In the past several years, significant progress has been

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1 Utility of Immunohistochemistry in the Diagnosis of Pleuropulmonary and Mediastinal Cancers A Review and Update Kai Zhang, MD; Hongbin Deng, MD, PhD; Philip T. Cagle, MD Context. Immunohistochemistry has become an indispensable ancillary tool for the accurate classification of pleuropulmonary and mediastinal neoplasms necessary for therapeutic decisions and predicting prognostic outcome in the era of personalized medicine. Diagnostic accuracy has significantly improved because of the continuous discoveries of tumor-associated biomarkers and the development of effective immunohistochemical panels. Objective. To increase the accuracy of diagnosis and classify pleuropulmonary neoplasms through immunohistochemistry. Data Sources. Literature review, authors research data, and personal practice experience. In the past several years, significant progress has been made in the field of immunohistochemistry (IHC) in the diagnosis of pleuropulmonary and mediastinal tumors. The emergence of novel and better squamous cell carcinoma (SCC) markers, such as p40 and desmoglein-3, coupled with double staining, enhances the possibility of effectively separating adenocarcinoma (ADC) from SCC on small biopsies and cytologic specimens, conserving tissue for molecular testing. Recognition of a new entity of invasive mucinous ADC (IMA) provides an opportunity to investigate new antibodies for diagnostic purposes. The study of IMA using anti-hepatocyte nuclear factor 4a (HNF4a) antibody has shown promising results, which may be useful in diagnosing a tumor in small biopsies or cytologic specimens. We have learned a great deal about the genetic features of neuroendocrine tumors (NETs), and there is mounting evidence to suggest that small cell carcinoma and large cell neuroendocrine carcinoma (LCNEC) share more similarities than differences. Although the diagnosis of undifferentiated large cell carcinoma (LCC) and sarcomatoid carcinoma remains challenging, with the aid of IHC, a Conclusions. This review article has shown that appropriately selecting immunohistochemical panels enables pathologists to effectively diagnose most primary pleuropulmonary neoplasms and differentiate primary lung tumors from a variety of metastatic tumors to the lung. The discovery of new mutation-specific antibodies identifying a subset of specific gene-arranged lung tumors provides a promising alternative and cost-effective approach to molecular testing. Knowing the utilities and pitfalls of each tumor-associated biomarker is essential to avoiding potential diagnostic errors. (Arch Pathol Lab Med. 2014;138: ; doi: /arpa RA) substantial number of these cases can be accurately classified as SCC or ADC. Investigation of IHC markers for predicting underlying epithelial growth factor receptor (EGFR) mutations is actively ongoing, and the data suggest that several mutation-specific antibodies seem promising. Immunohistochemical analysis using new anti anaplastic lymphoma kinase (ALK) or reactive oxygen species 1 (ROS1) antibodies has demonstrated their efficacy in identifying ALK- or ROS1-rearranged lung ADCs. Relatively new IHC markers such as transducin-like enhancer split 1 (TLE1) and friend leukemia virus integration 1 (Fli-1) have been used in the evaluation of certain sarcomas, such as synovial sarcoma (SS) or angiosarcoma. More recently, the antibodies glucose transporter 1 (GLUT1), insulin-like growth factor II messenger RNA binding protein 3 (IMP3), and cluster of differentiation (CD) 146 have been shown to be helpful in differentiating reactive from malignant mesothelial proliferations, especially in cytologic specimens. Finally, many new organ-specific antibodies, such as GATA binding protein 3 (GATA3), paired box gene (PAX) 8, PAX2, and arginase-1 have greatly facilitated the diagnosis of metastatic carcinoma to the lung. Accepted for publication March 27, From the Department of Laboratory Medicine, Geisinger Medical Center, Danville, Pennsylvania (Drs Zhang and Deng); and the Department of Pathology and Genomic Medicine, The Methodist Hospital, Houston, Texas (Dr Cagle). The authors have no relevant financial interest in the products or companies described in this article. Reprints: Kai Zhang, MD, Department of Laboratory Medicine, MC 01-31, Geisinger Medical Center, 100 N Academy Ave, Danville, PA ( kzhang1@geisinger.edu). PRIMARY LUNG ADC AND SCC Thyroid transcription factor 1 (TTF1) and napsin A are routinely used in the diagnosis of ADC; they are found in 85% to 90% of ADC cases Similarly, IHC markers, including p40, cytokeratin (CK) 5/6, CK5, and p63, are used in supporting the diagnosis of SCC More than 90% of SCC cases are positive for each of these markers. 1,3,6,7,11 16 Squamous cell carcinoma and ADC markers are mutually Arch Pathol Lab Med Vol 138, December 2014 Immunohistochemistry in Pulmonary and Mediastinum Zhang et al 1611

2 Table 1. Markers Commonly Used for Separating Squamous Cell Carcinoma (SCC) From Adenocarcinoma (ADC) Antibody SCC ADC TTF1 þ Napsin A þ p40 þ CK5/6 þ CK5 þ p63 þ (diffuse pattern) Desmoglein 3 þ Abbreviations: CK, cytokeratin; TTF1, thyroid transcription factor 1; þ, greater than 75% of cases are positive;, less than 5% of cases are positive. exclusive (Table 1; Figure 1, a through d). Use of the IHC panels in Table 1 allows subtyping of about 80% of moderately and poorly differentiated non small cell lung carcinomas (NSCLCs) as either SCC or ADC on small biopsies. The percentage could be higher on large resected specimens. Recommended rules for interpreting IHC results are as follows: the carcinoma will be considered ADC or containing the presence of an ADC component if the tumor is positive for either TTF1 or napsin A, irrespective of p63, CK5/6, or CK5 staining. The carcinoma will be considered SCC if the tumor is positive for p40 or either CK5/6 or CK5, or diffusely and strongly positive for p63 in the absence of CK5/6 or CK5, TTF1, and napsin A expression. 3,6,7 Because approximately 10% to 20% of NSCLC cases cannot be further classified using current panels, and because more than half of lung cancer diagnoses are made on small biopsy and cytologic samples, selecting and employing effective antibody cocktails or developing double immunostaining methods to separate SCC and ADC for conservation of small tissue for molecular testing seems durable and necessary. 3,6,7,14,15 Brown et al, 3 using antibody cocktails, such as a combination of desmoglein-3 (one of the major glycoproteins of desmosomal structures), CK5, and napsin A, or double labeling with p40 and TTF1, provided diagnostic accuracy with a sensitivity and specificity of 100% in SCCs and sensitivity of 86% and specificity of 100% in ADCs. Several recent studies have shown that both rabbit polyclonal p40 (RP40) and mouse monoclonal p40 (MMp40) (BC28) antibodies and desmoglein-3 antibody may be better lung SCC markers than anti-p63 antibody (4A4) because they have been shown to achieve equal sensitivity (85% 92%) in lung SCC, but RP40 and MMp40 antibodies have exhibited superior specificity (99% 100%) versus 90% for Figure 1. Adenocarcinoma positive for thyroid transcription factor 1 (a) and napsin A (b), and squamous cell carcinoma positive for p40 (c) and p63 (d) (immunohistochemistry, original magnifications 340 [a through c] and 310 [d]) Arch Pathol Lab Med Vol 138, December 2014 Immunohistochemistry in Pulmonary and Mediastinum Zhang et al

3 p63 antibody in lung SCC Tacha et al 15 demonstrated that using a 6-antibody panel (TTF1, napsin A, CK5, p63, desmoglein-3, and tripartite motif containing 29) in conjunction with the proposed algorithm allows classification of more than 96% and 87% of cases of moderately differentiated and poorly differentiated NSCLC, respectively. Adenosquamous cell carcinoma of the lung is rare. Its diagnosis can be challenging histologically in a small biopsy. The previously described sensitive and specific SCC and ADC markers may help in this setting. The ADC component of adenosquamous carcinoma is positive for TTF1, carcinoembryonic antigen, and CK7, whereas SCC is positive for CK5/6 and p63. Other SCC markers such as p40, CK5, and desmocollin 3 should produce similar results to CK5/6 and p63. Adenocarcinoma variants (fetal type, colloid, signet cell, and clear cell) are usually positive for CK7 and TTF1 and variably positive for CK20 and caudal type homeobox 2 (CDX2). 1,16 Rare cases of human papillomavirus (HPV) related lung SCC may exist. However, current data suggest that most of these HPV þ lung SCCs may represent metastasis from previous head and neck HPV þ SCC and uterine or cervical HPV þ lesions and SCC. 17,18 Basaloid carcinoma of the lung is an uncommon poorly differentiated NSCLC, probably akin to SCC. Histologically, the tumor is composed of relatively small cells with high numbers of mitotic figures showing a lobular pattern with peripheral palisading and comedo-type necrosis. There are 2 histologic variants of basaloid carcinoma. Large cell carcinoma variant shows no intercellular bridging and individual cell keratinization; however, foci of abrupt pearl formation without progressive squamous maturation are seen. Squamous cell carcinoma variant shows the presence of squamous differentiation in less than 50% of cases. Basaloid carcinoma is positive for 34bE12 and negative for TTF1 and neuroendocrine markers Knowing the pitfalls and merits of these markers is essential for correct interpretation. Adenocarcinomas positive for TTF1 or napsin A in the lung or extrapulmonary sites do not necessarily indicate a lung primary, because TTF1 þ or napsin A þ ADCs have been described in ADCs originating from other organs, particularly endometrial and endocervical ADCs, renal papillary or clear cell, and papillary thyroid carcinomas Entrapped benign bronchiolar epithelial cells, alveolar pneumocytes, and alveolar macrophages are positive for napsin A, and these napsin A þ cells in small biopsies or cytologic samples associated with SCC must not be misinterpreted as napsin A þ tumor cells. 8,9 The staining for p63 is relatively less specific for SCC unless the staining is diffusely and strongly positive. 6,7 A small subset of ADCs and neuroendocrine carcinomas (NECs) can be focally positive for p63. This should be kept in mind when interpreting p63 staining in small biopsies and cytologic samples. It is worth noting that approximately 8% of SCC cases were positive for 1 of 3 neuroendocrine markers (CD56, synaptophysin, and chromogranin) and about 12% of ADC cases were positive for 2 of 3 of the markers. 22 IMA AND ADC IN SITU, MUCINOUS TYPE The 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification of lung ADC 23 has identified 2 new entities: IMA and ADC in situ, mucinous type (,3.0 cm). Invasive mucinous ADC comprises approximately 4.9% of Table 2. Markers for Invasive Mucinous Adenocarcinoma (IMA) and Adenocarcinoma In Situ, Mucinous Type (AIS-M) Versus Adenocarcinoma (ADC) (Nonmucinous) Antibody IMA and AIS-M ADC (Nonmucinous) CK7 þ þ CK20 þ (focal)/ þ TTF1 /þ þ Napsin A /þ þ CDX2 /þ HNF4a þ Abbreviations: CK, cytokeratin; CDX2, caudal type homeobox 2; HNF4a, hepatocyte nuclear factor 4a; TFF1, thyroid transcription factor 1; þ, greater than 75% of cases are positive;, less than 5% of cases are positive; /þ, less than 50% of cases are positive. lung ADCs and is seen more frequently in female nonsmokers. The tumor typically features multiple nodules (.3.0 cm in size) or lacks a circumscribed border, with miliary spread to adjacent lung parenchyma. Histologically, the tumor shows invasion of greater than 0.5 cm, characterized by scattered, well-differentiated tumor cells in large mucin pools and growth along alveolar walls. The tumor cells are tall columnar or goblet cells with minimal atypia. Some cases overlap with columnar (not cuboidal) acinar ADC histologically. The tumor harbors Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation (52%) and rarely EGFR mutation (3%). 23 On a small biopsy or fineneedle aspiration sample, it is sometimes difficult to make a definitive diagnosis. 23 The major differential diagnosis in the clinical setting is to rule out other types of primary ADCs and metastatic mucinous ADCs, particularly those from the gastrointestinal tract The suggested IHC panel is shown in Table 2 and Figure 2, a through f. Invasive mucinous ADC and ADC in situ, mucinous type, are diffusely positive for CK7, and many cases are also positive for CK20 with a focal or patchy pattern. The staining for TTF1, napsin A, or CDX2 shows more variations, with many cases exhibiting the absence of these markers, and a small subset of positive cases tends to be focally and weakly positive. Care should be taken not to interpret entrapped TTF1 þ and napsin A þ benign alveolar and bronchiolar epithelial cells beneath tumor cells as positive on tumor cells. 25 Recently a new marker, HNF4a (a nuclear transcription factor expressed in the liver, kidney, and intestine), was shown to be a sensitive and specific marker for IMA. 24 However, more studies are needed before a definitive conclusion can be reached regarding the value of this marker in facilitating the diagnosis of IMA on a small biopsy. PRIMARY PULMONARY NECS Demonstration of neuroendocrine differentiation by IHC is often required to make the diagnosis of NEC. The markers include chromogranin, synaptophysin, and CD56. 23,30 CD56 is generally considered the most specific and sensitive marker, because more than 90% of NEC cases are positive. It should be kept in mind that about 5% to 10% of small cell lung carcinomas (SCLCs) and LCNECs lack expression of the 3 neuroendocrine markers. 31,32 Other IHC markers routinely used in the diagnostic workup of NECs include CKAE1/3, CAM5.2, CK7/CK20, TTF1, p63, and Ki-67 (MIB- 1 labeling index) Table 3 shows the percentage range of positive cases for these markers in various types of NETs, Arch Pathol Lab Med Vol 138, December 2014 Immunohistochemistry in Pulmonary and Mediastinum Zhang et al 1613

4 Figure 2. a, Hematoxylin-eosin stained section of invasive mucinous adenocarcinoma (IMA) with proliferating tall columnar with some scattered goblet cells. b, IMA showing diffuse and strong CK7 positivity. c, IMA showing patchy strong CK20 positivity. d, IMA showing few residual napsin A þ benign pneumocytes beneath overlying napsin A mucinous columnar cells. e, Mucinous adenocarcinoma in situ (AIS-M) showing napsin A þ preexisting benign alveolar pneumocytes beneath overlying napsin A mucinous columnar cells by immunostaining. f, AIS-M showing similar staining pattern for thyroid transcription factor 1 (TTF1) with preexisting benign alveolar pneumocytes beneath overlying TTF1 mucinous columnar cells, similar to staining pattern of napsin A (original magnification 340 [A]; immunohistochemistry, original magnifications 340 [b, d, and f] and 310 [c and e]) Arch Pathol Lab Med Vol 138, December 2014 Immunohistochemistry in Pulmonary and Mediastinum Zhang et al

5 Table 3. Markers for Typical Carcinoid, Atypical Carcinoid, Large Cell Neuroendocrine Carcinoma (LCNEC), and Small Cell Lung Carcinoma (SCLC) Antibody Typical Carcinoid Atypical Carcinoid LCNEC SCLC CD56 þ þ þ þ Synaptophysin þ þ þ/ þ/ Chromogranin þ þ þ/ þ/ CKAE1/3 þ þ þ (often weak with dotlike pattern) CAM 5.2 þ þ þ þ (often weak with dotlike pattern) CK7 þ/ þ/ þ/ TTF1 /or þ (weak) þ/ /þ þ in.90% of cases p63 /þ /þ /þ /þ Ki-67 (MIB1 labeling index) Low Moderate to high High High Abbreviations: CD, cluster of differentiation; CK, cytokeratin; TTF1, thyroid transcription factor 1; þ, greater than 75% of cases are positive; þ/, usually more than 50% of cases are positive;, less than 5% of cases are positive; /þ, less than 50% of cases are positive. including typical carcinoid (TC) and atypical carcinoid (AC), LCNEC, and SCLC (Figure 3, a and b). Positive staining for CK7 is seen in about 38% of NECs, 16% of TCs, 56% of LCNECs, and 25% of SCLCs. Positive staining for TTF1 (clone SPT24) is seen in 54% of NECs, 24% of TCs, 47% of LCNECs, and 86% of SCLCs. Thyroid transcription factor 1 positive NECs appear to correlate with primary lung origin, although this is not specific as it is in ADC. 30,33 The MIB-1 labeling index has proven to be very useful in separating low- from high-grade NETs. The labeling index for TC is less than 5%, for AC less than 10%, and for LCNEC/SCLC more than 50% 30 (Figure 3, a and b). Positive staining for Figure 3. a, Low MIB-1 labeling index in typical carcinoid (,5%). b, High MIB-1 labeling index in large cell neuroendocrine carcinoma (50%) (immunohistochemistry, original magnifications 310 [a] and 340 [b]). Figure 4. Lung adenocarcinoma (ADC; hematoxylin-eosin section) (a) and the same tumor showing cytoplasmic anaplastic lymphoma kinase 1 (ALK1) þ (Dako clone) (b). Note: ALK staining tends to be weaker in ADC than ALK þ anaplastic large cell lymphoma (immunohistochemistry, original magnification 340 [a and b]). Arch Pathol Lab Med Vol 138, December 2014 Immunohistochemistry in Pulmonary and Mediastinum Zhang et al 1615

6 p63 is seen in about 15% of NECs, less than 10% of TCs, less than 15% of LCNECs, and 22% of SCLCs. Positive staining for CK5/6 has been found in 1% of NECs (focal positivity), 0% of TCs, 2% of LCNECs, and 1.5% of SCLCs. Positive staining for desmocollin 3 is less than 1% in NECs. Napsin A is absent in TC, AC, and SCLC, but about 10% of LCNECs have been found to be strongly and diffusely positive. 22 Positive staining for sex-determining region Y box 2 (SOX2) is found in about 72% of NECs, 32% of TCs, 81% of LCNECs, and 87% of SCLCs. The positive staining pattern of SOX2 can be either diffusely or focally positive. 38 CDX2 þ NECs are more suggestive of intestinal origin. 39,40 Occasional cases of NSCLC (ADC, SCC, and undifferentiated LCC) can focally express CD56 and synaptophysin (uncommonly chromogranin), although these carcinomas lack histologic neuroendocrine features (such as growth pattern and nuclear features). These cases have been designated as NSCLC with neuroendocrine differentiation. Further studies of the tumor are needed to clarify the identity and clinical significance. 21,30 Recent studies have shown that there are more similarities than differences between SCLC and LCNEC. Large cell NEC is more likely to respond to chemotherapeutic strategies targeting SCLC or show no difference in therapeutic response or natural history compared with SCLC. About 50% of both SCLCs and LCNECs have shown a positive p53 mutant immunophenotype as compared with no TCs and 20% of ACs. Inactivated and dysfunctional retinoblastoma (RB) serves as a hallmark of high-grade NETs. Upregulation of E2F1 is seen at a high level in neuroendocrine lung tumors. B-cell CLL lymphoma 2 (Bcl2) is shown to be highly expressed as compared with Bcl2- associated X protein (Bax) in both high-grade NETs, with a Bcl2:Bax ratio of greater than 1, especially in the p53 mutant cases. 40 UNDIFFERENTIATED LCC AND SARCOMATOID CARCINOMA Large cell carcinoma is a heterogeneous group of histologically undifferentiated carcinomas. Large cell carcinoma with clear cell features and LCC with rhabdoid phenotype are histologic patterns of LCC and should not be considered as defining categories. 21 With additional immunostaining using common SCC and ADC IHC markers, about 34% and 25% of LCCs can be further subclassified as ADC and SCC, respectively. 41,42 In a small-sample setting, IHC analysis for p40, p63, CK5/6, CK5, desmocollin 3, TTF1, or napsin A is used as a predictive tool when the tumor shows no morphologic evidence of differentiation (NSCLC not otherwise specified); predictive accuracy is around 80% to 85% in most unselected studies. 3,6,7,11 16 A subset of cases may even focally express neuroendocrine markers, including chromogranin (9%) and synaptophysin (15%). 7,22 Patterns of multi-messenger RNA expression in some LCCs may match those in differentiated SCC or ADC. Adenocarcinoma-associated mutations (EGFR, KRAS, BRAF, MAP2K1/ MEK1, neuroblastoma RAS viral [v-ras] oncogene homolog [NRAS], and ERBB2/HER2, as well as ALK) or SCCassociated mutations (phosphoinositide 3 kinase [PI3K] and AKT1) may be found in LCC, and tend to segregate with an IHC profile associated with the corresponding tumor subtype. 43 Although the majority (80%) of LCCs can be classified by immunomarkers in conjunction with molecular analysis into clinically relevant ADCs and SCCs, these molecular profiles are not specific enough to replace a histologic diagnosis of LCC, and a significant percentage of LCCs will remain undifferentiated carcinomas. Similarly, none of these SCC and ADC markers are specific and sensitive enough to subtype these tumors; care should be taken when interpreting small samples. Clear cell carcinoma of the lung, though controversial, is a form of LCC listed in the 2008 World Health Organization classification. 21 Clear cell carcinoma of the lung is different from clearly evident ADC or SCC with clear cell change. Clear cell carcinoma of the lung lacks histologic features of ADC or SCC differentiation and is positive for CK7, with a subset positive for TTF1 and negative for p63 and CK5/6. It displays more frequent and usual KRAS mutations than expected in most ADCs of the lung as reported in a study with a small number of cases. 44 The diagnosis of lymphoepithelioma-like carcinoma variant of LCC of the lung requires the demonstration of Epstein-Barr virus on tumor cells. 45 Primary pulmonary sarcomatoid carcinoma is defined as a poorly differentiated NSCLC with 10% sarcoma or sarcomatoid elements (pleomorphic, spindle cell, and giant cell). 21 Sarcomatoid refers to cytologic features of tumor cells with pleomorphic, spindle cell, and giant cell appearance. The tumor comprises 0.3% to 1.3% of all lung malignancies, with an average patient age of 60 years, a male to female ratio of 4:1, and 90% associated with smoking. This entity can be reliably diagnosed only in surgically resected specimens. 21,46 50 About 43% of sarcomatoid carcinomas are sarcomatoid and LCC, 31% sarcomatoid and LCC plus ADC, and 26% sarcomatoid and LCC plus SCC. 21,46 49 The EGFR and KRAS mutations have been found in about 15% to 20% and 9.8% of pleomorphic carcinomas, respectively. 50 Table 4 shows markers commonly used in supporting the diagnosis of sarcomatoid carcinoma. Expression of epithelial markers in the spindle and giant cell component is not required for the diagnosis of sarcomatoid carcinoma so long as there is a component of ADC or SCC. Because cytokeratin staining is often weak or even absent, multiple anti-cytokeratin antibodies are needed. Generally, IHC profiles tend to correspond with SCC areas better than ADC areas. It should be pointed out that no reliable IHC markers exist or have been developed for diagnosing sarcomatoid carcinoma, and a wide immunostaining overlap exists among sarcomatoid carcinoma, sarcomatoid mesothelioma, sarcoma, and metastatic sarcomatoid renal cell carcinoma. IHC FOR PREDICTING UNDERLYING EGFR MUTATIONS, ALK1 REARRANGEMENT, AND OTHER GENE ALTERATIONS IN LUNG ADC AND NSCLC WITH ADC COMPONENT Clinical evidence has demonstrated that EGFR mutation analysis is the most reliable method of determining which NSCLCs have EGFR mutation and predicting response to first-generation tyrosine kinase inhibitor therapy Analysis of EGFR copy number by fluorescence in situ hybridization (FISH) reveals that tumors with a high number of copies of EGFR show slightly better response to tyrosine kinase inhibitors, but it is not nearly as good for activating mutations by molecular methods The IHC data using anti phospho-egfr antibodies are limited. 50 Recent IHC studies using mutation-specific antibodies for exon 19 deletion mutations to predict underlying EGFR mutations have shown 100% sensitivity for 15-bp mutations 1616 Arch Pathol Lab Med Vol 138, December 2014 Immunohistochemistry in Pulmonary and Mediastinum Zhang et al

7 Table 4. Markers for Sarcomatoid Carcinoma (Pleomorphic, Spindle, and Giant Cell) Antibodies Pancytokeratin CKAE1/3 CK7 EMA CEA TTF1 SP-A Vimentin Smooth muscle actin Literature þ/ þ/ þ/ þ/ þ/ (but can be þ in giant cell carcinoma) or þ (epithelial component) þ /þ Abbreviations: CEA, carcinoembryonic antigen; CK, cytokeratin; EMA, epithelial membrane antigen; SP-A, surfactant protein A; TTF1, thyroid transcription factor 1; þ, greater than 75% of cases are positive; þ/, usually more than 50% of cases are positive;, less than 5% of cases are positive; /þ, less than 50% of cases are positive. and 98.8% specificity for predicting underlying mutations Strong protein expression has 100% positive predictive value for underlying mutations However, analysis of the antibody shows less than 50% sensitivity for detecting mutations of other sizes or activating mutations outside of these hot spots, which makes it insufficiently sensitive as a standard single assay The antibody for L858R mutation has been shown to have a sensitivity of 95% and a specificity of 98.8% for predicting underlying mutations of the gene locus. These antibodies may be useful in screening for most EGFR mutations but will not detect the less frequent ones. 55,56 EML4-ALK translocation in lung ADC by IHC was first identified in 2007, and it was rapidly moved to clinical practice within 3 years Detecting ALK translocations can be done by FISH, reverse transcription polymerase chain reaction, and IHC. The antibody (Dako clone ALK1, Carpinteria, California) used as screening test was found to be insufficiently sensitive in detecting ALK protein expression in most rearranged tumors compared with ALK FISH (Vysis LSI ALK dual color, Break Apart Rearrangement Probe, FDA-approved, Abbott Molecular Inc, Des Plaines, Illinois), which was considered the gold standard diagnostic test to enroll patients for clinical trials and for treatment with crizotinib Subsequently Mino-Kenudson et al 61 in 2010 reported that IHC analysis using the new anti-alk antibody clone D5F3 (Cell Signaling Technology, Danvers, Massachusetts) showed excellent sensitivity and specificity of 100% and 99%, respectively, for the detection of ALKrearranged lung ADCs in biopsy specimens. Another antibody, clone 5A4 (Leica Biosystems Newcastle Ltd, Newcastle, United Kingdom), has been shown to achieve sensitivity, specificity, and positive and negative predictive values of 93.3%, 96.0%, 93.3%, and 96%, respectively. 58 Despite some variability of the data in studies using ALK1, D5F3, and 5A4 antibodies (Ventana Medical System, Tucson, Arizona) for the detection of ALK-rearranged lung ADCs, these studies demonstrate that ALK IHC can achieve greater than 90% sensitivity and greater than 85% specificity compared with ALK FISH (Figure 4, a and b). There are rare cases in which there are discordant results between ALK IHC and ALK FISH, that is, FISH false-negative but IHC ALKþ tumors. 58,61 Compared to IHC, cons of using FISH are that it is technically challenging, with many levels such as interpretation (subtle signals difficult to interpret) and about 10% failure rate because of improper fixation, poorly preserved cells, and insufficient tumor cellularity requiring greater than or equal to 100 tumor cells for ALK FISH testing Although ALK expression is known to correlate with ALK translocation with these antibodies, clinical trials validating ALK IHC as a predictive biomarker for patient outcome with crizotinib therapy have not been performed. 51 ROS1 gene rearrangement is reported in 1% to 2% of lung ADCs and is associated with response to the multitargeted tyrosine kinase inhibitor crizotinib. Lung ADCs with ROS1 gene arrangement can be detected by FISH or IHC. Sholl et al 63 demonstrated that IHC using the antibody clone D4D6 showed 100% sensitivity and 92% specificity for ROS1 gene rearrangement compared with ROS1 FISH; they concluded that ROS1 IHC is an effective screening tool for this rare but clinically important subset of lung ADCs. RET gene rearrangement is rare, reported in 1% of lung ADCs resulting from small intrachromosomal rearrangement (inv[10](p11.22q11.2). RET-rearranged ADCs are more common in nonsmokers and respond to cabozantinib (c-met and vascular endothelial growth factor receptor 2 inhibitor). RET IHC showed positive staining in normal kidney tubules and overexpression of RET in papillary renal cell carcinoma; however, overexpression in papillary renal cell carcinoma does not appear to correlate with underlying RET translocation. Currently RET antibodies are insufficiently sensitive to detect RET rearrangements in lung cancer. 64,65 Immunohistochemistry using BRAF V600E mutationspecific antibodies has established good sensitivity and specificity in melanoma, thyroid, and colon cancers. However, lung ADC-harboring BRAF mutations are rare, only about half of them being V600E. Others are non-v600e exon 15 or exon 11 variants. Studies of the clinical efficacy of BRAF inhibitors in lung cancer are ongoing. 66 Other gene rearrangements include the following: (1) ERBB2 (Her2) IHC: Protein overexpression highly correlated with gene amplification in breast and upper GI cancers; 2% of lung cancers harbor in-frame (activating) insertion mutations in exon 20 of ERBB2. In lung cancers, no correlation is found between protein expression using commercially available HER2 antibodies and gene amplifications. Antibodies for exon 20 of ERBB2 tested are negative; (2) MET activation appears to represent a major resistance mechanism in patients receiving EGFR tyrosine kinase inhibitors. The role of de novo MET activation in lung ADCs is under investigation. Clinical trials of MET inhibitors are ongoing. MET protein overexpression correlates with copy number gain. MET amplification can be detected by FISH and IHC using antibody clone SP44 with ultraview detection (Ventana Medical System). It remains unclear which MET testing (FISH or IHC) is the predictor of response to inhibitors. Several trials are currently ongoing; (3) Common tumor suppressor genes are reported in lung ADCs. TP53 is very commonly inactivated in lung cancer. Immunohistochemistry for p53 is uncommonly performed. Inhibitors are under investigation. Loss of phosphatase and tensin homolog (PTEN), predicting activation of pathway/ phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit a (PIK3CA) pathway activation, is an uncommon alteration in lung ADC, but relatively common in SCC. Inhibitors are undergoing trials. Immunohistochemistry PTEN for is available using the antibody clone 138G6 (Cell Signaling Technology). Loss of STK11/LKB1, inactivated in Arch Pathol Lab Med Vol 138, December 2014 Immunohistochemistry in Pulmonary and Mediastinum Zhang et al 1617

8 25% to 30% of lung ADCs, is commonly associated with KRAS mutations. Gene alteration is predictive of resistance to mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitors. Immunohistochemistry using the antibody clone Ley37D/G6 (Abcam, Cambridge, Massachusetts) is available, and gives a cytoplasmic granular staining pattern. PRIMARY PLEUROPULMONARY SARCOMAS Primary pulmonary sarcoma is rare, comprising 0.2% of lung cancers. Diagnostic challenges often arise because of histologically similar but diverse tumors. Immunohistochemical analysis coupled with molecular testing is usually needed to confirm the diagnosis with certainty. 20 Pulmonary sarcomas can be generally grouped into 4 categories: (1) spindle cell sarcomas, including SS, malignant peripheral nerve sheath tumor, leiomyosarcoma, inflammatory myofibroblastic tumor, solitary fibrous tumor, and undifferentiated pleomorphic sarcoma; (2) vascular tumors, including angiosarcoma, Kaposi sarcoma, epithelioid hemangioendothelioma, and pulmonary artery intima sarcoma; (3) primitive/embryonal tumors, including primary pleuropulmonary blastoma, primary NET, and rhabdomyosarcoma; and (4) others, including primary pulmonary myxoid sarcoma, angiomatoid fibrous histiocytoma, and alveolar soft part sarcoma. 21 Pleuropulmonary SS is histologically similar to its soft tissue counterparts and usually presents at a younger age than mesothelioma. Primary pleural SSs are usually biphasic, and primary pulmonary SSs are often more sarcomatous monophasic. Immunohistochemistry for pan- CK, CKAE1/3, epithelial membrane antigen (EMA), and Bcl2 is typically performed for the diagnosis of SS. The positivity for each marker depends on whether SS is biphasic or monophasic. Most cases are positive for Bcl2 and one epithelial marker; they are more often positive for EMA (76%) than cytokeratin 7 (60%). A new marker, TLE1, was found in 95% of the tumors. Biphasic SS shows a higher percentage of positivity for cytokeratins, mesothelial-related markers (calretinin and CK5/6), and epithelial adhesion molecule antibody (Ber-EP4) than monophasic or spindle cell SS. More than 9% of cases have detectable SYT-SSX1 or SYT-SSX2 fusion genes: t(x18)(p11.2;q11.2) by reverse transcription polymerase chain reaction and FISH. 21,67 69 Pulmonary malignant peripheral nerve sheath tumor is very rare. The tumor shows extensive morphologic overlap with monophasic SS and is associated with neurofibromatosis type 1. The tumor is variably positive for S100 (often focal positivity in residual Schwann cells only); diffuse S100 positivity should raise suspicion for melanoma. The tumor is often positive for CD56 and CD57 and negative for desmin, keratin, human melanoma black 45 (HMB45), and melanoma-associated antigen recognized by T cells (Mart-1). Negative testing for SS fusion genes is very helpful in the diagnosis of malignant peripheral nerve sheath tumor. 21,67 Leiomyosarcoma, besides being positive for desmin, a- smooth muscle actin, and muscle-specific actin, is also positive for calponin and h-caldesmon, 2 relatively new markers. 21,67 Pleural solitary fibrous tumor is usually a benign, slowly enlarging mass. The tumor may recur and occasionally undergo metastasis; it shows variegated spindle cells (pericytic and storiform patterns). The tumor is positive for CD34, CD99, and Bcl2 and negative for cytokeratin, EMA, desmin, S100, and CD31. 21,67 Pleural desmoplastic small round cell tumor is very rarely seen. The tumor often shows focal patchy positivity for pan- CK and CKAE1/3, and is characteristically positive for Wilms tumor 1 (WT1) and desmin (dotlike pattern). The tumor harbors t(11;22)/wt1-ews fusion. 21,67,70 Inflammatory myofibroblastic tumor is uncommon. The tumor can be focally positive for CKAE1/3, and the spindle cell component is positive for vimentin and smooth muscle actin. About 40% of inflammatory myofibroblastic tumors show ALK1 positivity. Inflammatory myofibroblastic tumor is negative for S100, desmin, and myogenin. 1,67,71,72 Malignant vascular neoplasm rarely arises in the pleura, spreading diffusely to mimic pleural mesothelioma. Angiosarcoma, Kaposi sarcoma, epithelioid hemangioendothelioma, and epithelioid hemangioma are positive for CD31, CD34, and Fli-1, and well-differentiated tumors are also positive for factor VIII. Kaposi sarcoma is characteristically positive for human herpes virus 8/latency-associated nuclear antigen. 1,67,73 Pleuropulmonary blastoma is an uncommon tumor with type 1 (cystic type) and types 2 and 3 (cystic plus solid and solid types, respectively). Pleuropulmonary blastoma type 1 comprises 14% of pleuropulmonary blastomas, and patients have a median age of 9 months. Embryonal cells can show myogenic markers. Twenty-five percent of pleuropulmonary blastoma patients have germline mutations in DICER1. Pleuropulmonary blastoma types 2 and 3 comprise 48%, with a mean patient age of 36 months, and 38%, with a mean patient age of 43 months, respectively. The tumor contains small round blue cells, fibrosarcoma-like foci, benign or malignant cartilage, rhabdomyosarcomatous areas, and pleomorphic multinucleated tumor giant cells. The tumor has a 45% recurrence rate, with a 5-year survival rate of 42%. About 25% of tumors can have distal metastasis (brain, spinal cord, or bone). Glands in blastoma stain with antibodies to keratin, carcinoembryonic antigen, EMA, and TTF1, whereas the epithelial cells in morules are positive for TTF1. The stromal cells are stained with smooth muscle actin and vimentin. The morules and glands show staining for chromogranin, synaptophysin, or neuron-specific enolase. Suspected rhabdomyosarcomatous elements can be confirmed with antibodies for myogenin, myogenic differentiation 1 (MyoD1), and desmin. 1,21 Pulmonary artery intimal sarcomas occur in the pulmonary trunk, frequently presenting as chronic thromboembolic events. Many cases are high-grade undifferentiated sarcomas, and some can show areas of osteosarcoma, chondrosarcoma, or rhabdomyosarcoma. Some cases show a myofibroblastic phenotype. 1,21 Primary lung Ewing sarcoma/primary NET is very rare, commonly occurring in the chest wall of children or young adults with rare reports with endobronchial presentation. Immunohistochemical markers for this tumor are similar to the markers of the tumor in other sites. The tumor is positive for CD99, Fli-1, and vimentin, and less than 50% are positive for synaptophysin, neuron-specific enolase, keratin, CD117, and S100. The tumor is negative for CD56, desmin, chromogranin, terminal deoxynucleotidyl transferase (Tdt), and CD45. 1,21,67 t(11;22)(q24;q12)/ewsr1-ffl1 and t(21;22)(q22;q21)/ewsr1-erg are found in 90% of Ewing sarcomas and 5% of primary NETs. 1,21,67,73 Primary lung rhabdomyosarcoma is very rare; however, metastatic rhabdomyosarcoma to the lung is occasionally 1618 Arch Pathol Lab Med Vol 138, December 2014 Immunohistochemistry in Pulmonary and Mediastinum Zhang et al

9 seen. Distinction of the tumor from other sarcomas can usually be done by IHC. Rhabdomyosarcoma is positive for myogenin and MyoD1, whereas SS, Ewing sarcoma/primary NET, neuroblastoma, and desmoplastic small round cell tumor are typically negative for these markers. Primary pulmonary myxoid sarcoma with EWSR1-CREB1 fusion is a newly described rare tumor entity. Mean age of patients is 45 years, and there are more female than male patients. The tumor is well circumscribed, lobulated, and related to the bronchus. It is composed of spindle, stellate, and polygonal cells that are arranged in strands and cords with myxoid stroma. The tumor cells show variable atypia, generally mild to moderate, and generally few mitotic figures. There is often patchy chronic inflammation and necrosis. The tumor is positive for vimentin, and some cases show focal and weak EMA positivity. The tumor has been found to be negative for CKAE1/3, CK5/6, 34bE12, CK7, CK20, p63, desmin, CD31, CD34, TTF1, Melan-A, and HMB45. Eight of 9 cases and 5 of 6 cases were negative for S100, respectively, whereas 1 of 9 and 1 of 6 cases showed very focal S100 nuclear staining and staining for neuroendocrine markers. EWSR1-CREB fusion was noted in 79% of cases. 21,67 Pulmonary angiomatoid fibrohistiocytoma is very rare, is seen in middle-aged and older adults, and is characterized by nodular growth with pronounced lymphoplasmacytic infiltrates. The tumor is positive for EMA and CD68, and is often positive for desmin and negative for myogenin/ MyoD1, CD31, and CD34. The tumor frequently harbors EWSR1-CREB1, FUS-ATF1 or ERSR1-ATF1 fusions. 74 PLEURAL MESOTHELIOMA In the past decades, a number of mesothelial markers and epithelial markers have been developed that show very good specificity and sensitivity for the diagnosis of mesothelioma and/or differentiation between malignant mesothelioma (MM) and metastatic epithelial tumors. Although there is currently no single immunohistochemical marker that shows 100% specificity and sensitivity for mesothelioma, immunohistochemical panels including 2 or more positive mesothelial markers and 2 or more positive carcinoma markers can usually provide necessary support for the diagnosis or exclusion of MM. Given the availability of a number of mesothelial and epithelial markers, instead of reviewing individual antibodies, this section focuses on the application of immunohistochemistry in diagnosing MM, including distinguishing MM from reactive mesothelial proliferations and differentiation of epithelioid mesothelioma from metastatic carcinoma and sarcomatoid or biphasic mesothelioma from other spindle cell malignancies. APPLICATION OF IHC FOR DISTINGUISHING MM FROM REACTIVE MESOTHELIAL PROLIFERATIONS The distinction between MM and reactive mesothelial proliferation on routine histologic preparation can be extremely challenging. Because some mesotheliomas can be cytologically bland, it is not uncommon for reactive mesothelial proliferations to show various degrees of cytologic atypia. Organizing pleuritis is a great mimic of desmoplastic mesothelioma, especially in small pleural samplings. Current mesothelial markers are not helpful in this distinction because they are positive in both benign and malignant mesothelial cells. Because of these challenges, a number of immunohistochemical markers have been Table 5. Antibodies Markers for Distinguishing Malignant Mesothelioma From Reactive Mesothelial Proliferations Malignant Mesothelioma Reactive Mesothelial Proliferations GLUT1 þ or þ/ /þ CD146 þ IMP3 þ or þ/ /þ EMA þ /þ Desmin /þ þ p53 þ /þ Abbreviations: CD146, cluster of differentiation 146; EMA, epithelial membrane antigen; GLUT1, glucose transporter 1; IMP3, insulin-like growth factor II messenger RNA binding protein 3; þ, greater than 75% of cases are positive; þ/, 50% 75% of cases are positive;, less than 5% of cases are positive; /þ, 5% 50% of cases are positive. proposed to assist in the distinction, as shown in Table 5. Epithelial membrane antigen, desmin, and p53 are the markers that have previously been investigated Epithelial membrane antigen has been found to be more frequently expressed in mesotheliomas (70% 100%) than in reactive mesothelial cells (0% 70%), whereas desmin is more frequently positive in reactive mesothelial cells (about 84%) than in mesotheliomas (about 21%). Positive p53 staining favors a diagnosis of mesothelioma over reactive mesothelial cells, with sensitivity of 41% to 61% and specificity of 91% to 98%. GLUT1, IMP3, and CD146 are recently emerging markers that show promising sensitivity and specificity in distinguishing mesotheliomas from benign mesothelial cells (Figure 5, a through c). GLUT1 is a highaffinity glucose transporter; its expression in MM has been reported in a few studies. 75,78 82 Kato et al 79 found that GLUT1 was positive in 100% of tissue sections of pleural mesothelioma (48 of 48 cases) but not detectable in any reactive mesothelial proliferations (0 of 40 cases). Ikeda et al 80 reported that 100% of mesotheliomas from cytologic effusion specimens stained with GLUT1, compared with 20% of benign mesothelial cells. However, 2 other studies performed by Lee et al 81 and Lagana et al 82 reported lower overall sensitivity (53%) and specificity (90% 98%) for GLUT1. The different data reported can probably be attributed to the technical approach used and also to differences in the selection of antibodies. 81 However, all 4 studies reveal high specificity of GLUT1 in the distinction between MM and reactive mesothelial proliferation. Thus, positive GLUT1 stain can be a strong suggestion of a MM. GLUT1 also stains erythrocytes, a fact that should be kept in mind when interpreting cell block sections of a cytologic specimen containing blood. Insulin-like growth factor II messenger RNA binding protein 3 is found to be upregulated in a wide variety of malignant tumors. Its expression in MM has been investigated in several studies that demonstrated that IMP3 was positive in 36% to 91% of MMs and 0% to 31% of benign mesothelial proliferations. 80,83 Initial exploration of the possibility of using CD146, a cell adhesion molecule, as a diagnostic marker for distinguishing MM from reactive mesothelial proliferation started a couple years ago, and few studies have been published. 84,85 One study performed by Sato et al 84 evaluated the expression of 2 clones of CD146 (OJ79 and EPR3208) in 23 pleural MMs and 28 reactive mesothelial proliferation cases. They found that the sensitivities of OJ79 and EPR3208 were 94% and 90%, respectively, and the Arch Pathol Lab Med Vol 138, December 2014 Immunohistochemistry in Pulmonary and Mediastinum Zhang et al 1619

10 Figure 5. a, Pleural epithelioid mesothelioma showing membranous and some cytoplasmic staining for glucose transporter 1 (GLUT1) in tissue section. b, Epithelioid mesothelioma showing membranous and some cytoplasmic staining for GLUT1 in pleural effusion cytology cell block. c, Epithelioid mesothelioma showing membranous staining for CD146 in tissue section (immunohistochemistry, original magnification 340 [a through c]). Figure 6. a, Pleural epithelioid mesothelioma showing nuclear and cytoplasmic staining for calretinin. b, Pleural epithelioid mesothelioma shows nuclear staining for Wilms tumor 1. c, Pleural epithelioid mesothelioma showing membranous staining for podoplanin (D2-40) (immunohistochemistry, original magnification 340 [a through c]) Arch Pathol Lab Med Vol 138, December 2014 Immunohistochemistry in Pulmonary and Mediastinum Zhang et al

11 Table 6. Markers Commonly Used for Diagnosing Epithelioid Mesothelioma Antibodies Epithelioid MM Lung ADC Other Carcinomas Calretinin þ a /þ Breast CA /þ; SCC /þ; RCC /þ; SCLC /þ D2-40 þ b SCC /þ; RCC CK5/6 þ SCC þ; RCC ; UCþ WT1 þ c Breast CA þ/ ; SCC ; RCC Mesothelin þ þ/ SCC /þ; pancreatic ADC þ; UCþ; RCC Thrombomodulin þ þ/ SCC þ/ ; RCC ; UCþ MOC31 /þ (small area or sparse) þ Breast þ; SCC þ; UC /þ; RCC /þ Ber-EP4 /þ (small area or a few) þ SCC þ; RCC þ/ pcea and mcea þ Breast CA þ; SCC /þ; RCC ; colon ADC þ B72.3 þ breast CA þ; SCC þ/ BG8 /þ (small area or sparse) þ breast CA þ; SCC þ/ CD15 þ/ RCC þ; SCC /þ Claudin-4 þ Breast CA þ; SCC þ; RCC þ/ ; UCþ/ Abbreviations: ADC, adenocarcinoma; Ber-EP4, epithelial cell adhesion molecule antibody; BG8, anti blood group Lewis Y; CA, carcinoma; CD15, cluster of differentiation 15; CK5/6, cytokeratin 5/6; D2-40, podoplanin; mcea, monoclonal carcinoembryonic antigen; MM, malignant mesothelioma; pcea, polyclonal carcinoembryonic antigen; RCC, renal cell carcinoma; SCLC, small cell lung carcinoma; SCC, squamous cell carcinoma; UC, urothelial carcinoma; WT1, Wilms tumor 1; þ, greater than 75% of cases are positive; þ/, 50% 75% of cases are positive; /þ, 5% 50% of cases are positive;, less than 5% of cases are positive. a Both nuclear and cytoplasmic stains. b Membranous stain, usually along the apical surface of the cells. c Nuclear stain. specificity was 100% for both clones. The data are very encouraging, suggesting that CD146 is a sensitive and specific immunohistochemical marker enabling differential diagnosis of MM from reactive mesothelium. Many mesotheliomas show deletion of the p16/cdkn2a gene, and this is associated with poorer survival Some studies have proposed that detection of p16 deletions by in situ hybridization is a useful approach to differentiate between MM and reactive mesothelial proliferations. 86,87,89 Currently few data are available regarding the detection of p16 protein using immunohistochemistry. In a study presented by Aydin et al 90 at a cytopathology meeting in 2010, it was demonstrated that a majority of benign and reactive mesothelial cells (80% 100%) had strong expression of p16, and 78% of MMs lost p16 expression. Although these data suggest that p16 immunohistochemistry could be useful in distinguishing MMs from benign mesothelial proliferations, additional studies are needed to fully determine its practical value. APPLICATION OF IHC FOR DIAGNOSIS OF MM AND ITS DIFFERENTIATION FROM METASTATIC CARCINOMAS Metastatic carcinomas can be great mimics of MM, making it difficult to accurately classify these tumors based on morphologic features alone. Immunohistochemistry always plays an important role in distinguishing MM from metastatic carcinoma. Currently, many mesothelial markers and epithelial markers showing variable sensitivity and specificity in the differential diagnosis of epithelioid MM and metastatic lung ADC (Table 6) are available. In order to provide reliable information, an immunohistochemical panel instead of 1 or 2 markers should be used. The panel usually includes 2 or more markers that are frequently expressed in mesothelium and not in carcinomas, and 2 or more markers that are frequently expressed in carcinomas and not in mesothelium. Calretinin has been one of the primary mesothelial markers recommended in various immunochemical panels because it is expressed in all histologic types of mesotheliomas. 91 As indicated by Ordóñez 91,92 in his comprehensive studies and recent review, calretinin is expressed in nearly all epithelioid mesotheliomas when polyclonal antibodies against recombinant human calretinin are used. The immunoreactions are usually strong and diffuse in both nuclei and cytoplasm. The lower sensitivities reported by other studies are most likely due to the type of antibody used. 93,94 Mesothelin also exhibits 100% sensitivity in staining epithelioid mesothelioma despite its low specificity. 94,95 Therefore, negative staining for calretinin and/or mesothelin should be regarded as a strong indication against the diagnosis of epithelioid mesothelioma. Cytokeratin 5/6 is another commonly used mesothelial marker, with reported positivity in epithelioid mesothelioma ranging from 65% to 100% in different studies. 94,96,97 Podoplanin is a recently recognized mesothelial marker that shows a high sensitivity and specificity for epithelioid mesotheliomas 91,98 (Figure 6, a through c). Several studies have reported that it is expressed in about 86% to 100% of epithelioid mesothelioma and 57% of sarcomatoid mesotheliomas. 94,99,100 All mesothelial markers also exhibit variable immunoreactions to different carcinomas. A number of studies demonstrate that calretinin positivity can be seen in 6% to 23% of lung ADCs, 94,96 15% to 74% of breast carcinomas, 101,102 23% to 40% of SCCs, 93,103 41% to 49% of SCLCs of lung, and 0% to 10% of renal cell carcinomas. 100,104 Podoplanin appears to be one of the most specific mesothelial markers in differentiating epithelioid mesothelioma from metastatic ADC. 98,100 Several publications have reported that it showed no immunostaining in lung ADCs 97,99,105 but immunoreactivity was demonstrated in 50% of 30 cases of SCC of the lung in 1 study. 103 Other mesothelial markers show lower sensitivities and specificities in diagnosing MM than the markers discussed above. However, they are still useful when additional mesothelial markers are needed in an immunohistochemical panel. In Table 6, several epithelial markers commonly used in the differentiation of mesothelioma and metastatic carcinomas are included. Among them, MOC31 has been demonstrated to be one of the best epithelial markers expressed in a broad spectrum of carcinomas. It is positive in 86% to 100% of lung ADCs, 94,106 97% to 100% of breast Arch Pathol Lab Med Vol 138, December 2014 Immunohistochemistry in Pulmonary and Mediastinum Zhang et al 1621

12 Figure 7. a, Metastatic lung exhibiting positivity for MOC31. b, Squamous cell carcinoma (SCC) exhibiting positivity for Ber-EP4 (immunohistochemistry, original magnification 340 [a and b]). Figure 8. a, Metastatic breast carcinoma showing nuclear staining for GATA3. b, Metastatic colonic adenocarcinoma showing nuclear staining for SATB2 (immunohistochemistry, original magnification 340 [a and b]). carcinomas, % to 100% of SCCs of lung, 102 and 100% of pancreas and colon carcinomas; 106,107 it stains 2% to 15% of epithelioid mesotheliomas in small areas and sparse cells. 94 Epithelial cell adhesion molecule antibody (Ber-Ep4) is another popular marker used in this situation that shows 91% to 100% positivity for ADC of lung, 74% to 100% positivity for SCC, and 13% to 26% positivity for epithelioid mesothelioma 99,108,109 (Figure 7, a and b). Carcinoembryonic antigen (both polyclonal and monoclonal) and B72.3 are 2 epithelial markers that are usually negative in epithelioid mesothelioma but are expressed in 50% to 90% and 75% to 85% of ADCs of the lung, respectively. 94,96,103,104 Claudin-4, a transmembrane protein in tight junctions, is a newly emerging epithelial marker reported to have high sensitivity and specificity in the differential diagnosis of epithelioid mesothelioma and carcinomas In a recent study published by Ordóñez, 111 none of 60 mesotheliomas (40 epithelioid, 10 biphasic, 10 sarcomatoid) were positive for claudin-4; however, its positivity was demonstrated in all lung ADCs (100% of 25 cases), most SCCs of the lung (95%; 19 cases), and all breast carcinomas (100%; 7 cases). The data indicate that claudin-4 appears to be an excellent epithelial marker that can be included in an immunohistochemistry panel for the diagnosis and exclusion of mesotheliomas. In addition to including generalized epithelial markers in the immunohistochemical panel, some organ-specific markers can be added if histologic features and clinical history indicate a possible primary. Many organ-associated immunomarkers are very specific and show no expression in epithelioid mesothelioma. Distinction between mesothelioma and metastatic renal cell carcinoma to the pleura can be difficult, in part because mesothelioma can express renalrelated markers such as renal cell carcinoma marker, CD10, and EMA. 92,104,112,113 Recently recognized renal cell carcinoma markers PAX8 and PAX2 have been reported to be absent in pleural epithelioid mesothelioma, indicating their great usefulness in confirming metastatic renal cell carcinoma involving the pleura In Table 7, several immunohistochemical panels are suggested for the differentiation of epithelioid mesothelioma and commonly encountered metastatic carcinomas involving the pleura Arch Pathol Lab Med Vol 138, December 2014 Immunohistochemistry in Pulmonary and Mediastinum Zhang et al