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Application of Immunohistochemistry in the Diagnosis of Pulmonary and Pleural Neoplasms.

Biopsies of lung and pleural neoplasms play an important role in the characterization and staging of lung cancers, particularly for patients with advanced disease (stages III and IV) and for patients who are not candidates for surgical procedures. Clinical and imaging correlation in conjunction with morphologic assessment by routine hematoxylin-eosin staining is crucial in the interpretation of small biopsies. However, immunohistochemistry (IHC) may be a valuable diagnostic tool in the workup of challenging cases. For example, it can help differentiate between lung adenocarcinoma and squamous cell carcinoma (SqCC), lung adenocarcinoma and malignant mesothelioma (MM), primary and metastatic carcinomas, and small cell lung carcinoma (SCLC) and carcinoid tumor. It can also help detect oncogenic mutants, such as endothelial growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), and repressor of silencing 1 (ROS1), and immune checkpoint molecules, such as programmed death ligand-1 (PD-L1). This article provides an up-to-date review of the role of IHC in the workup of common entities seen in the small lung/ pleural biopsy setting while using examples.


An 82-year-old nonsmoking man presented with a 4.7 X 3.6-cm right upper lobe mass found on computed tomography (CT) chest imaging. A CT-guided biopsy demonstrated a sheetlike proliferation of poorly differentiated malignant epithelial cells (Figure 1, A). The tumor cells were positive for p40 (Figure 1, B) and cytokeratin 5/6 (CK5/ 6) (Figure 1, C), but negative for thyroid transcription factor 1 (TTF-1) (Figure 1, D) and napsin A (Figure 1, E). The final diagnosis was poorly differentiated SqCC.

A 66-year-old Asian woman presented with a poorly circumscribed, solid 2.6 X 2.5-cm right upper lobe mass found on CT chest imaging. A CT-guided biopsy demonstrated a solid growth pattern of poorly differentiated malignant epithelial cells (Figure 2, A). The tumor cells were positive for TTF-1 (Figure 2, B) and napsin A (Figure 2, C), but negative for p40 (Figure 2, D) and CK5/6 (Figure 2, E). The final diagnosis was poorly differentiated adenocarcinoma with a solid pattern of growth. Molecular studies showed that the tumor cells were positive for an EGFR exon 19 deletion.

Historically, the most clinically significant distinction among lung tumors was the distinction between non-small cell lung carcinoma (NSCLC) and SCLC. However, because of markedly different prognostic and treatment implications among different lung tumors, accurate subtyping with molecular characterization is critical. This is especially true for the most common NSCLC types, lung adenocarcinoma and SqCC, as treatment options and the role of ancillary molecular and cytogenetic studies widely differ for both tumors. Specifically, EGFR inhibitors such as erlotinib (OSI Pharmaceuticals, Melville, New York; Hoffmann-La Roche, Basel, Switzerland; Genentech, South San Francisco, California) and gefitinib (AstraZeneca, London, United King dom) have been shown to be effective in EGFR-mutated tumors, (1) and the ALK inhibitor crizotinib (Pfizer, New York, New York) has been shown to be effective in tumors with EML4-ALK fusion, (2) which are both predominantly seen with adenocarcinomas. Additionally, several therapeutic agents used in the treatment of adenocarcinoma are contraindicated in SqCC, including pemetrexed (Alimta, Eli Lilly and Company, Indianapolis, Indiana), because of the lack of effectiveness, (3) and the vascular endothelial growth factor (VEGF) inhibitor bevacizumab (Avastin, Genentech), because of the risk of life-threatening pulmonary hemorrhage. (4) Two genetic alterations have been recently identified in pulmonary SqCC, discoidin domain receptor tyrosine kinase 2 (DDR2) mutation (5) and fibroblast growth factor receptor 1 (FGFR1) amplification, (6) both of which have potential as novel therapeutic targets.

In most instances, the morphologic distinction between adenocarcinoma and SqCC is relatively straightforward: the formation of glands and the production of cytoplasmic mucin are characteristic of adenocarcinoma, whereas the production of keratin and the presence of intercellular bridges are characteristic of SqCC. However, the pathologist may run into diagnostic challenges in the setting of a poorly differentiated tumor, or tumors with nonspecific morphologic findings, especially in the biopsy setting. In this case, the judicious use of IHC markers may be helpful for further characterization (Table 1).

The most useful IHC markers for pulmonary adenocarcinoma include TTF-1 and napsin A. TTF-1 is a homeodomain-containing transcription factor that is predominantly found in normal type II alveolar pneumocytes. (7) With a reported sensitivity ranging from 75% to 80%, (8, 9) TTF-1 has long been the predominant nuclear IHC marker used to identify cells of lung origin. However, TTF-1 expression has been shown to decrease inversely with the degree of tumor differentiation. (8-10) Of the 2 main commercial TTF-1 monoclonal antibodies available for IHC staining (8G7G1/1 and SPT24), the SPT24 clone has been shown to have a stronger affinity for TTF-1. However, the SPT24 TTF-1 clone may also show an affinity for colorectal adenocarcinomas. (11)

Napsin A, a relatively new cytoplasmic IHC marker for pulmonary adenocarcinoma, is an aspartic proteinase involved (12) in the maturation of surfactant protein B that has an expression thought to be regulated by TTF-1. (13) Napsin A has been shown to be superior to TTF-1 in distinguishing primary lung adenocarcinomas from other carcinomas (except for renal cell carcinoma, (14, 15) clear cell carcinomas of the gynecologic tract, (16, 17) and thyroid carcinomas (15)). Napsin A, with a higher specificity but a lower sensitivity for adenocarcinoma than TTF-1, was also found to be a useful marker in cases of poorly differentiated lung adenocarcinoma or an unknown primary tumor. (17)

Another IHC marker for glandular origin may be the cytoplasmic immunostain cytokeratin 7 (CK7). However, CK7 reactivity can be seen in most poorly differentiated adenocarcinomas and has been reported in 9 of 15 morphologically challenging cases of poorly differentiated SqCCs (60%), which limits its diagnostic utility. (18)

Immunohistochemical markers for SqCC include p63, CK5/6, cytokeratin 34bE12 (CK903), and p40. The p63 antibody, with numerous studies showing excellent sensitivity as a marker for SqCC, (19-21) has long been the most commonly used nuclear marker for squamous origin. However, p63 has been shown to be positive in 16% to 65% of lung adenocarcinoma20, (22-24) and in 55 of 172 cases (32%) of diffuse large B cell lymphoma. (25)

p63 consists of several isoforms. The 2 major groups include DNp63 and TAp63. (26) DNp63 is the predominant p63 transcript in SqCC of lung and functions as an oncogene, (27, 28) whereas TAp63 has been shown to function as a p53-like tumor suppressor. (26) In the majority of pathology laboratories, the routine p63 antibody is 4A4, which recognizes both TAp63 and [DELTA]Np63 isoforms. The p40 antibody, which exclusively recognizes the [DELTA]Np63 isoform, may have a superior specificity (29) but inferior sensitivity (30-32) compared with p63 in the diagnosis of pulmonary SqCC.

Previous studies have demonstrated that a majority of poorly differentiated NSCLCs can be subclassified as adenocarcinoma or SqCC by IHC using a simple panel of IHC markers. (19, 33, 34) For limited biopsy samples, we recommend an initial IHC panel of TTF-1 and p40. If needed, additional squamous markers (p63 and CK5/6) or glandular markers (napsin A and CK7) may be added. The distinction between adenocarcinoma and SqCC is generally straightforward when following this IHC approach. One or more markers of glandular differentiation (TTF-1 or napsin A) with negative squamous markers is supportive of adenocarcinoma. Conversely, one or more positive squamous markers (p40 or p63), in the context of negative glandular markers, is suggestive of SqCC. Cases of TTF-1- and/or napsin A-positive adenocarcinoma may show focal p63 expression. (19) Interestingly, diffuse coexpression of TTF-1 and p63 may be seen in adenocarcinomas with signet ring cell morphology (many of which contain EML4-ALK translocations). (35) Adenosquamous carcinoma requires the presence of 2 separate components demonstrating opposite and mirror-image staining patterns (napsin A and TTF-1 positive and p40 and CK5/6 negative in the glandular component, with napsin A and TTF-1 negative and p40 and CK5/6 positive in the squamous component). (36) Nevertheless, in small biopsy samples, the ability to definitively diagnose adenosquamous carcinoma may be particularly challenging.

Isolated p63 positivity in scattered cells is a nonspecific staining pattern that does not contribute to the further classification of the lesion. Similarly, isolated CK5/6 staining in scattered cells, in the absence of positivity for additional squamous or glandular markers, is also a nonspecific finding. In these cases, or in cases with nonreactivity to the entire proposed IHC panel, the most suitable diagnosis would be NSCLC, not otherwise specified. (37)

A major pitfall in interpreting IHC-stained slides of small lung biopsy samples is the positivity of normal alveolar epithelium for TTF-1 and/or napsin A. Therefore, the proper interpretation of immunomarkers requires morphologic correlation. In addition, alveolar macrophages show cytoplasmic napsin A staining. Prior to the availability of IHC stains, pathologists heavily relied on the histochemical mucin stain to confirm a diagnosis of adenocarcinoma. Even today, despite a low sensitivity, (38-40) special stains for cytoplasmic mucin may still prove to be useful in particularly challenging cases that show nonspecific staining patterns.

Furthermore, in this age of targeted therapies, molecular and cytogenetic studies play a critical role in the workup of primary lung malignancies, especially in NSCLC. Although molecular and cytogenetic testing will remain the gold-standard methodology for the detection of specific somatic genetic mutations, IHC has emerged as a fairly effective and rapid means for identifying these genetic variants. One of the most significant mutations in NSCLC includes sensitizing mutations of EGFR, particularly in exons 18-21. EGFR mutations are important to identify, as neoplasms demonstrating these mutations may be susceptible to EGFRtyrosine kinase inhibitor (TKI) therapy (ie, erlotinib; gefitinib; afatinib, Boehringer Ingelheim, Ingelheim, Germany]). The 2 most common EGFR mutations include inframe deletions in exon 19 (E746_A750del) and a point mutation at codon 858 in exon 21 (L858R), together representing 85% to 90% of EGFR mutations in NSCLC patients. (41, 42) Mutation-specific antibodies for these 2 genetic mutations were developed (43) in 2009, and numerous studies have since demonstrated that IHC may be used as a reliable prescreening test for detecting EGFR mutations in NSCLC. (44-47) A moderately differentiated adenocarcinoma of the lung shows an acinar pattern of growth (Figure 3, A) and contains an EGFR mutant with E746-A750 (Figure 3, B) detected by using rabbit monoclonal antibody against EGFR E746-A750 (clone 6B6; Cell Signaling Technology, Danvers, Massachusetts); a poorly differentiated adenocarcinoma of the lung exhibits a solid pattern of growth (Figure 4, A) and harbors an EGFR mutant with L835R (Figure 4, B) detected by using rabbit monoclonal antibodies against EGFR L835R (clone 43B2; Cell Signaling Technology).

Chromosomal rearrangements of the ALK gene, although found in a minority (0.4%-15%) of NSCLCs, (48, 49) are also important to identify, as these tumors may be susceptible to ALK inhibitors such as crizotinib. Recent studies have also shown that the use of ALK antibodies may also be an effective prescreening tool to detect the presence of ALK rearrangements in addition to the conventional ALK fluorescence in situ hybridization testing. (50, 51) A moderately differentiated adenocarcinoma of the lung shows an acinar pattern of growth (Figure 5, A) and expresses ALK protein (Figure 5, B) detected by using a rabbit monoclonal antibody against ALK (clone D5F3; Cell Signaling Technology).

Immunohistochemical stains have also been developed to detect rearrangements of ROS1 (present in 1%-2% of NSCLC), (52, 53) which encodes for a receptor tyrosine kinase. Several studies suggest that ROS1 may represent another therapeutic target of the ALK inhibitor crizotinib, (52-54) with a recent study showing a marked antitumor activity by crizotinib in patients with ROS1-rearranged advanced NSCLC. (55) Similar to IHC for ALK, IHC stains for ROS1 have been shown to be an effective and cost-effective means to screen for ROS1 rearrangements. (56-58) A poorly differentiated adenocarcinoma of the lung shows acinar and micropapillary patterns of growth (Figure 6, A) and expresses ROS1 (Figure 6, B), detected by using a rabbit monoclonal antibody against ROS1 (clone D4D6; Cell Signaling Technology). Therefore, the judicious use of mutation-specific IHC stains may be used as a practical screening tool to detect certain actionable mutations (EGFR, ALK, and ROS1) amenable to targeted therapy.

In addition to the ROS1, ALK, and EGFR pathways, programmed death receptor-1 (PD-1), an immunoregulatory receptor expressed by activated T cells, (59) and its ligand, PD-L1 in cancer cells, have been shown to be promising targets in NSCLC. Targeted therapies against PD-1 have been postulated to treat cancers by restoring proper effector T-cell function. Recent clinical trials have shown that anti-PD-1 therapy in NSCLC demonstrates antitumor activity in patients with advanced NSCLC. (60-64) The US Food and Drug Administration has approved inhibitors targeting PD-1 for patients with NSCLC. Currently, a number of different anti-PD-L1 antibodies by various vendors have been formally studied. In the future, pathologists may expect to become more familiar with PD-L1 as an important NSCLC predictive or companion biomarker. Figure 7, A, shows a pleomorphic carcinoma with a prominent spindle cell component that is strongly positive for TTF-1 (Figure 7, B) and PD-L1 (Figure 7, C) by using a mouse monoclonal antibody against PD-L1 (clone 22C3; DAKO, Carpinteria, California).


A 60-year-old man presented with multiple right pleural masses on CT imaging. A right pleural biopsy was performed at an outside institution and a diagnosis of epithelioid MM was rendered. The patient was evaluated for a decortication of pleural mesothelioma and a rebiopsy was performed. Biopsies of the right pleural masses demonstrated a biphasic MM (75% epithelioid type, 25% sarcomatoid type) (Figure 8, A). The tumor cells were positive for AE1/AE3 (Figure 8, B), calretinin (Figure 8, C), and Wilms tumor 1 (WT-1) (Figure 8, D).

Malignant mesothelioma of the pleura is a rare neoplasm arising from mesothelial cells. Although known for a great diversity of histologic patterns, MMs are generally divided into 3 major histologic types: epithelioid, sarcomatoid, and biphasic (mixed epithelioid and sarcomatoid). (65) Given the wide range of morphologic features, the differential diagnosis of MM may significantly vary depending on the histologic type. For epithelioid MM, the differential diagnosis may include carcinoma (including primary adenocarcinoma, metastatic adenocarcinoma, or SqCC) as well as other tumors with epithelioid features. Sarcomatoid MM raises the differential diagnosis of sarcomatoid carcinoma, malignant tumors with sarcomatoid features, and a variety of malignant sarcomas, including undifferentiated pleomorphic sarcoma (formerly malignant fibrous histiocytoma), osteosarcoma, and chondrosarcoma. Lymphoma, melanoma, angiosarcoma, and epithelioid hemangioendothelioma are also included in the differential diagnosis, as are benign mesothelial proliferations. Biphasic patterns should raise the differential diagnosis of other biphasic tumors such as synovial sarcoma.

The most important consideration for the pathologist is the patient's clinical and radiologic features in correlation with the hematoxylin-eosin morphology. However, IHC can be helpful in confirming MM, and the initial selection of stains will vary depending on the histologic type. In general, nearly all mesothelial cells (including mesotheliomas) are positive for pancytokeratin (including AE1/AE3). (66) However, sarcomatoid MM may show loss of keratin reactivity. In these cases, a cocktail of keratins may improve detection. (67) Furthermore, sarcomatoid MM with either osteosarcomatous or chondrosarcomatous differentiation is known to be typically negative for keratin staining.

Other mesothelial markers include calretinin (strong and diffuse nuclear and cytoplasmic staining), CK5/6 (cytoplasmic staining), D2-40 (membranous staining), and WT-1 (nuclear staining). (66) It must be noted that because of the variability of staining qualities among different antibody clones and IHC laboratories, no single specific panel of IHC antibodies can be recommended universally. In general, the pathologist should become familiar with the staining pattern at his or her performing laboratory and take into account this knowledge when selecting the initial panel of stains. Immunohistochemistry laboratories should aim for a sensitivity of at least 80% when validating immunostains for this purpose. (68)

For the initial workup of an epithelioid MM, where the main differential diagnoses are MM versus carcinoma, a reasonable panel may include 2 mesothelial markers and 2 carcinoma markers (Table 2). An expanded panel may be necessary if the initial panel reveals discordant results or if a metastatic adenocarcinoma is suspected. Immunostains positive for adenocarcinoma include TTF-1, napsin A, Ber-EP4, carcinoembryonic antigen (CEA), Leu-M1, and MOC-31. TTF-1 and napsin A have the additional benefit of helping to confirm a primary lung origin. In differentiating MM from adenocarcinoma, calretinin, CK5/6, D2-40, and WT-1 are very useful mesothelial markers. However, in differentiating MM from SqCC, the role of mesothelial markers is more limited, owing to the fact that SqCCs may be positive for CK5/6, D2-40, and (to a lesser extent) calretinin. Therefore, WT-1 is the most clinically useful immunostain in differentiating MM from SqCC. Markers positive in SqCC include p40, p63, MOC-31, and Ber-EP4. (69)

Additional immunomarkers that may be helpful, depending on the differential diagnosis, include CD45 (for lymphoma); S100, HMB-45, MART1, and SOX10 (for melanoma; both S100 and SOX10 are more sensitive for spindle cell melanoma) (70); and CD31, CD34 and ERG1 (for angiosarcoma and epithelioid hemangioendothelioma).

For sarcomatoid MM, an initial panel may include multiple cytokeratins, calretinin, and D2-40. Multiple keratin antibodies including AE1/AE3, CAM 5.2, and CK7 are recommended, as cytokeratin expression may be absent or variable in sarcomatoid mesothelioma. (71, 72) However, because sarcomatoid carcinoma and metastatic sarcomatoid renal cell carcinoma may show the same staining pattern, diffuse keratin positivity with sarcomatoid morphology is not by itself diagnostic for sarcomatoid mesothelioma. Metastatic renal cell carcinoma may be highlighted with paired box gene 2 (PAX2) or PAX8 staining. Moreover, focal keratin positivity must be interpreted with caution, as a number of sarcomas may demonstrate focal keratin positivity. (67) For this reason, markers for sarcomatous differentiation, such as smooth muscle actin (SMA), desmin, and myoglobin, may be helpful.

Useful markers for sarcomatoid MM include calretinin and D2-40, (73, 74) although the number of tumor cells positive for these markers is variable. Additional immunostains to distinguish between MM and other sarcomas may include SMA, desmin, myoglobin and myogenin. Overall, no single immunostain by itself is particularly helpful for the diagnosis of sarcomatoid mesothelioma. A carefully chosen panel of immunostains is the key in the workup of such cases. Immunohistochemical staining with equivocal or nondiagnostic results may benefit from ultrastructural examination by electron microscopy. However, the diagnostic utility of electron microscopy may be more limited in the setting of poorly differentiated tumors. (75, 76)

Benign mesothelial proliferations are also in the differential diagnosis of MM. Morphologic features favoring malignancy include dense cellularity, complex papillae, tubules, cellular stratification, and necrosis. (67) However, because benign and malignant mesothelial cells may share overlapping cytologic features, the presence of stromal invasion is considered the most reliable feature for separating benign versus MM proliferations. (77-80) The invasion of stroma or fat by atypical mesothelial cells may be highlighted by immunostains including AE1/AE3 or calretinin. In this case, the pathologist must determine whether mesothelial cells represent entrapped benign cells or benign cells cut en face versus true invasive malignancy. To distinguish fatlike spaces that may be seen in organizing pleuritis, (81) S100, laminin, and collagen IV stains may be performed to highlight adipocytes, whereas fatlike spaces will be negative.

Immunohistochemical stains to distinguish between benign and malignant mesothelial proliferations may be helpful, but are often not entirely definitive for malignancy or benignity (Table 3). Epithelial membrane antigen (EMA) and p53 have been described as immunomarkers for malignancy, (82-85) whereas desmin has been described as an indicator of benign mesothelial cells. (84, 85) A review by King et al (84) demonstrated sensitivity and specificity of desmin and EMA to be less than 90%, which may be deemed not sufficient enough when distinguishing benign from malignant.

Several recent studies have reported insulin-like growth factor II messenger ribonucleic acid-binding protein 3 (IMP3) and glucose transporter 1 (GLUT1) to be useful biomarkers for differentiating MM from reactive mesothelial cells. IMP3 is an oncofetal protein involved in embryogenesis. GLUT-1 is a member of the GLUT family of passive carriers, which functions as an energy-independent system for transport of glucose. Both IMP3 and GLUT1 have been reported in a variety of carcinomas. (86, 87) Multiple studies suggest that both epithelioid and sarcomatoid MMs are more likely to be positive for IMP3 (diffuse dark brown cytoplasmic staining), whereas reactive mesothelial proliferations are more likely to be negative for IMP3. (88-91) A consistent difference in staining intensity of IMP3 between benign and malignant proliferations has not been reported. (88)

Overall, positive staining for IMP3 and/or GLUT1 may be helpful to confirm the diagnosis of malignancy; however, negative staining for IMP3 and/or GLUT1 does not completely rule out MM. BRCAl-associated protein 1 (BAPl) has most recently emerged as a promising biomarker for pleural MM, with loss of nuclear BAPl staining seen in some mesotheliomas but none of the benign cases. (92, 93) BAPl IHC has been shown to have a relatively high specificity (100%) but low sensitivity (27%). (93)

Homozygous p16 (CDKN2A) gene deletion identified using fluorescence in situ hybridization techniques has also been described as an indicator of malignancies, including MM. (94, 95) However, fluorescence in situ hybridization testing has a number of limitations, such as artifactual loss of p16 due to section thickness of formalin-fixed, paraffin-embedded tissue (tumor cells may not be in the plane of section and may appear to lose one or more copies of the gene) and difficulty of picking out individual cells of interest. (80) Therefore, a fresh nodule of cells selected for fluorescence in situ hybridization studies may be more reliable. Additionally, homozygous p16 deletion is present in only a subset of mesotheliomas. Therefore, the absence of p16 gene deletion cannot entirely exclude the diagnosis of malignancy. (96)

In summary, the role of IHC can be helpful to confirm a diagnosis of MM, and the precise IHC panel should be selected with a careful consideration of the differential diagnosis. However, caution should be made when interpreting immunostains, because of staining variability among different antibody clones and individual IHC laboratories. Because of this, IHC laboratories should aim for a sensitivity of at least 80%. (67) Electron microscopy and cytogenetic studies may be helpful in difficult circumstances, although their diagnostic ability may be limited in the setting of poorly differentiated tumors.


A 76-year-old woman with a remote history of breast carcinoma and bilateral lung adenosquamous carcinoma presented with an enlarging pleural nodule at the lingula. A CT-guided biopsy of the pleural nodule revealed adenocarcinoma (Figure 9, A) positive for GATA-binding protein 3 (GATA3) (Figure 9, B), progesterone receptor (PR) (Figure 9, C), estrogen receptor (ER) (Figure 9, D), mammaglobin (Figure 9, E), and gross cystic disease fluid protein 15 (GCDFP-15) (focal), but negative for TTF-1 (Figure 9, F) and p40. The overall findings were consistent with metastatic breast carcinoma to the pleura.

Metastases from the extrapulmonary sites represent the most common form of pulmonary neoplasm, as the lungs are one of the most common sites of distant metastases. (97) Accordingly, although pathologists may more frequently encounter primary lung neoplasms, differentiating primary lung malignancies from metastatic neoplasms, especially from poorly differentiated extrapulmonary adenocarcinomas, can be particularly challenging.

TTF-1 and napsin A are 2 widely used markers to confirm adenocarcinomas of pulmonary origin. (15, 98) TTF-1 is a nuclear protein involved in the organogenesis of the lung and thyroid, and is expressed in approximately 75% of lung adenocarcinomas. (99) However, it is also highly sensitive and specific for thyroid carcinomas, with the exception of anaplastic carcinoma and a small proportion of other extrapulmonary adenocarcinomas (including ovarian serous carcinomas, endometrial and endocervical adenocarcinomas, and colonic adenocarcinomas). (99, 100) Napsin A is predominantly expressed in the lung and kidney, and demonstrates granular and cytoplasmic staining. (101) It has a comparable sensitivity to that of TTF-1 in identifying lung adenocarcinomas. It is important to be aware that the 2 markers may not always be coexpressed. (101) Similar to TTF-1, napsin A is not entirely specific for lung adenocarcinomas, as its expression is seen in a majority of papillary renal cell carcinomas (75%-80%), a subset of clear cell renal cell carcinomas (approximately 30%), rare cases of papillary thyroid carcinomas, and a significant proportion of ovarian and endometrial clear cell carcinomas. (15, 100, 102 ) Therefore, although TTF-1 and napsin A may be used to support a diagnosis of primary lung adenocarcinoma, in cases where morphology or clinical history may implicate an extrapulmonary primary, other organ-specific biomarkers may be needed.

The above clinical example describes the frequent challenge of differentiating a primary lung adenocarcinoma from a metastatic breast carcinoma. Women with breast cancer have a 30% higher risk than the general population of developing a second primary malignancy, with approximately 4% to 9% developing lung cancer. (103) In this case, a panel of TTF-1, napsin A, mammaglobin, GCDFP-15, and GATA3 was used to support the diagnosis of breast carcinoma metastatic to the lung. Both TTF-1 and napsin A have been shown to be negative in breast adenocarcinoma. (103) Mammaglobin and GCDFP-15 are established cytoplasmic markers positivity in 85% and 53% of breast carcinomas and up to 17% and for metastatic breast carcinomas, with 2% of lung carcinomas, respectively. (9) GATA3, a nuclear marker, was found to be expressed in 90 of 99 of breast ductal carcinomas (91%) and 48 of 48 breast lobular carcinomas (100%), but was also seen in 62 of 72 urothelial carcinomas (86%), (104) 61 of 62 basal cell carcinomas of the skin (98%), 25 of 31 SqCCs of the skin (81%), 11 of 11 choriocarcinomas (100%), 6 of 6 endodermal sinus tumors (100%), 37 of 64 MMs (58%), 18 of 22 extra-adrenal paragangliomas (82%), and 22 of 24 pheochromocytomas (92%). (105) GATA3 expression may also have prognostic implications in patients with breast cancer, where some studies have suggested that higher levels predict improved survival. (103, 106) GATA3 may also stain background lymphocytes. Lastly, the use of this panel of immunostains can also aid with the diagnosis in cytology specimens. Mammaglobin and GCDFP-15 have high specificities for breast carcinomas on cell block (88% and 96%, respectively), but have suboptimal sensitivities (26% and 14%, respectively), especially relative to that of GATA3 (86%). (107, 108) A combined double stain of TTF-1 and napsin A has also been proposed for these limited specimens, and it has been demonstrated to be diagnostically useful in identifying pulmonary adenocarcinomas on cell block. (109)

In addition to breast cancer, prostate and colorectal cancers are 2 of the most common malignancies that lead to pulmonary metastases. The common IHC stains used to differentiate primary lung carcinoma from carcinomas of breast, prostate, and colorectal origin are summarized in Table 4. In the United States, prostatic adenocarcinoma is the most common type of cancer in males and is estimated to metastasize to the lungs in 46% of cases of metastatic prostate cancer. In this setting, it is helpful to note that metastatic prostate cancer rarely presents as an isolated metastasis. (110,111) Cytokeratin 7 can be used to differentiate lung adenocarcinoma from prostatic acinar carcinoma (more than 90% of which are negative for CK7 and cytokeratin 20 [CK20]), with the exception of prostatic ductal carcinoma, which can be CK7 positive. (101) Prostate-specific antigen and prostate-specific acid phosphatase are sensitive and specific cytoplasmic markers for identifying more than 90% of metastatic prostate adenocarcinomas, although both may demonstrate weaker expression in poorly differentiated tumors. (100,112) Although NK3 homeobox 1 (NKX3.1), a highly sensitive nuclear marker for primary prostatic adenocarcinoma, was previously thought to be lost in a majority of metastatic disease, newer antibodies have demonstrated near-100% sensitivity and specificity for high-grade and metastatic prostate adenocarcinomas. (112) Lastly, v-ets avian erythroblastosis virus E26 oncogene homolog (ERG) immunostain has been recently described as a surrogate marker for the TMPRESS2-ERG fusion transcript that is reported in 40% to 70% of prostate cancer. (113,114) Several studies have shown ERG antibodies to be highly accurate in identifying prostate cancers with ERG overexpression, in both primary and metastatic disease. (113-115)

Colorectal cancer metastasizes to the lungs in 10% to 20% of patients. (116) CK7 and CK20 are frequently used as the first-line markers to differentiate lung (CK7 positive, CK20 negative) from metastatic colorectal adenocarcinomas (CK7 negative, CK20 positive). (100) TTF-1 and napsin A can also be included in the panel to support a lung primary, along with caudal type homeobox 2 (CDX2), a highly sensitive nuclear marker of intestinal adenocarcinomas, to support a colorectal origin. (117) However, caution should be taken when interpreting this panel on mucinous adenocarcinoma of the lung, as this subtype frequently shifts away from its lung phenotype, and may be negative for TTF-1 and napsin A and positive for CK20 and CDX2. (100, 118) Moreover, TTF-1 has also been found to be positive in a subset of colorectal carcinomas. (11) In a study of 555 colorectal adenocarcinomas, TTF-1 was positive in 18 (3.2%) and 24 cases (4.3%), using the 8G7G3/1 and SPT24 clones, respectively. (119)

For other extrapulmonary adenocarcinomas, PAX8 has also been recently proposed as a third immunomarker used alongside TTF-1 and napsin A to distinguish primary lung adenocarcinomas from metastatic neoplasms. (120) PAX8 is a nuclear protein that is negative in primary lung adenocarcinomas, and has positive expression restricted to epithelial tumors of the thyroid, kidney, Mullerian system, and thymus. (121)

In summary, to distinguish a primary pulmonary adenocarcinoma from a metastatic malignancy, an initial panel of immunostains should include TTF-1, napsin A, CK7, and CK20. Organ-specific markers should be added according to clinical suspicion and morphologic impression. Breast cancer markers include GATA3, mammaglobin, and GCDFP-15. For prostate cancer, prostate-specific antigen and prostate-specific acid phosphatase are established markers capable of identifying more than 90% of metastatic tumors. Newer markers for prostate cancer include NKX3.1, as well as ERG, a highly specific marker in identifying prostate tumors affected by a TMPRSS2-ERG gene fusion, although it is a less sensitive marker overall. CDX2 may be added to identify metastatic colorectal adenocarcinomas, although caution should be taken when primary mucinous adenocarcinomas of the lung is in the differential diagnosis, as this subtype may mimic the immunophenotype of colorectal carcinoma. PAX8 offers high utility in identifying metastatic adenocarcinomas to the lung, because it is negative in primary lung adenocarcinoma but positive in epithelial tumors of the thyroid, kidney, Mullerian system, and thymus.


A 72-year-old man with a 150-pack-year smoking history presented with significant weight loss and CT chest imaging that showed a right upper lobe lung mass and multiple enlarged mediastinal lymph nodes. Microscopic examination of the endobronchial ultrasound biopsy revealed sheets and nests of loosely cohesive, small, round to fusiform hyperchromatic cells with finely granular chromatin, inconspicuous nucleoli, and scant cytoplasm (Figure 10, A and B). There was prominent crush artifact. In better preserved areas, the mitotic count ranged from 6 to 8 per high-power field. The neoplastic cells were positive for CK7 (Figure 10, C), synaptophysin (Figure 10, D), and TTF-1 (Figure 10, E); they were negative for CK20 and chromogranin A. The Ki67 labeling index was greater than 90% (Figure 10, F). The final diagnosis was SCLC.

With an incidence of more than 30 000 cases per year in the United States, SCLC accounts for about 14% of all lung cancers. (122-125) Nearly all cases involve individuals with a history of smoking. (126) Only a minority of cases present with a small, localized lesion; most cases present with an advanced stage and metastatic disease. (126) Imaging studies can reveal a large mass demonstrating mediastinal invasion or compression with regional lymphadenopathy; superior vena cava syndrome can occur in some instances. (126)

Small cell lung carcinoma can be easily recognized by its distinct morphology. (127, 128) The neoplastic cells usually form sheets and nests. Cytologically, they are small (less than 3 times the diameter of a resting lymphocyte), hyperchromatic, and round to fusiform; they contain inconspicuous nucleoli, finely granular chromatin, and scant cytoplasm. (127, 128) Mitotic figures may be difficult to identify on small biopsies, but when present, they average about 80 mitoses per 2 [mm.sup.2]. (127, 129, 130)

Small cell lung carcinoma is one subclassification of neuroendocrine tumors of the lung, which also include typical carcinoid, atypical carcinoid, and large cell neuroendocrine carcinoma (LCNEC). Typical carcinoid is characterized by a mitotic count of less than 2 per 2 [mm.sup.2], atypical carcinoid is characterized by a mitotic count of 2 to 10 per 2 [mm.sup.2], and high-grade neuroendocrine carcinomas (SCLC and LCNEC) are characterized by a mitotic count of more than 10 per 2 mm2. Necrosis is typically frequent, but it may be missed because of limited sampling. (128) Biopsy samples may be subject to extensive crush artifact, which may limit the ability to render a definitive diagnosis. In these cases, cytology preparations may be better preserved and diagnostic. (128) In addition, in the larger resected specimens, because of better fixation, the neoplastic cells tend to appear bigger, (131) which may pose confusion with LCNEC or other malignancies.

In histologically challenging cases, especially in cases of extensive crush artifact, a core panel of immunostains may prove helpful to confirm the diagnosis: pan-cytokeratin AE1/AE3 (AE1/AE3), CD56, chromogranin A, synaptophysin, TTF-1, and Ki-67 (Table 5). (127, 128, 132, 133)

AE1/AE3 is useful to confirm an epithelial origin, because a keratin-negative SCLC is extremely unusual. (128) Although CK7 is positive in about half of cases, CK20 is positive in less than 10% of cases. (134, 135) Although CD56 is a highly sensitive marker (positive in 90%-100% of cases of SCLC), (134, 136, 137) it lacks specificity. Therefore, the diagnosis of SCLC in the context of isolated CD56 positivity requires morphologic correlation. (128) Synaptophysin and chromogranin A are typically positive in SCLC, but in a study, no neuroendocrine IHC activity was identified in 5 of 21 transbronchial biopsy specimens (24%) and 4 of 20 open lung biopsy specimens (20%) of SCLC. (138) In these cases negative for neuroendocrine markers, a morphology highly characteristic of SCLC can support the diagnosis. (128) Although TTF-1 can be positive in 70% to 90% of SCLCs, (127, 139-143) a study showed that 7 of 16 extrapulmonary small cell carcinomas (44%) were also positive for TTF-1. (144) Thus, TTF-1 has little to no utility in determining the primary origin of the tumor. A high Ki67 proliferation index can be very useful to differentiate carcinoids from SLCL and LCNEC. Ki-67 proliferation index is usually up to 5% in typical carcinoid, up to 20% in atypical carcinoid, 50% to 100% in SCLC, and 40% to 80% in LCNEC. (133) It should be noted, however, that after chemotherapy, the high Ki-67 proliferation index of SCLC can be reduced to levels more characteristic of carcinoid tumors and thus be a source of confusion. (128, 145)

The immunophenotypic profiles of SCLC and LCNEC are highly similar. Thus, the distinction between the two ultimately relies on morphologic examination. Small cell lung carcinoma contains cells that are smaller (less than the diameter of 3 small resting lymphocytes), have a higher nuclear to cytoplasmic ratio, contain finely granular and uniform chromatin, and have absent to inconspicuous nucleoli, fusiform shape with scant cytoplasm, and crush artifact. (128) In contrast, LCNEC is less uniform than SCLC and contains polygonal-shaped cells with coarsely granular to vesicular chromatin, prominent nucleoli, and abundant pink cytoplasm. LCNEC is also less likely to demonstrate nuclear molding.

Nonneuroendocrine mimickers of SCLC that are keratin positive include Merkel cell carcinoma and various subtypes of SqCC (Table 6). Merkel cell carcinoma has a very similar immunophenotypic profile to that of SCLC; however, although Merkel cell carcinoma is typically positive for CK20 (perinuclear dotlike pattern) and negative for TTF-1, SCLC typically demonstrates the opposite profile (negative for CK20 and positive for TTF-1). (132, 146) Although SqCC is typically negative for neuroendocrine markers (CD56, chromogranin A, and synaptophysin) and positive for CK903 and p63, SCLC also typically demonstrates the opposite profile (positive for CD56, CG, and synaptophysin and negative for CK903 and p63). (132, 146, 147)

Nonneuroendocrine mimickers of SCLC that are keratin negative include lymphoid infiltrates/lymphoma, Ewing sarcoma, and melanoma (Table 7). CD45 can demonstrate hematolymphoid differentiation. CD99 and S100 can help screen for Ewing sarcoma and melanoma, respectively.


A 33-year-old woman with a history of familial adenomatous polyposis and abdominal desmoid tumor presented with a heterogeneously enhancing 2.0 X 1.8-cm right upper lobe nodule seen on chest CT. A CT-guided biopsy demonstrated a low-grade epithelioid neoplasm (Figure 11, A) positive for AE1/AE3 (focally positive in rare scattered cells) (Figure 11, B), Cam5.2 (Figure 11, C), and TTF-1 (Figure 11, D) and negative for CK7 (Figure 11, E). The neoplastic cells were also negative for synaptophysin, chromogranin, HMB-45, MART1, S100, and PAX8. The histologic features and IHC staining results supported a final diagnosis of sclerosing pneumocytoma.

Sclerosing pneumocytoma (formerly pulmonary sclerosing hemangioma) is an uncommon lung tumor arising from primitive lung epithelium. (148) It is typically seen in middle-aged adults, with a female predilection (5:1 female to male ratio). (148) On imaging studies, sclerosing pneumocytoma usually consists of a solitary (rarely multiple) nodule/mass that is well defined, oval, and homogeneous. (149)

By histology, sclerosing pneumocytoma consists of 2 major cell types: surface cells and round cells. Surface cells resemble reactive type II pneumocytes and are cuboidal, whereas round cells consist of cells with well-defined borders, fine chromatin, and inconspicuous nucleoli. (150) Sclerosing pneumocytomas demonstrate 4 major patterns: papillary, sclerotic, solid, and hemorrhagic, the former 3 patterns of which are the most common. (148) Other histologic findings may include chronic inflammation, mast cells, xanthomatous histiocytes, hemosiderin, calcifications, cholesterol clefts, and large lamellar structures. (148) Cytologic atypia of the surface and round cells is usually not seen, although moderate to marked atypia can occur on rare occasions. (150)

The largest IHC study of sclerosing pneumocytoma demonstrated that both surface and round cells stain for TTF-1 and EMA. (148) The surface cells are characteristically positive for AE1/AE3 and also for CK7 and CAM 5.2. In contrast, round cells are characteristically negative for AE1/ AE3, with only a few cases showing scattered cells positive for CK7 and CAM 5.2 (Table 8). Antibodies for surfactant proteins A and B are also positive in surface cells and negative in round cells. (148)

The differential diagnosis of sclerosing pneumocytoma comprises both benign and malignant entities (Table 9). Differentiating sclerosing pneumocytoma from malignant lung tumors (eg, adenocarcinoma and carcinoid tumor) and metastatic carcinomas (eg, metastatic papillary thyroid carcinoma and metastatic renal cell carcinoma) is especially critical.

Distinguishing sclerosing pneumocytoma from lung adenocarcinoma may be particularly challenging in cases with subtle nuclear atypia. In this scenario, the presence of 2 distinct epithelial cell populations seen in the context of 1 of the 4 classic patterns of sclerosing pneumocytoma may be sufficient for a morphologic diagnosis. TTF-1 (staining both surface and round cells) and AE1/AE3 (staining surface cells only) can differentially highlight the 2 distinct epithelial cell populations. Sclerosing pneumocytoma is negative for the neuroendocrine markers chromogranin and synaptophysin. Because sclerosing pneumocytoma can assume a papillary configuration, a metastatic papillary thyroid carcinoma or renal cell carcinoma may enter the differential diagnosis. Although cytologic features seen in papillary thyroid carcinoma can be helpful, immunostains for thyroglobulin and/or PAX8 can also be used to exclude the possibility of a metastatic papillary thyroid carcinoma. Similarly, the marked cytologic atypia of a metastatic renal cell carcinoma may favor a malignant process. Immunohistochemical staining for CD10, carbonic anhydrase IX, and/or PAX8 (all typically positive in metastatic renal cell carcinoma) may be helpful in these cases.

Benign tumors that enter the differential diagnosis of sclerosing pneumocytoma include clear cell tumor, hemangioma, and pulmonary hamartoma. Clear cell tumors typically demonstrate strong HMB-45 expression. Hemangiomas typically stain positively for vascular markers such as CD31, CD34, and ERG1. Pulmonary hamartoma, which demonstrates varying degrees of mature cartilage, smooth muscle, and/or adipose tissue, is usually diagnosed by morphology alone. Immunohistochemical markers are generally not needed, but S100 may be used to highlight mature chondrocytes.


In conclusion, the judicious use of IHC stains can be helpful in the small-biopsy setting. In addition to helping the pathologist further characterize the origin of the tumor cells in diagnostically challenging biopsies, IHC has also become increasingly important to identify diagnostic, therapeutic, and prognostic biomarkers in cases of NSCLC.

Please Note: Illustration(s) are not available due to copyright restrictions.


(1.) Shigematsu H, Lin L, Takahashi T, et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst. 2005; 97(5):339-346.

(2.) Kwak EL, Bang YJ, Camidge DR, et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl I Med. 2010; 363(18):1693-1703.

(3.) Scagliotti GV, Parikh P, von Pawel J, et al. Phase III study comparing cisplatin plus gemcitabine with cisplatin plus pemetrexed in chemotherapy-naive patients with advanced-stage non-small-cell lung cancer. J Clin Oncol. 2008; 26(21):3543-3551.

(4.) Johnson DF, Fehrenbacher L, Novotny WF, et al. Randomized phase II trial comparing bevacizumab plus carboplatin and paclitaxel with carboplatin and paclitaxel alone in previously untreated locally advanced or metastatic non-small-cell lung cancer. J Clin Oncol. 2004; 22(11):2184-2191.

(5.) Hammerman PS, Sos ML, Ramos AH, et al. Mutations in the DDR2 kinase gene identify a novel therapeutic target in squamous cell lung cancer. Cancer Discov. 2011; 1(1):77-87.

(6.) Weiss J, Sos ML, Seidel D, et al. Frequent and focal FGFR1 amplification associates with therapeutically tractable FGFR1 dependency in squamous cell lung cancer. Sci Transl Med. 2010; 2(62):62ra93.

(7.) Ikeda K, Clark JC, Shaw-White JR, et al. Gene structure and expression of human thyroid transcription factor-1 in respiratory epithelial cells. J Biol Chem. 1995; 270(14):8108-8114.

(8.) Lau S, Luthringer D, Eisen R. Thyroid transcription factor-1: a review. Appl Immunohistochem Mol Morphol. 2002; 10(2):97-102.

(9.) Jagirdar J. Application of immunohistochemistry to the diagnosis of primary and metastatic carcinoma to the lung. Arch Pathol Lab Med. 2008; 132(3):384-396.

(10.) Chuman Y, Bergman A, Ueno T, et al. Napsin A, a member of the aspartic protease family, is abundantly expressed in normal lung and kidney tissue and is expressed in lung adenocarcinomas. FEBS Lett. 1999; 462(1-2):129-134.

(11.) Comperat E, Zhang F, Perrotin C, et al. Variable sensitivity and specificity of TTF-1 antibodies in lung metastatic adenocarcinoma of colorectal origin. Mod Pathol. 2005; 18(10):1371-1376.

(12.) Ueno T, Linder S, Na C, Rice WR, Johansson J, Weaver TE. Processing of pulmonary surfactant protein B by napsin and cathepsin H. J Biol Chem. 2004; 279(16):16178-16184.

(13.) DeFelice M, Siberschmidt D, DiLaura R, et al. TTF-1 phosphorylation is required for peripheral lung morphogenesis, perinatal survival, and tissue-specific gene expression. J Biol Chem. 2003; 278(37):35574-35583.

(14.) Turner BM, Cagle PT, Sainz IM, Fukuoka J, Shen SS, Jagirdar J. Napsin A, a new marker for lung adenocarcinoma, is complementary and more sensitive and specific than thyroid transcription factor 1 in the differential diagnosis of primary pulmonary carcinoma: evaluation of 1674 cases by tissue microarray. Arch Pathol Lab Med. 2012; 136(2):163-171.

(15.) Bishop JA, Sharma R, Illei PB. Napsin A and thyroid transcription factor-1 expression in carcinomas of the lung, breast, pancreas, colon, kidney, thyroid, and malignant mesothelioma. Hum Pathol. 2010; 41(1):20-25.

(16.) Fadare O, Desouki MM, Gwin K, et al. Frequent expression of napsin A in clear cell carcinoma of the endometrium: potential diagnostic utility. Am J Surg Pathol. 2014; 38(2):189-196.

(17.) Kandalaft PL, Gown AM, Isacson C. The lung-restricted marker napsin A is highly expressed in clear cell carcinomas of the ovary. Am J Clin Pathol. 2014; 142(6):830-836.

(18.) Mukhopadhyay S, Katzenstein AL. Subclassification of non-small cell lung carcinomas lacking morphologic differentiation on biopsy specimens: utility of an immunohistochemical panel containing TTF-1, napsin A, p63, and CK5/6. Am J Surg Pathol. 2011; 35(1):15-25.

(19.) Kargi A, Gurel D, Tuna B. The diagnostic value of TTF-1, CK5/6, and p63 immunostaining in classification of lung carcinomas. Appl Immunohistochem Mol Morphol. 2007; 15(4):415-420.

(20.) Rekhtman N, Ang DC, Sima CS, Travis WD, Moreira AL. Immunohistochemical algorithm for differentiation of lung adenocarcinoma and squamous cell carcinoma based on large series of whole-tissue sections with validation in small specimens. Mod Pathol. 2011; 24(10):1348-1359.

(21.) Nicholson AG, Gonzalez D, Shah P, et al. Refining the diagnosis and EGFR status of non-small cell lung carcinoma in biopsy and cytologic material, using a panel of mucin staining, TTF-1, cytokeratin 5/6, and P63, and EGFR mutation analysis. J Thorac Oncol. 2010; 5:436-441.

(22.) Au NH, Gown AM, Cheang M, et al. P63 expression in lung carcinoma: a tissue microarray study of 408 cases. Appl Immunohistochem Mol Morphol. 2004; 12(3):240-247.

(23.) Bishop JA, Benjamin H, Cholakh H, Chajut A, Clark DP, Westra WH. Accurate classification of non-small cell lung carcinoma using a novel microRNA-based approach. Clin Cancer Res. 2010; 16(2):610-619.

(24.) Pelosi G, Pasini F, Olsen Stenholm C, et al. P63 immunoreactivity in lung cancer: yet another player in the development of squamous cell carcinomas? J Pathol. 2002; 198(1):100-109.

(25.) Hedvat CV, Teruya-Feldstein J, Puig P, et al. Expression of p63 in diffuse large B-cell lymphoma. Appl Immunohistochem Mol Morphol. 2005; 13(3):237-242.

(26.) Crum CP, McKeon FD. P63 in epithelial survival, germ cell surveillance, and neoplasia. Annu Rev Pathol. 2010; 5:349-371.

(27.) Sniezek JC, Matheny KE, Westfall MD, Pietenpol JA. Dominant negative p63 isoform expression in head and neck squamous cell carcinoma. Laryngoscope. 2004; 114(12):2063-2072.

(28.) Massion PP, Taflan PM, Jamshedur Rahman SM, et al. Significance of p63 amplification and overexpression in lung cancer development and prognosis. Cancer Res. 2003; 63(21):7113-7121.

(29.) Bishop JA, Teruya-Feldstein J, Westra WH, Pelosi G, Travis WD, Rekhtman N. p40 (DNp63) is superior to p63 for the diagnosis of pulmonary squamous cell carcinoma. Mod Pathol. 2012; 25(3):405-415.

(30.) Pelosi G, Sonzogni A, Papotti M, et al. Different prevalence of transactivating (TA) p63 and nonTAp63 isoforms in pulmonary adenocarcinomas: a useful diagnostic tool. Mod Pathol. 2010; 23(suppl 1): 411A-412A.

(31.) Pelosi G, Fabbri A, Rossi G, et al. A two-hit minimalist diagnostic algorithm based on p40 (deltaNp63) and TTF-1 immunostaining upon small biopsy/cellblock samples for differentiating main subtypes of non-small cell lung cancer and sparing material. J Thorac Oncol. 2011; 6(6):S335-S336.

(32.) Righi L, Graziano P, Fornari A, et al. Immunohistochemical subtyping of nonsmall cell lung cancer not otherwise specified in fine-needle aspiration cytology: a retrospective study of 103 cases with surgical correlation. Cancer. 2011; 117(15):3416-3423.

(33.) Stoll LM, Johnson MW, Gabrielson E, Askin F, Clark DP, Li QK. The utility of napsin-A in the identification of primary and metastatic lung adenocarcinoma among cytologically poorly differentiated carcinomas. Cancer Cytopathol. 201025; 118(6):441-449.

(34.) Whithaus K, Fukuoka J, Prihoda TJ, Jagirdar J. Evaluation of napsin A, cytokeratin 5/6, p63, and thyroid transcription factor 1 in adenocarcinoma versus squamous cell carcinoma of the lung. Arch Pathol Lab Med. 2012; 136(2):155-162.

(35.) Yoshida A, Tsuta K, Watanabe S, et al. Frequent ALK rearrangement and TTF-1/p63 co-expression in lung adenocarcinoma with signet-ring cell component. Lung Cancer. 2011; 72(3):309-315.

(36.) Zhang K, Hongbin D, Cagle PT. Utility of immunohistochemistry in the diagnosis of pleuropulmonary and mediastinal cancers: a review and update. Arch Pathol Lab Med. 2014; 138(12):1611-1628.

(37.) Travis W, Brambilla E, Noguchi M, et al. International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society international multidisciplinary classification of lung adenocarcinoma. J Thorac Oncol. 2011; 6(2):244-285.

(38.) Loo PS, Thomas SC, Nicolson MC, Fyfe MN, Kerr KM. Subtyping of undifferentiated non-small cell carcinomas in bronchial biopsy specimens. J Thorac Oncol. 2010; 5(4):442-447.

(39.) Terry J, Leung S, Laskin J, Leslie KO, Gown AM, lonescu DN. Optimal immunohistochemical markers for distinguishing lung adenocarcinomas from squamous cell carcinomas in small tumor samples. Am J Surg Pathol. 2010; 34(12):1805-1811.

(40.) McGregor DH, Dixon AY, McGregor DK. Adenocarcinoma of the lung: a comparative diagnostic study using light and electron microscopy. Hum Pathol 1988:19(8):910-913.

(41.) Kosaka T, Yatabe Y, Endoh H, Kuwano H, Takahashi T, Mitsudomi T. Mutations of the epidermal growth factor receptor gene in lung cancer: biological and clinical implications. Cancer Res. 2004; 64(24):8919-8923.

(42.) Riely GJ, Politi KA, Miller VA, Pao W. Update on epidermal growth factor receptor mutations in non-small cell lung cancer. Clin Cancer Res. 2006; 12(24): 7232-7241.

(43.) Yu J, Kane S, Wu J, et al. Mutation-specific antibodies for the detection of EGFR mutations in non-small-cell lung cancer. Clin Cancer Res. 2009; 15(9): 3023-3028.

(44.) Xiong Y, Bai Y, Leong N, et al. Immunohistochemical detection of mutations in the epidermal growth factor receptor gene in lung adenocarcinomas using mutation-specific antibodies. Diagn Pathol. 2013; 8:27.

(45.) Seo AN, Park Tl, Jin Y, et al. Novel EGFR mutation-specific antibodies for lung adenocarcinoma: highly specific but not sensitive detection of an E746_A750 deletion in exon 19 and an L858R mutation in exon 21 by immunohistochemistry. Lung Cancer. 2014; 83(3):316-323.

(46.) Chen Z, Liu HB, Yu CH, Wang Y, Wang L, Song Y. Diagnostic value of mutation-specific antibodies for immunohistochemical detection of epidermal growth factor receptor mutations in non-small cell lung cancer: a meta-analysis. PLoS One. 2014; 9(9):e105940.

(47.) Ragazzi M, Tamagnini I, Bisagni A, et al. Diamond: immunohistochemistry versus sequencing in EGFR analysis of lung adenocarcinomas. J Clin Pathol. 2016; 69(5):440-447.

(48.) Solomon B, Varella-Garcia M, Camidge DR. ALK gene rearrangements: a new therapeutic target in a molecularly defined subset of non-small cell lung cancer. J Thorac Oncol. 2009; 4(12):1450-1454.

(49.) Pao W, Girard N. New driver mutations in non-small-cell lung cancer. Lancet Oncol. 2012; 12(2):175-180.

(50.) Selinger CI, Rogers TM, Russell PA, et al. Testing for ALK rearrangement in lung adenocarcinoma: a multicenter comparison of immunohistochemistry and fluorescent in situ hybridization. Mod Pathol. 2013; 26(12):1545-1553.

(51.) To KF, Tong JH, Yeung KS, et al. Detection of ALK rearrangement by immunohistochemistry in lung adenocarcinoma and the identification of a novel EML4-ALK variant. J Thorac Oncol. 2013; 8(7):883-891.

(52.) Bergethon K, Shaw AT, Ou SH, et al. ROS1 rearrangements define a unique molecular class of lung cancers. J Clin Oncol. 2012; 30(8):863-870.

(53.) Davies KD, Le AT, Theodoro MF, et al. Identifying and targeting ROS1 gene fusions in non-small cell lung cancer. Clin Cancer Res. 2012; 18(17):4570-4579.

(54.) Rimkunas VM, Crosby K, Kelly M, et al. Analysis of receptor tyrosine kinase ROS1 positive tumors in non-small cell lung cancer: identification of a FIG-ROS1 fusion. Clin Cancer Res. 2012; 18(16):4449-4457.

(55.) Shaw AT, Ou SH, Bang YJ, et al. Crizotinib in ROS1-rearranged non-small-cell lung cancer. N Engl I Med. 2014; 371(21):1963-1971.

(56.) Yoshida A, Tsuta K, Wakai S, et al. Immunohistochemical detection of ROS1 is useful for identifying ROS1 rearrangements in lung cancers. Mod Pathol. 2014; 27(5):711-720.

(57.) Boyle TA, Masago K, Ellison KE, Yatabe Y, Hirsch FR. ROS1 immunohistochemistry among major genotypes of non-small-cell lung cancer. Clin Lung Cancer. 201 5; 16(2):106-111.

(58.) Shan L, Lian F, Guo L, et al. Detection of ROS1 gene rearrangement in lung adenocarcinoma: comparison of IHC, FISH and real-time RT-PCR. PLoS One. 2015; 10(3):e0120422.

(59.) Radziewicz H, Ibegbu CC, Fernandez ML, et al. Liver-infiltrating lymphocytes in chronic human hepatitis C virus infection display an exhausted phenotype with high levels of PD-1 and low levels of CD127 expression. J Virol. 2007; 81(6):2545-2553.

(60.) Gettinger S, Herbst RS. B7-H1/PD-1 blockade therapy in non-small cell lung cancer: current status and future direction. Cancer J. 2014; 20(4):281-289.

(61.) Brahmer JR, Tykodi SS, Chow LQ, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med. 2012; 366(26):2455-2465.

(62.) Topalian SL, Hodi FS, Brahmer JR, et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med. 2012; 366(26):2443-2454.

(63.) Herbst RS, Soria JC, Kowanetz M, et al. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature. 2014; 515(7528):563-567.

(64.) Garon EB, Rizvi NA, Hui R, et al. Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med. 2015; 372(21):2018-2028.

(65.) Allen TC. Recognition of histopathologic patterns of diffuse malignant mesothelioma in differential diagnosis of pleural biopsies. Arch Pathol Lab Med. 2005; 129(11):1415-1420.

(66.) Ordonez NG. The immunohistochemical diagnosis of mesothelioma: a comparative study of epithelioid mesothelioma and lung adenocarcinoma. Am J Surg Pathol. 2003; 27(8): 1031-1051.

(67.) Klebe S, Brownlee NA, Mahar A, et al. Sarcomatoid mesothelioma: a clinical-pathologic correlation of 326 cases. Mod Pathol. 2010; 23(3):470-479.

(68.) Husain AN, Colby T, Ordonez N, et al. Guidelines for pathologic diagnosis of malignant mesothelioma: 2012 update of the consensus statement from the International Mesothelioma Interest Group. Arch Pathol Lab Med. 2013; 137(5):647-667.

(69.) Ordonez NG. The diagnostic utility of immunohistochemistry in distinguishing between epithelioid mesotheliomas and squamous carcinomas of the lung: a comparative study. Mod Pathol. 2006; 19(3):417-428.

(70.) Weissinger SE, Keil P, Silvers DN, et al. A diagnostic algorithm to distinguish desmoplastic from spindle cell melanoma. Mod Pathol. 2014; 27(4): 524-534.

(71.) Mayall FG, Goddard H, Gibbs AR. The diagnostic implications of variable cytokeratin expression in mesotheliomas. J Pathol. 1993; 170(2):165-168.

(72.) Chirieac LR, Pinkus GS, Pinkus JL, Godleski J, Sugarbaker DJ, Corson JM. The immunohistochemical characterization of sarcomatoid malignant mesothelioma of the pleura. Am J Cancer Res. 2011; 1(1):14-24.

(73.) Chu AY, Litzky LA, Pasha TL, Acs G, Zhang PJ. Utility of D2-40, a novel mesothelial marker, in the diagnosis of malignant mesothelioma. Mod Pathol. 2005; 18(1):105-110.

(74.) Ordonez NG. D2-40 and podoplanin are highly specific and sensitive immunohistochemical markers of epithelioid malignant mesothelioma. Hum Pathol. 2005; 36(4):372-380.

(75.) Dardick I, Al-Jabi M, McCaughey WT, et al. Ultrastructure of poorly differentiated diffuse epithelial mesotheliomas. Ultrastruct Pathol. 1984; 7(2-3): 151-160.

(76.) Dardick I, Jabi M, McCaughey WT, et al. Diffuse epithelial mesothelioma: a review of the ultrastructural spectrum. Ultrastruct Pathol. 1987; 11(5-6):503-533.

(77.) Churg A, Colby TV, Cagle P, et al. The separation of benign and malignant mesothelial proliferations. Am I Surg Pathol. 2000; 24(9):1183-2000.

(78.) Cagle PT, Churg A. Differential diagnosis of benign and malignant mesothelial proliferations on pleural biopsies. Arch Pathol Lab Med. 2005; 129(11):1421-1427.

(79.) Churg A, Cagle PT, Roggli VL. Tumors of the Serosal Membranes. Washington, DC: American Registry of Pathology; 2006. Atlas of Tumor Pathology; 4th series, fascicle 3.

(80.) Churg A, Galateau-Salle F. The separation of benign and malignant mesothelial proliferations. Arch Pathol Lab Med. 2012; 136(10):1217-1226.

(81.) Churg A, Cagle P, Colby TV, et al. The fake fat phenomenon in organizing pleuritis: a source of confusion with desmoplastic malignant mesotheliomas. Am I Surg Pathol. 2011; 35(12):1823-1829.

(82.) Mayall FG, Goddard H, Gibbs AR. p53 immunostaining in the distinction between benign and malignant mesothelial proliferations using formalin-fixed paraffin sections. J Pathol. 1992; 168(4):377-381.

(83.) Cagle PT, Brown RW, Lebovitz RM. p53 immunostaining in the differentiation of reactive processes from malignancy in pleural biopsy specimens. Hum Pathol. 1994; 25(5):443-448.

(84.) King J, Thatcher N, Pickering C, Hasleton P. Sensitivity and specificity of immunohistochemical antibodies used to distinguish between benign and malignant pleural disease: a systematic review of published reports. Histopathology. 2006; 49(6):561-568.

(85.) Attanoos RL, Griffin A, Gibbs AR. The use of immunohistochemistry in distinguishing reactive from neoplastic mesothelium: a novel use for desmin and comparative evaluation with epithelial membrane antigen, p53, platelet-derived growth factor-receptor, P-glycoprotein and bcl-2. Histopathology. 2003; 43(3): 231-238.

(86.) Findeis-Hosey JJ, Xu H. The use of insulin like-growth factor II messenger RNA binding protein-3 in diagnostic pathology. Hum Pathol. 2010; 42(3):303-314.

(87.) Younes M, Lechago LV, Somoano JR, et al. Wide expression of the human erythrocyte glucose transporter Glut1 in human cancers. Cancer Res. 1996; 56(5): 1164-1167.

(88.) Lee AF, Gown AM, Churg A. IMP3 and GLUT-1 immunohistochemistry for distinguishing benign from malignant mesothelial proliferations. Am J Surg Pathol. 2013; 37(3):421-426.

(89.) Shi M, Fraire AE, Chu P, et al. Oncofetal protein IMP3, a new diagnostic biomarker to distinguish malignant mesothelioma from reactive mesothelial proliferation. Am J Surg Pathol. 2011; 35(6):878-882.

(90.) Ikeda K, Tate G, Suzuki T, Kitamura T, Mitsuya T. Diagnostic usefulness of EMA, IMP3, and GLUT-1 for the immunocytochemical distinction of malignant cells from reactive mesothelial cells in effusion cytology using cytospin preparations. Diagn Cytopathol. 2011; 39(6):395-01.

(91.) Minato H, Kurose N, Fukushima M, et al. Comparative immunohistochemical analysis of IMP3, GLUT1, EMA, CD146, and desmin for distinguishing malignant mesothelioma from reactive mesothelial cells. Am J Clin Pathol. 2014; 141(1):85-93.

(92.) Sheffield BS, Hwang HC, Lee AF, et al. BAP1 immunohistochemistry and p16 FISH to separate benign from malignant mesothelial proliferations. Am J Surg Pathol. 2015; 39(7):977-982.

(93.) Bott M, Brevet M, Taylor BS, et al. The nuclear deubiquitinase BAP1 is commonly inactivated by somatic mutations and 3p21.1 losses in malignant pleural mesothelioma. Nat Genet. 2011; 43(7):668-672.

(94.) Illei PB, Ladanyi M, Rusch VW, Zakowski MF. The use of CDKN2A deletion as a diagnostic marker for malignant mesothelioma in body cavity effusions. Cancer. 2003; 99(1):51-56.

(95.) Chiosea S, Krasinskas A, Cagle PT, Mitchell KA, Zander DS, Dacic S. Diagnostic importance of 9p21 homozygous deletion in malignant mesotheliomas. Mod Pathol. 2008; 21(6):742-747.

(96.) Monaco SE, Shuai Y, Bansal M, Krasinskas AM, Dacic S. The diagnostic utility of p16 FISH and GLUT-1 immunohistochemical analysis in mesothelial proliferations. Am J Clin Pathol. 2011; 135(4):619-627.

(97.) Raab SS, Allen TC, Leslie KO, Wick MR. Metastatic tumors in the lung. In: Leslie KO, Wick MR, eds. Practical Pulmonary Pathology: A Diagnostic Approach. Philadelphia, PA: Elsevier; 2011:597-644.

(98.) Ye J, Findeis-Hosey JJ, Yang Q, et al. Combination of napsin A and TTF-1 immunohistochemistry helps in differentiating primary lung adenocarcinoma from metastatic carcinoma in the lung. Appl Immunohistochem Mol Morphol. 2011; 19(4):313-317.

(99.) Ordonez NG. Value of thyroid transcription factor-1 immunostaining in tumor diagnosis. Appl Immunohistochem Mol Morphol. 2012; 20(5):429-444.

(100.) Lin F, Liu H. Immunohistochemistry in undifferentiated neoplasm/tumor of uncertain origin. Arch Pathol Lab Med. 2014; 138(12):1583-1610.

(101.) Ordonez NG. Napsin A expression in lung and kidney neoplasia. Adv Anat Pathol. 2012; 19(1):66-73.

(102.) Iwamoto M, Nakatani Y, Fugo K, Kishimoto T, Kiyokawa T. Napsin A is frequently expressed in clear cell carcinoma of the ovary and endometrium. Hum Pathol. 2015; 46(7):957-962.

(103.) Yang M, Nonaka D. A study of immunohistochemical differential expression in pulmonary and mammary carcinomas. Mod Pathol. 2010; 23(5): 654-661.

(104.) Liu H, Shi J, Wilkerson ML, Lin F. Immunohistochemical evaluation of GATA3 expression in tumors and normal tissues: a useful immunomarker for breast and urothelial carcinomas. Am J Clin Pathol. 2012; 138(1):57-64.

(105.) Miettinen M, McCue PA, Sarlomo-Rikala M, et al. GATA3: a multispecific but potentially useful marker in surgical pathology: a systematic analysis of 2500 epithelial and nonepithelial tumors. Am J Surg Pathol. 2014; 38(1):13-22.

(106.) Yoon NK, Maresh EL, Shen D, et al. Higher levels of GATA3 predict better survival in women with breast cancer. Hum Pathol. 2010; 41(12):1794-1801.

(107.) Yan Z, Gidley J, Horton D, Roberson J, Eltoum IE, Chhieng DC. Diagnostic utility of mammaglobin and GCDFP-15 in the identification of metastatic breast carcinoma in fluid specimens. Diagn Cytopathol. 2009; 37(7): 475-478.

(108.) Braxton DR, Cohen C, Siddiqui MT. Utility of GATA3 immunohistochemistry for diagnosis of metastatic breast carcinoma in cytology specimens. Diagn Cytopathol. 2015; 43(4):271-277.

(109.) Fatima N, Cohen C, Lawson D, Siddiqui MT. TTF-1 and napsin A double stain. Cancer Cytopathol. 2011; 119(2):127-133.

(110.) Bubendorf L, Schopfer A, Wagner U, et al. Metastatic patterns of prostate cancer: an autopsy study of 1,589 patients. Hum Pathol. 2000; 31(5):578-583.

(111.) Wallis CJD, English JC, Goldenberg SL. The role of resection of pulmonary metastases from prostate cancer: a case report and literature review. Can Urol Assoc J. 2011; 5(6):E104-E108.

(112.) Gurel B, Ali TZ, Montgomery EA, et al. NKX3.1 as a marker of prostatic origin in metastatic tumors. Am I Surg Pathol. 2010; 34(8):1097-1105.

(113.) Chaux A, Albadine R, Toubaji A, et al. Immunohistochemistry for ERG expression as a surrogate for TMPRSS2-ERG fusion detection in prostatic adenocarcinomas. Am J Surg Pathol. 2011; 35(7):1014-1020.

(114.) van Leenders GJLH, Boormans JL, Vissers CJ, et al. Antibody EPR3864 is specific for ERG genomic fusions in prostate cancer: implications for pathological practice. Mod Pathol. 2011; 24(8):1128-1138.

(115.) Braun M, Goltz D, Shaikhibrahim Z, et al. ERG protein expression and genomic rearrangement status in primary and metastatic prostate cancer--a comparative study of two monoclonal antibodies. Prostate Cancer Prostatic Dis. 2012; 15(2):165-169.

(116.) Zisis C, Tsakiridis K, Kougioumtzi I, et al. The management of the advanced colorectal cancer: management of the pulmonary metastases. J Thorac Dis. 2013; 5(suppl 4):S383-S388.

(117.) Barbareschi M, Murer B, Colby TV, et al. CDX-2 homeobox gene expression is a reliable marker of colorectal adenocarcinoma metastases to the lungs. Am J Surg Pathol. 2003; 27(2):141-149.

(118.) Inamura K, Satoh Y, Okumura S, et al. Pulmonary adenocarcinomas with enteric differentiation. Am J Surg Pathol. 2005; 29(5):660-665.

(119.) Dettmer M, Kim TE, Jung CK, Jung ES, Lee KY, Kang CS. Thyroid transcription factor-1 expression in colorectal adenocarcinomas. Pathol Res Pract. 2011; 207(11):686-690.

(120.) Ye J, Hameed O, Findeis-Hosey JJ, et al. Diagnostic utility of PAX8, TTF-1 and napsin A for discriminating metastatic carcinoma from primary adenocarcinoma of the lung. Biotech Histochem. 2012; 87(1):30-34.

(121.) Ordonez NG. Value of PAX 8 immunostaining in tumor diagnosis: a review and update. Adv Anat Pathol. 2012; 19(3):140-151.

(122.) Altekruse SF, Kosary CL, Krapcho M, et al. SEER cancer statistics review, 1975-2007, National Cancer Institute. Accessed January 16, 2017.

(123.) Travis WD, Travis LB, Devesa SS. Lung cancer [published erratum appears in Cancer. 1995; 75(12):2979]. Cancer. 1995; 75:191-202.

(124.) Krug LM, Pietanza MC, Kris MG, et al. Small cell and other neuroendocrine tumors of the lung. In: DeVita VT, Lawrence TS, Rosenberg SA, eds. DeVita, Hellman and Rosenberg's Cancer: Principles and Practice of Oncology. 9th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2011:848870.

(125.) Siegel R, Ward E, Brawley O, et al. Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths. CA Cancer J Clin. 2011; 61(4):212-236.

(126.) Krug LM, Kris MG, Rosenzweig K, et al. Cancer of the lung: small cell and other neuroendocrine tumors of the lung. In DeVita VT, Lawrence TS, Rosenberg SA, eds. DeVita, Hellman and Rosenberg's Cancer: Principles and Practice of Oncology. 8th ed. Philadelphia, PA: LippincottWilliams and Wilkins; 2008:946971.

(127.) Travis WD. Advances in neuroendocrine lung tumors. Ann Oncol. 2010; 21(suppl 7):vii65-vii71.

(128.) Travis WD. Update on small cell carcinoma and its differentiation from squamous cell carcinoma and other non-small cell carcinomas. Mod Pathol. 2012; 25(suppl 1):S18-S30.

(129.) Travis WD, Brambilla E, Muller-Hermelink HK, et al. Pathology and Genetics: Tumors of the Lung, Pleura, Thymus and Heart. Vol 1. Lyon, France: IARC; 2004.

(130.) Nicholson SA, Beasley MB, Brambilla E, et al. Small cell lung carcinoma (SCLC): a clinicopathologic study of 100 cases with surgical specimens. Am J Surg Pathol. 2002; 26(9):1184-1197.

(131.) Vollmer RT. The effect of cell size on the pathologic diagnosis of small and large cell carcinomas of the lung. Cancer. 1982; 50(7):1380-1383.

(132.) Lin F, Prichard J. Handbook of Practical Immunohistochemistry: Frequently Asked Questions. 2nd ed. New York, NY: Springer; 2015.

(133.) Travis WD, Brambilla E, Burke AP, Marx A, Nicholson AG. WHO Classification of Tumors of the Lung, Pleura, Thymus and Heart. Lyon, France: IARC; 2015.

(134.) Bobos M, Hytiroglou P, Kostopoulos I, et al. Immunohistochemical distinction between merkel cell carcinoma and small cell carcinoma of the lung. Am J Dermatopathol. 2006; 28:99-104.

(135.) Chu PG, Wu E, Weiss LM. Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms: a survey of 435 cases. Mod Pathol. 2000; 13:962-972.

(136.) Kontogianni K, Nicholson AG, Butcher D, Sheppard MN. CD56: a useful tool for the diagnosis of small cell lung carcinomas on biopsies with extensive crush artefact. J Clin Pathol. 2005; 58(9):978-980.

(137.) Hiroshima K, Iyoda A, Shida T, et al. Distinction of pulmonary large cell neuroendocrine carcinoma from small cell lung carcinoma: a morphological, immunohistochemical, and molecular analysis. Mod Pathol. 2006; 19(10):1358-1368.

(138.) Guinee Jr DG, Fishback NF, Koss MN, Abbondanzo SL, Travis WD. The spectrum of immunohistochemical staining of small cell lung carcinoma in specimens from transbronchial and open-lung biopsies. Am J Clin Pathol. 1994; 102(4): 406-414.

(139.) Folpe AL, Gown AM, Lamps LW, et al. Thyroid transcription factor-1: immunohistochemical evaluation in pulmonary neuroendocrine tumors. Mod Pathol. 1999; 12(1):5-8.

(140.) Sturm N, Rossi G, Lantuejoul S, et al. 34BetaE12 expression along the whole spectrum of neuroendocrine proliferations of the lung, from neuroendocrine cell hyperplasia to small cell carcinoma. Histopathology. 2003; 42(2):156166.

(141.) Sturm N, Rossi G, Lantuejoul S, et al. Expression of thyroid transcription factor-1 in the spectrum of neuroendocrine cell lung proliferations with special interest in carcinoids. Hum Pathol. 2002; 33(2):175-182.

(142.) Travis WD. Neuroendocrine lung tumors. Pathol Case Rev. 2006; 11:235-242.

(143.) Travis WD. Lung tumours with neuroendocrine differentiation. Eur J Cancer. 2009; 45(suppl 1):251-266.

(144.) Agoff SN, Lamps LW, Philip AT, et al. Thyroid transcription factor-1 is expressed in extrapulmonary small cell carcinomas but not in other extrapulmonary neuroendocrine tumors. Mod Pathol. 2000; 13(3):238-242.

(145.) Brambilla E, Moro D, Gazzeri S, et al. Cytotoxic chemotherapy induces cell differentiation in small cell lung carcinoma. J Clin Oncol. 1991; 9(1):50-61.

(146.) Asioli S, Righi A, Volante M, Eusebi V, Bussolati G. p63 expression as a new prognostic marker in Merkel cell carcinoma. Cancer. 2007; 110(3):640-647.

(147.) Sturm N, Lantuejoul S, Laverriere MH, et al. Thyroid transcription factor 1 and cytokeratins 1, 5, 10, 14 (34betaE12) expression in basaloid and large-cell neuroendocrine carcinomas of the lung. Hum Pathol. 2001; 32(9):918-925.

(148.) Devouassoux-Shisheboran M, Hayashi T, Linnoila RI, Koss MN, Travis WD. A clinicopathologic study of 100 cases of pulmonary sclerosing hemangioma with immunohistochemical studies: TTF-1 is expressed in both round and surface cells, suggesting an origin from primitive respiratory epithelium. Am J Surg Pathol. 2000; 24(7):906-916.

(149.) Shin SY, Kim MY, Oh SY, et al. Pulmonary sclerosing pneumocytoma of the lung: CT characteristics in a large series of a tertiary referral center. Medicine (Baltimore). 2015; 94(4):e498.

(150.) Keylock JB, Galvin JR, Franks TJ. Sclerosing hemangioma of the lung. Arch Pathol Lab Med. 2009; 133(5):820-825.

Jennifer S. Woo, MD; Opal L. Reddy, MD; Matthew Koo, MD; Yan Xiong, MD; Faqian Li, MD, PhD; Haodong Xu, MD, PhD

Accepted for publication December 15, 2016.

Published as an Early Online Release June 23, 2017.

From the Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California (Drs Woo, Reddy, Koo, and Xu); the Department of Pathology, Peking University First Hospital, Beijing, China (Dr Xiong);and the Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis (Dr. Li).

The authors have no relevant financial interest in the products or companies described in this article.

Presented at the First Chinese American Pathologists Association (CAPA) Diagnostic Pathology Course: Best Practices in Immunohistochemistry in Surgical Pathology and Cytopathology; Flushing, New York; August 22-24, 2015.

Reprints: Haodong Xu, MD, PhD, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, 10833 Le Conte Ave, CHS 13-145E, Los Angeles, CA 90095-1732 (email:

Caption: Figure 1. Poorly differentiated squamous cell carcinoma of the lung. The needle core biopsy shows sheets of malignant large cells without obvious squamous differentiation (A). The tumor cells are positive for p40 (B) and cytokeratin 5/6 (C). The tumor cells are negative for both thyroid transcription factor 1 (D) and napsin A (E) (hematoxylin-eosin, original magnification X400 [A]; original magnification X400 [B through E]).

Caption: Figure 2. Poorly differentiated adenocarcinoma of the lung. The needle core biopsy shows a solid pattern of malignant large cells without glandular formation (A). The tumor cells are positive for thyroid transcription factor 1 (B) and napsin A (C). The tumor cells are negative for both p40 (D) and cytokeratin 5/6 (E) (hematoxylin-eosin, original magnification X400 [A]; original magnification X400 [B through E]).

Caption: Figure 3. Moderately differentiated adenocarcinoma of the lung. The biopsy shows a moderately differentiated adenocarcinoma with an acinar pattern (hematoxylin-eosin, (A). The tumor cells express the EGFR mutant protein, E746-A750 (B) (hematoxylin-eosin, original magnification X200 [A]; original magnification X200 [B]).

Caption: Figure 4. Poorly differentiated adenocarcinoma of the lung. The biopsy shows poorly differentiated adenocarcinoma with a solid pattern (A). The tumor cells express the EGFR mutant protein L858R (B) (hematoxylin-eosin, original magnification X200 [A]; original magnification X200 [B]).

Caption: Figure 5. Moderately differentiated adenocarcinoma of the lung. The biopsy shows a moderately differentiated adenocarcinoma with an acinar pattern (A). The tumor cells express ALK protein (B) (hematoxylin-eosin, original magnification X200 [A]; original magnification X200 [B]).

Caption: Figure 6. Poorly differentiated adenocarcinoma of the lung. The biopsy shows a poorly differentiated adenocarcinoma with acinar and micropapillary patterns (A). The tumor cells express ROS1 protein (B) (hematoxylin-eosin, original magnification X200 [A]; original magnification X200 [B]).

Caption: Figure 7. Pleomorphic carcinoma with prominent spindle cell component of the lung. The biopsy shows a proliferation of malignant spindle cells (A), with tumor cells positive for thyroid transcription factor 1 (B). The tumor cells are strongly positive for programmed death receptor-1 (C) (hematoxylin-eosin, original magnification X200 [A]; original magnification X200 [B and C]).

Caption: Figure 8. Biphasic pleural malignant mesothelioma. The biopsy shows a sheet of malignant epithelioid cell proliferation with focal malignant spindle cells (A). The tumor cells are positive for AE1/AE2 (B), calretinin (C), and Wilms tumor 1 (D) (hematoxylin-eosin, original magnification X400 [A]; original magnification X400 [B through D]).

Caption: Figure 9. Metastatic breast carcinoma of the lung. The biopsy shows a moderately differentiated adenocarcinoma with glandular formation (A). The tumor cells are positive for GATA3 (B), progesterone receptor (C), estrogen receptor (D), and mammaglobin (E). The tumor cells are negative for thyroid transcription factor 1 (F) (hematoxylin-eosin, original magnification X200 [A]; original magnification X200 [B through F]).

Caption: Figure 10. Small cell lung carcinoma. The biopsy shows a sheet of small round hyperchromatic cells with peripheral crush artifact at low power (A). At high power, the biopsy shows small round cells with scant cytoplasm, even and finely granular chromatin, inconspicuous nucleoli, and high mitotic activity (B). The tumor cells are positive for cytokeratin 7 (C), synaptophysin (D), and thyroid transcription factor 1(E). The Ki67 proliferative index is greater than 90% of the tumor cells (F) (hematoxylin-eosin, original magnifications X40 [A] and X400 [B]; original magnifications X400 [D] and X100 [C, E, and F]).

Caption: Figure 11. Sclerosing pneumocytoma. The biopsy shows a low-grade epithelial neoplasm (A) positive for AE1/AE3 (focal) (B), Cam5.2 (C), and thyroid transcription factor 1 (D) and negative for cytokeratin 7 (E) (hematoxylin-eosin, original magnification X400 [A]; original magnification X400 [B through E]).
Table 1. Common Immunohistochemical Stains
Used to Differentiate Pulmonary Squamous Cell
Carcinoma From Adenocarcinoma

Squamous Cell
Carcinoma                            Adenocarcinoma

CK5/6                      TTF-1-SPT24 (lower specificity)
p40 (higher specificity)   TTF-1-8G7G3/1 (higher specificity)
p63 (lower specificity)    Napsin A

Abbreviations: CK, cytokeratin; TTF-1, thyroid
transcription factor 1.

Table 2. Common Immunohistochemical Stains
Used to Differentiate Pulmonary Malignant
Mesothelioma From Adenocarcinoma

Malignant Mesothelioma   Adenocarcinoma

CK5/6                    TTF-1
Calretinin               Napsin A
D2-40                    Ber-EP4
WT-1                     B72.3

Abbreviations: CEA, carcinoembryonic antigen; CK, cytokeratin; TTF-1,
thyroid transcription factor-1; WT-1, Wilms tumor 1.

Table 3. Common Immunohistochemical Stains
Used to Differentiate Pulmonary Malignant
Mesothelioma From Reactive Mesothelial cells

Malignant                      Reactive
Mesothelioma                   Mesothelial Cells

p53                            Desmin
EMA                            IMP3--less frequently
IMP3-frequently positive       GLUT1--less frequently
GLUT1-frequently positive
p16 gene homozygous
BAP1 (loss of nuclear stain)

Abbreviations: BAP1, BRCA1 associated protein 1; EMA, epithelial
membrane antigen; GLUT1, glucose transporter 1; IMP3, IG2BP3,
insulin-like growth factor mRNA-binding protein.

Table 4. Common Immunohistochemical Stains
Used to Differentiate Primary Lung Carcinoma From
Carcinoma of Breast, Prostate, and Colorectal Origin

Lung          Breast      Prostatic   Colorectal
Origin        Origin       Origin       Origin

TTF-1 (a)   Mammaglobin   PSA         CDX2 (b)
Napsin A    GCDFP-15      PSAP
            GATA3         NKX3.1

Abbreviations: ERG, v-ets erythroblastosis virus E26 oncogene
homologue; GCDFP-15, gross cystic disease fluid protein 15; PSA,
prostate-specific antigen; PSAP, prostate-specific acid phosphatase;
TTF-1, thyroid transcription factor 1.

(a) TTF-1 may be positive in a subset of colorectal carcinomas.

(b) CDX2 may be positive in mucinous adenocarcinoma of the lung.

Table 5. Immunohistochemistry of Neuroendocrine Tumors of the Lung

Immunostain             TC           AC        LCNEC      SCLC

AE1/AE3 (a)         POS          POS          POS       POS
CAM5.2              NEG/POS      POS/NEG      POS       POS
CK7                 NEG          NEG          POS       NEG/POS
CK20                NEG          NEG          NEG       NEG
CD56 (a)            POS          POS          POS/NEG   POS
Synaptophysina      POS          POS          POS       POS/NEG
Chromogranin A (a)  POS          POS          POS/NEG   NEG/POS
TTF-1 (a)           NEG          NEG          POS       POS
Ki-67               [less than   [less than   40%-80%   50%-100%
(proliferation      or equal     or equal
index) (a)          to] 5%       to] 20%

Abbreviations: AC, atypical carcinoid; CK, cytokeratin; LCNEC, large
cell neuroendocrine carcinoma; NEG, negative; POS, positive; SCLC,
small cell lung carcinoma; TC, typical carcinoid; TTF-1, thyroid
transcription factor 1.

(a) Especially useful stains.

Table 6. Immunohistochemistry of Small Cell Lung
Carcinoma (SCLC) Versus Keratin-Positive Mimics

Immunostain       SCLC       MCC      SqCC

AE1/AE3          POS       POS       POS
CD56             POS       POS       NEG (a)
Synaptophysin    POS       POS       NEG (a)
Chromogranin A   POS/NEG   POS       NEG (a)
TTF-1            POS (a)   NEG (a)   NEG
CK20             NEG (a)   POS (a)   NEG
CK903            NEG       ...       POS (a)
p63              NEG       POS/NEG   POS (a)

Abbreviations: CK, cytokeratin; MCC, Merkel cell carcinoma; NEG,
negative; POS, positive; SqCC, squamous cell carcinoma; TTF-1,
thyroid transcription factor 1.

(a) Especially useful results.

Table 7. Immunohistochemistry of Small Cell Lung Carcinoma (SCLC)
Versus Keratin-Negative Mimics

Immunostain   SCLC   Lymphoid Infiltrate   Ewing Sarcoma   Melanoma

AE1/AE3       POS            NEG                NEG          NEG
CD45          NEG            POS                NEG          NEG
CD99          NEG            ...                POS          ...
S100          NEG            ...                ...          POS

Abbreviations: NEG, negative; POS, positive.

Table 8. Immunohistochemical Differences
Between Surface Cells and Round Cells
of Sclerosing Pneumocytoma

Surface Cells   Round Cells

TTF-1              TTF-1
EMA                 EMA

Abbreviations: EMA, epithelial membrane antigen; TTF-1, thyroid
transcription factor 1.

Table 9. Differential Diagnosis of Sclerosing

Differential Diagnosis           Immunostain

  Clear cell tumor         HMB-45
  Hemangioma               Vascular markers (CD31,
                             CD34, ERG)
  Hamartoma                S100 (mature
  Adenocarcinoma in situ   TTF-1
  Carcinoid tumor          Synaptophysin,
                             chromogranin A
  Metastatic papillary     Thyroglobulin, PAX8
    thyroid carcinoma
  Metastatic renal cell    CD10, CA IX, PAX8

Abbreviations: CA IX, carbonic anhydrase IX; ERG, v-ets avian
erythroblastosis virus E26 oncogene homolog; PAX8, paired box gene 8;
TTF-1, thyroid transcription factor 1.
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Author:Woo, Jennifer S.; Reddy, Opal L.; Koo, Matthew; Xiong, Yan; Li, Faqian; Xu, Haodong
Publication:Archives of Pathology & Laboratory Medicine
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Date:Sep 1, 2017
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