX2. Large-cell carcinomas are negative for CK5/CK6 and positive for CK7; several cells are positive for p63. Neuroendocrine carcinomas will stain for NCAM (CD56), chromogranin A, and synaptophysin. NCAM (140 kDa variant) is most useful, because the intensity of the stain increases in high-grade carcinomas and is faintly positive in carcinoids (Table 17.4).
Table 17.4
Useful markers for the differentiation of common lung carcinomas in biopsies, cytology, and resection specimen
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