Immunohistology of Pulmonary and Pleural Neoplasms

Primary Pulmonary Adenocarcinoma

Whether one follows a specific classification, the reality is that T1N0M0 tumors are currently treated with surgery alone. The term “in situ adenocarcinoma” defines a tumor that exists only when the tumor is T1N0M0; if the same histology is associated with a tumor that is not within these parameters, then the tumor is designated as adenocarcinoma, irrespective of whether it shows the same histology. In any case, the use of IHC remains valid for those tumors whether one wants to classify them as in situ adenocarcinoma, bronchioloalveolar carcinomas, or well-differentiated adenocarcinoma with bronchioloalveolar or lepidic growth pattern. These entities will show a similar immunophenotype, which should not be used to promote a specific classification schema.

• Bronchioloalveolar carcinoma—in situ adenocarcinoma—well-differentiated adenocarcinoma

• A tumor with a lepidic or bronchioloalveolar growth pattern, ranging in size from 0.5 to 3.0 cm in greatest diameter, without pleural, interstitial, or lymphatic invasion—essentially T1N0M0 ( Fig. 12.4A and B ).

  

• Similar histology in a lesion that is 0.5 cm or less will be regarded as adenomatous hyperplasia.

• Similar histology of a lesion that is more than 3.0 cm is considered adenocarcinoma.

The immunophenotype of these tumors for the most part will be similar. Essentially, the tumors show positive staining for thyroid transcription factor 1 (TTF-1), napsin A, and keratin 7. Needless to say, because a tumor has a bronchioloalveolar pattern does not mean that the tumor is a primary lung adenocarcinoma, as metastatic adenocarcinomas from extrathoracic origin may also show similar growth pattern. In this setting, the use of a wider panel of IHC is not only advisable but is also warranted.

It is important to be mindful that even though lung carcinoma is among the most common malignancies worldwide, lung is also one of the most common sites for metastatic disease, and in many cases, patients debut with metastatic disease before the primary tumor becomes clinically apparent. Thus, it is always important to keep in mind the possibility of metastatic disease even in cases in which the histology of the tumor may suggest primary disease. Therefore, it is important to recognize that not all adenocarcinomas will show the same IHC profile, and the distinction among those different growth patterns is crucial in order to have an idea of the differences that may exist.

To provide more insights into the daily practice and the pitfalls and shortcomings of the diagnosis of primary pulmonary adenocarcinoma, it is essential that we make an attempt to separate the pulmonary adenocarcinomas by the growth pattern that they may show, as it will largely lead the IHC panel that one is likely to request. Importantly, there are many carcinoma epitopes that can aid in the diagnosis of adenocarcinoma, but most of them are used in the broad sense, leaving only a few that may help in providing a site of origin. It is in this setting in which one will make the determination of the type of IHC studies to use.

Adenocarcinomas are commonly grouped by a grading system, which, although not universally agreed upon, is commonly used to provide a status of the growth pattern that these tumors will show ( Fig. 12.5A and B ).

• Well-differentiated adenocarcinoma —a tumor that at low-power magnification shows an atypical glandular proliferation replacing normal lung parenchyma. The glands are formed of columnar or mucinous type of epithelium, round to oval cells, ample cytoplasm, round nuclei, and prominent nucleoli. Although mitotic activity is present, it is not marked.

• Moderately differentiated adenocarcinoma —similar features as the well-differentiated counterpart; however, in this tumor the glands vary in size and shape and mitotic activity is more easily identifiable.

• Poorly differentiated adenocarcinoma —contrary to the previous tumors, this adenocarcinoma characteristically shows solid areas with areas of abortive glandular structures. In addition, the tumor shows more nuclear atypia and mitotic activity. Areas of necrosis can be present.

• The immunophenotype of these tumors as primary lung adenocarcinoma will be similar to that previously stated, namely positive nuclear staining for TTF-1, and cytoplasmic staining for napsin A and keratin 7 ( Fig. 12.6A and B ; Table 12.1 ). Having this particular immunophenotype in a patient who radiologically presents with an intrapulmonary mass without any other pertinent clinical history will likely lead to the interpretation of a primary lung adenocarcinoma. It is important to highlight, however, that the current IHC diagnosis of pulmonary adenocarcinoma is heavily supported by the presence of positive staining for TTF-1, napsin, and cytokeratin 7. In a recent study by Fiala and colleagues on the expression of TTF-1 and its association with outcome of patients with nonsquamous non–small cell carcinoma, the authors retrospectively analyzed 463 patients with advanced lung carcinoma and identified 76% of the tumors to express TTF1 and concluded that such expression is associated with better outcome. In addition, Yoshimura and colleagues, in a study to determine prognostic roles and heterogeneity of TTF-1 copy number and TTF-1 protein expression in non–small cell lung cancer (NSCLC), found that TTF-1 expression was identified in 85% of adenocarcinomas. In this study, the authors reviewed 423 adenocarcinomas and 171 squamous cell carcinomas. TTF-1 copy number alterations were observed in 23% of adenocarcinomas and in 20% of squamous cell carcinomas. However, despite the strong association between positive nuclear staining of tumor cell for TTF-1 in lung adenocarcinomas, it is also important to note that TTF-1 may also show positive staining in other extrathoracic tumors including tumors of the gastrointestinal, genitourinary, gynecologic, thyroid, and even central nervous system. On the other hand, as napsin A is considered to be rather specific for lung adenocarcinoma, this antibody can also be expressed in other extrapulmonary carcinomas, including tumors of genitourinary origin. Therefore, if a clinical history is provided or the histologic features of the tumor raise any suspicion about the possibility of metastatic disease, it is incumbent on the pathologist to widen the IHC study panel. In the setting of possible metastatic disease, taking into consideration the most common metastatic diseases, we recommend the following ( Table 12.2 ):

  
  TABLE 12.1

  Commonly Used Immunohistochemical Markers in the Diagnosis of Lung Adenocarcinoma and Squamous Carcinoma

  	TTF-1	Napsin A	Ker7	Ker 20	P63	Ker 5/6	P40
  Adenocarcinoma
  Conventional	+	+	+	−/+	−/+	−	−
  Colloid carcinoma	+/−	−	+	+	−	−	−
  Signet ring cell	+/−	+/−	+	−	−	−	−
  Papillary	+	+	+	−	−/+	−	−
  Squamous Carcinoma	−	−	−/+	−	+	+	+

  TTF-1 , Thyroid transcription factor 1.

  TABLE 12.2

  Commonly Used Immunohistochemical Markers in Primary Lung Versus Metastatic Carcinoma

  	TTF-1	Napsin A	GATA-3	CDX2	PAX8	NKX-3	Uroplakin	GCDFP1	Thyroglobulin
  Lung Adenocarcinoma
  Conventional	+	+	−	+	−	−	−	−	−
  Colloid carcinoma	±	−	−	±	−	−	−	−	−
  Signet ring cell	±	+/−	−	±	−	−	−	−	−
  Papillary	+	+	−	−	−	−	−	−	−
  Metastatic Carcinoma
  G.I. origin	−	−	−	+	−	−	−	−	−
  GYN origin	±	−	−	±	+	−	−	−	+
  G.U. origin	−	±	+	−	+	+	+	−	−
  Breast origin	−	−	+	−	−	−	−	+	−
  Thyroid origin	+	±	−	−	+	−	−	−	+
  	P16	P40	Ker 5/6	P63
  Lung squamous carcinoma	−	+	+	+
  Metastatic squamous carcinoma	±	+	+	+

  TTF-1 , Thyroid transcription factor 1.

• Metastatic adenocarcinoma of gastrointestinal origin : the use of TTF-1, napsin A, and keratin 7 should also be accompanied by keratin 20 and CDX2/ SATB2. The presence of strong positivity for keratin 20 and nuclear staining for CDX2/SATB2 in the absence of positive staining for TTF-1 and napsin A should lead to the interpretation of metastatic disease. However, it is important to highlight that adenocarcinomas of the upper digestive tract may show positive staining for both keratin 7 and keratin 20. In addition, CDX2 is also commonly positive in primary mucinous adenocarcinomas of the lung ( Fig. 12.7A–D ).

  

• Metastatic adenocarcinoma of gynecologic origin : it is known that TTF-1 may show positive staining in about 10% of endometrial adenocarcinomas. Therefore, in this setting, the use of PAX8 may be of great importance, as positive nuclear staining for PAX8 essentially rules out any primary lung carcinoma and would lead to the interpretation of metastatic disease from a carcinoma of müllerian origin ( Fig. 12.8A–C ).

  

• Metastatic adenocarcinoma of genitourinary origin : in this particular setting, the use of additional stains such as GATA-3, PAX8, uroplakin, and NKX3 should be of help in identifying a possible site of origin in the kidney, bladder, or prostate ( Fig. 12.9A–C ).

  

• Metastatic adenocarcinoma of breast origin : breast carcinoma ( Fig. 12.10A–C ) shares with lung adenocarcinoma the strong positive staining for keratin 7; however, the majority of breast carcinomas are negative for TTF-1 (exception is primary breast small cell carcinoma) and positive for GATA-3. The use of keratin 5/6 in defining a specific site of origin between breast and lung adenocarcinoma should be done in the context of a good clinical and histologic evaluation of the tumor, as primary lung adenocarcinoma may show positive staining for keratin 5/6, whereas the majority of squamous cell carcinomas of the lung are positive for keratin 5/6. Other stains that could be of aid in the differential between lung and breast primary would include gross cystic disease fluid protein (GCDFP), mammaglobin, and estrogen and progesterone receptors (ER/PR). Positive staining for the former two stains GCDFP and mammaglobin would lead to the interpretation of breast primary. However, the ER and PR may be positive in lung adenocarcinoma. Raso and colleagues reported a study of 317 cases of non–small cell lung carcinoma and the immunohistochemical expression of ER and PR. The authors found that ER α and β could be expressed in lung carcinoma (nuclear staining) in as many as 56% of the cases (range, 5% to 56%). Also, the expression of PR was seen in 63% of the cases. The ER-α nuclear expression was correlated with adenocarcinoma histology, female sex, and history of never smoking.

  

Variants of Primary Adenocarcinoma of the Lung

Mucin-Rich, “Colloid” Carcinoma

Historically, there has been considerable debate about not only the name of this particular tumor but also the malignant potential. Most of the controversy regarding this tumor is in part due to initial descriptions of mucinous lesions in the lung under the designation of mucinous cystadenoma, multilocular cystic carcinoma, mucinous cystic tumor, adenocarcinoma arising in mucinous cystadenoma, and pulmonary mucinous cystic tumors of borderline malignancy, which represent the same entity described as colloid carcinoma of the lung. The macroscopic and histologic features of all of these descriptions are similar; however, it is important to highlight that the tumor is a malignant neoplasm of low malignant potential. The most salient features of these tumors and the specific histologic and molecular features have been recently reported.

Histologically, colloid carcinoma, as its name implies, represents a mucin-rich adenocarcinoma in which the tumor shows extensive pools of mucinous material and only focal areas in which the alveolar lining is replaced by mucin-rich epithelium ( Fig. 12.11A–G ). In some cases, focal solid areas of adenocarcinoma may also be present. However, the hallmark of the tumor is the extensive mucin deposition. The immunohistochemical features of these tumors are somewhat different from the conventional adenocarcinomas, as this particular variant of adenocarcinoma may show positive staining for CDX2 as well as weak positive staining for TTF-1. In addition, keratin 7 is positive and keratin 20 may also be positive. In the report by Zenali and colleagues, the authors found that the tumor expressed positive staining in 13 cases evaluated for keratin 7, keratin 20, and CDX2, whereas staining for TTF-1 was present in about 50% of the cases. Interestingly, napsin A was negative in all cases studied and surfactant apoprotein was positive in only one case. Molecular analysis of the cases reported showed KRAS in two, whereas none of the cases analyzed showed epidermal growth factor receptor EGFR or EML4-ALK mutations. Based on the IHC profile and molecular findings, this particular variant of pulmonary adenocarcinoma can be easily confused with a primary mucinous carcinoma of colorectal origin. Therefore it is important to properly correlate not only the histologic, IHC, and molecular features of these tumors, but also to properly analyze the clinical setting in which the tumor occurs.

Because there are other tumors that may share similar histopathologic features as colloid carcinoma, it is important to properly exclude tumors such as mucinous carcinoma of colorectal origin, mucinous carcinoma of breast origin, and mucinous carcinoma of the bladder (urachal carcinoma). These tumors potentially can pose serious difficulties in separating them from pulmonary colloid carcinoma. In this setting, the use of a more extensive panel of IHC, such as mammaglobin, GCDFP, GATA-3, PAX8, and uroplakin, can provide some aid in arriving at a proper interpretation.

Signet-Ring Cell Adenocarcinoma

The signet ring feature is commonly associated with tumors of gastrointestinal origin. However, this variant of primary lung adenocarcinomas is well recognized. The tumor may show an acinar or solid growth pattern, which can be arranged in a subtle nested pattern and composed of medium-sized cells with clear cytoplasm, and nuclei displaced toward the periphery of the cells, imparting the so-called signet-ring cell appearance ( Fig. 12.12A–C ). Marked nuclear atypia and increased mitotic activity are not common in these tumors. Histochemical stains for mucicarmine and periodic acid–Schiff (PAS), with or without diastase, will show the presence of intracellular and extracellular mucin in these tumors. Immunohistochemical studies performed in these tumors have shown that MUC-1 and MUC-2 may show positive staining in tumor cells. Castro and colleagues, in their report of signet ring cell adenocarcinomas of the lung, reported TTF-1 positive in 100% of their cases, whereas keratin 7 was positive in about 50% and keratin 20 was negative. Once again, due to the histologic similarity with tumors of gastrointestinal origin, a wider IHC profile including CDX2, GATA-3, GCDFP may help in properly determining primary site for these tumors.

Papillary Adenocarcinoma

This variant of primary pulmonary adenocarcinoma is not very common and may represent a challenge in diagnosis mainly for separating the tumor from metastatic thyroid carcinoma. Silver and Askin defined this particular variant as one that requires at least 75% of papillary histology to be considered “true” papillary carcinoma. A variant of this growth pattern, such as the micropapillary variant, has also been described as primary lung carcinoma.

Histologically, papillary adenocarcinoma is characterized by the presence of papillary cords of different sizes, with a delicate fibrovascular core. The neoplastic cells may show clearing of the cytoplasm and grooving similar to the features described in papillary thyroid carcinoma ( Fig. 12.13A and B ). Mitotic activity and nuclear pleomorphism are not common in these tumors. Interestingly, primary papillary carcinomas of the lung may also show the presence of psammoma bodies, which adds another element to the challenge in properly designating these tumors as primary lung carcinomas. Immunohistochemically, the tumor is positive for TTF-1, napsin A, and keratin 7, but is negative for thyroglobulin and PAX8, which, if positive, would lead to the diagnosis of metastatic disease from thyroid ( Fig. 12.14A–E ).

Primary Mammary-Like Carcinoma

This is an unusual primary lung carcinoma recently described by Lindholm as a single case report in an adult male individual. The tumor is composed of a homogeneous cellular proliferation of small to medium-size cells with round nuclei and inconspicuous nucleoli. By IHC the tumor shows strong positive staining for ER, PR, and GATA3 while negative for other pneumocytic or squamous markers ( Fig. 12.15 ). In addition, by molecular means the tumor shows a somatic mutation in ARID1A while negative for EGFR. The importance of this report is to highlight the existence of primary lung carcinomas that may show similar IHC features as a breast carcinoma, but yet, the tumor is of pulmonary origin. In addition, as it is stated in this particular report, the tumor in question was in a male patient without prior or current breast neoplasm.

Squamous Cell Carcinoma

Even though there may not be a universal grading system for squamous cell carcinomas, one usually designates a degree of differentiation to these tumors as a way of predicting aggressiveness.

In general, primary squamous cell carcinoma of the lung may present in different ways, either obstructing the lumen of the airway in a polypoid fashion or as an intraparenchymal mass that may extend into the pleura and chest wall soft tissues. In both of these presentations, the basic histopathologic features of the tumor do not change, but the staging of the tumor is different.

Histologically, squamous cell carcinomas can be separated into three main categories:

• Well-differentiated squamous cell carcinoma —keratinization is easily identified as well as the presence of intercellular bridges. The tumor may show different growth patterns including sheets of neoplastic cells composed of medium-sized cells, moderate amount of eosinophilic cytoplasm, small nuclei, and inconspicuous nucleoli. Mitotic activity and cellular pleomorphism is variable. However, areas of necrosis and hemorrhage are absent or only focal.

• Moderately differentiated squamous cell carcinoma —the tumor may show areas of keratinization admixed with nonkeratinizing areas alternating with areas of hemorrhage or necrosis. Mitotic activity and cellular pleomorphism are present.

• Poorly differentiated squamous cell carcinoma —solid growth of sheets of neoplastic cells in which the tumor does not display keratinization and intercellular bridges are not easily identified. Mitotic activity and cellular pleomorphism are easily identified, which may be associated with areas of hemorrhage and/or necrosis. In some cases, focal areas of more conventional features of squamous carcinoma may be seen.

Squamous cell carcinomas, regardless of the anatomic site, may not only show similar histologic features but also show similar IHC profiles. The challenge is in providing a primary site of origin, mainly in those cases in which there is previous history of squamous cell carcinoma. Thoracic pathologists often rely on the presence of multiple bilateral pulmonary nodules to determine whether a tumor is more likely to be primary or metastatic; however, such a finding is not present in many cases in which the patient has a history of extrathoracic squamous cell carcinoma.

For poorly differentiated tumors, the confirmation of the diagnosis of squamous cell carcinoma may include the use of p63, keratin 5/6, and p40 ( Fig. 12.16A–D ). The use of broad-spectrum keratin and keratin 7 is not completely reliable as other carcinomas may show positive staining for these markers. However, it is important to highlight that some of those markers, which at one time were regarded as specific for squamous cell carcinomas, have also been described as positive in adenocarcinomas. For instance, p63, although useful in the diagnosis of squamous cell carcinoma, also immunostains approximately one third of adenocarcinomas, limiting its diagnostic value when one is dealing with small biopsies. Similarly, keratin 5/6, an important marker for squamous cell carcinoma, also decorates a subset of adenocarcinomas and pleural mesotheliomas; p40 is more specific for squamous cell carcinoma. In our experience, we have observed only a minority of cases with morphology of adenocarcinoma and TTF-1 positivity that have also expressed p40. The series presented by Bishop and colleagues studied 81 squamous cell carcinomas, 237 adenocarcinomas, and 152 large cell carcinoma and identified only three cases of adenocarcinoma that also expressed p40, whereas 31% of adenocarcinomas expressed p63. Also, Tatsumori and colleagues reported their experience with p40 immunostain and compared it with adenocarcinomas and neuroendocrine carcinomas. In their report 96.8% of squamous cell carcinomas stained for p40, whereas 4.6% of adenocarcinomas also expressed p40. The authors reported that 3.6% of large cell neuroendocrine carcinomas, 1.5% of small cell carcinomas, and 2.4% of mesotheliomas also expressed positive nuclear staining for p40. Contrary to the percentages provided in these two studies on p40, Setodeh and colleagues reported a higher incidence of adenocarcinomas positive for p40, at 18%.

However, one important challenge is to determine primary site in those cases in which there is previous history of extrapulmonary squamous cell carcinoma, or in cases in which there are mimickers of squamous cell carcinomas that may also show similar immunophenotype. Head and neck squamous cancers metastatic in the lung may be distinguished by their strong P16 nuclear/cytoplasmic phenotype; however, studies on p16 in primary pulmonary squamous cell carcinomas have shown that approximately 25% of these tumors may also show positive staining for p16 ( Fig. 12.17A and B ). In this situation, further testing with in situ hybridization probes for HPV would be prudent to make the diagnostic distinction.

Commonly Used Immunohistochemical Biomarkers in Non–Small Cell Carcinoma and Molecular Biology

Even though there are many potential biomarkers that can be tested by IHC, there are only a few that have the potential to define the course of treatment that oncologists may use to determine the best possible treatment options. In practice, the immunohistochemical analysis of non–small cell carcinomas with cellular-mesenchymal-epithelial transition (c-MET), BRAF, and programmed death-ligand 1 (PD-L1) are probably the most commonly requested and useful biomarkers that currently play an important role in selecting treatment options. Below we will discuss each one of these biomarkers and their use in clinical practice.

c-MET is a protein that has recently been identified as a novel target in non–small cell carcinoma ( Fig. 12.18 ). Pyo and colleagues evaluated the significance and concordance of the expression of c-MET in non–small cell carcinoma in a meta-analysis of 4454 cases. The authors identified that c-MET immunostaining was high in nonsquamous cell carcinomas and tumors in stages III and IV. The authors did not encounter any correlations between positive results and sex, smoking history, and lymph node metastasis. In addition, the positivity for c-MET was significantly correlated with poor overall survival. The authors concluded that c-MET IHC could be useful for screening of c-MET genetic alteration in non–small cell carcinoma. In a different study by Park and colleagues, the authors evaluated the MET protein expression by IHC and MET amplification by fluorescence in situ hybridization (FISH) in 316 surgically resected lung adenocarcinomas. The tumors were divided into four different groups depending on the positive and negative MET IHC and FISH positivity or negativity, and then 15 to 20 tumors of each category were randomly selected for mutation analyses. The authors identified that MET amplification was significantly associated with IHC score, thus concluding that there is a significant relationship between MET amplification and protein expression, and selection of tumors with amplification using IHC is effective. However, Li and colleagues in a review of 158 patients with advance non–small cell carcinoma, observed no significant difference in overall survival between MET-positive and -negative patients, concluding that c-MET is not a predictive or prognostic factor for stage IV non–small cell lung carcinoma. In addition, Watermann and colleagues evaluated 222 tumors using microarray IHC and observed no significant association between c-MET amplification, c-MET protein expression, and phosphorylation. Therefore, the authors concluded that neither expression of c-MET nor gene amplification status might be the best way to select patients for MET targeting therapies.

BRAF is also another immunostain that is commonly requested in the panel of biomarkers for non–small cell lung carcinoma. There appear to be two different monoclonal antibodies available for the detection of BRAF V600E mutation. Routhier and colleagues evaluated the sensitivity and specificity of these monoclonal antibodies in 152 tumors including 25 lung carcinomas. The authors concluded that IHC with monoclonal VE1 has better performance compared with the anti-BRAF in an automated staining platform. In that regard, Marchetti and colleagues documented in a study of 1046 cases of non–small cell lung carcinoma that BRAF mutations are present in about 4.9% of adenocarcinomas and 0.3% of squamous cell carcinomas. Interestingly, the authors observed that V600E-mutated tumors showed an aggressive histotype characterized by micropapillary features in 80% of cases, which were also associated with shorter disease-free and overall survival rates. All non-V600E mutations were found in smokers and were not associated with prognosis. In a comparative study between KRAS and BRAF by IHC and genotyping, Piton and colleagues observed that BRAF had 100% specificity and sensitivity in detecting V600E mutations. Also Hayashi and colleagues, in a study on ovarian serous borderline tumors, found high concordance between CAST-PCR and IHC using VE1 clone and FLEX linker and concluded that it is a specific method for the detection of BRAF V600E and may be an alternative to molecular-biological techniques for the detection of mutations.

More recently, PD-L1 expression has become an important tool in the evaluation of lung carcinoma for treatment purposes ( Fig. 12.19 ). PD-L1 expression in tumor and immune cells is believed to be induced by Th1 cytokine released by infiltrating tumor immune cells, thus treatments directed to target that pathway appear to offer an alternative treatment for patients with non–small cell carcinoma. When using the PD-L1, it is important to know the antibody characteristics and the percentage of positive membranous staining. The reading of positive or negative is not useful for PD-L1. The minimum number of tumor cells present for performing this immunostain has been stated to be at least 100 cells so that a percentage of positive membranous staining can be provided. Cooper and colleagues evaluated the expression of PD-L1 by IHC in 678 cases of non–small cell carcinoma stages I to III and 52 paired nodal metastases using tissue microarrays. In this study, tumors showing at least 50% of cells with membranous staining of any intensity were considered PD-L1 positive. In addition, the authors identified that PD-L1 expression of any intensity was present in 32.8% of cases, with high expression in 7.4%. PD-L1 expression was observed in squamous cell carcinoma, large cell carcinoma, and adenocarcinoma in 7.4%, 12.1%, and 5.1%, respectively. High expression was associated with younger patient’s age and high tumor grade. No sex, tumor size, stage, nodal status, or EGFR or KRAS mutation status was correlated with the PD-L1 expression. The authors concluded that PD-L1 is expressed at high levels in a significant proportion of non–small cell lung carcinomas and appears to be a favorable prognostic factor in early-stage disease. However, the authors also warned about the issue that their study used tissue microarrays instead of whole tissue sections.

The study by Scheel and collegues was a review of inter-laboratory agreement of PD-L1 IHC for NSCLC. The fundamental conclusion of the paper is that reproducibility among multiple sites is excellent for previously described staining patterns of the assays, as well as the use of the recommended current clinical cutoffs for interpretation.

Scheel and colleagues compared the IVD assays DAKO 22C3 pharmDx, DAKO 28-8 pharmDx, Ventana Roche SP263, and Ventana Roche SP142, interpreted according to the manufacturer’s package insert. For agreement purposes, the analysis included previously described categorical scoring systems based on six steps: less than 1%, 1% to 4%, 5% to 9%, 10% to 24%, 25% to 49%, and greater than or equal to 50%. The clinical cutoffs for clinical relevance of greater than or equal to 1% and greater than or equal to 50% were also compared. Regardless of the scoring method, the agreement among observers and sites was very good to excellent.

Currently, there are three checkpoint inhibitors that are FDA-approved for NSCLC treatment. For second-line treatment, nivolumab and atezolizumab are approved with PD-L1 IHC, and pembrolizumab is approved for PD-L1–positive carcinomas defined as greater than or equal to 1% tumor cells with membrane staining with DAKO 22C3 pharmDx assay. For first-line treatment, pembrolizumab is approved for carcinomas defined as greater than 50% of tumor cells staining. Each assay that exists is a predictive test for different anti-PD1 or anti-PD-L1 antibody and therefore has a different purpose. ^, Currently, the 23C3 pharmDx is a mandatory predictive test, whereas assays 28-8 and SP142 are optional tests.

Scheel and colleagues found that immune cell scoring was somewhat more difficult, with half of the testing sites using SP142 finding concordant scores with a moderate ϰ coefficient for the greater than or equal to 1% cutoff. The work by Scheel, and Rimm and colleagues suggests that standardization of PD-L1 immunostaining and scoring is achievable and likely will lead to incorporation as a comprehensive panel for predictive therapeutic purposes.

ROS1 and ALK have become additional immunohistochemical tests that appear to be helpful in selecting therapies for patients with lung carcinoma. The IHC for both of these markers may be viewed as a screening tool, as it is generally agreed that molecular analysis by FISH remains the gold standard. Nevertheless, in cases in which the molecular technique is not readily available, the use of IHC may provide important information. In that regard, Huang et al. evaluated 122 non–small cell lung carcinomas in which there were both ROS1 IHC stains and FISH analysis. The authors used a quantitative method in which tumors with cytoplasmic staining of 2+ or above in 30% of the tumor cells had a better correlation with FISH positive results in 97% of the cases evaluated.

More recently, another IHC approach is to evaluate non–small cell carcinomas for microsatellite instability (MSI). High MSI is commonly associated in patients with Lynch syndrome; however, a study by Bonneville et al. identified that MSI may affect non-Lynch syndrome tumor types, and in their study mesothelioma was one of those neoplasms. The exact incidence of MSI in lung non–small cell carcinomas is likely low.

Regarding molecular phenotype in lung carcinoma, recent advances have suggested the possibility of specific gene mutations as prognostic indicators. Thus, evaluation of different pathways including EGFR, ALK, ROS1, K-RAS, BRAF, AKT1, PIK3CA, and HER2 are currently used in the evaluation of lung cancer. One important issue regarding the molecular analysis of lung carcinoma is to determine whether the primary lung carcinoma or the metastatic deposit is the best tissue to test. Bittar and colleagues, in a small study of ALK FISH, concluded that ALK FISH results show more differences between primary and metastatic deposits than ALK IHC.

Although the vast majority of molecular mutations are associated with adenocarcinomas of different histologies, a small percentage of squamous cell carcinomas may also show some of the mutations that are commonly associated with adenocarcinomas.

Large Cell Carcinoma, Pleomorphic Carcinoma, Sarcomatoid Carcinoma, and Giant Cell Carcinoma

Although all of these tumors histologically may appear to show some histopathologic differences, more recent developments suggest similarities (see Figs. 12.1–12.3, 12.20 ). Therefore, these tumors will be discussed as a group.

Sarcomatoid Carcinoma, Pleomorphic Carcinoma, and Giant Cell Carcinoma

The term “sarcomatoid carcinoma” historically has been used to refer to spindle cell carcinomas—that is, carcinomas that have a “sarcoma-like” spindle cell morphology. Most of these cases can be categorized among the conventional subtypes of carcinoma. In addition, even if one cannot further define some of these tumors with spindle cell morphology, the reality is that those tumors in particular may also represent a histologic variant of large cell carcinomas.

The term “pleomorphic carcinoma” has been used as a synonym for tumors similar to spindle cell carcinoma. ^, Introduced in 1994 as a description of 78 tumors with spindle cell and giant cell components, 45% of the cases showed histologic evidence of adenocarcinoma and 8% histologic evidence of squamous cell carcinoma. However, by current standards, adequate IHC analysis was not performed. More recently, we have evaluated the IHC profile of 86 cases that had been diagnosed either as spindle cell carcinomas, sarcomatoid carcinoma, or pleomorphic carcinoma with the concept of identifying specific differentiation within the spindle cell component. The study included a wide panel of antibodies, including CAM5.2, keratin 7, TTF-1, napsin A, keratin 5/6, p40, desmocollin 3, Sox2, calretinin, and D2-40. The spindle cell component in these tumors was positive for TTF-1 in 41% of the cases, napsin A was present in 20%, keratin 5/6 in 9%, p40 in 8%, and desmocollin in 3%. Forty-two percent of the tumors initially labeled as sarcomatoid carcinoma were adenocarcinomas, whereas 14% were squamous carcinoma. The authors further proposed abandonment of the term “sarcomatoid” carcinoma, which is not helpful for targeted treatment purposes. Flow charts for the interpretation and proper classification of non–small cell carcinomas, and more specifically those with spindle cell component, are presented (see Figs. 12.1–12.3 ).

Once the spindle cell component of these tumors is identified, then the other component of giant cells also needs to be properly identified. There are essentially four types of giant cells that may be seen in carcinomas of the lung: (1) pneumocytic type, (2) syncytiotrophoblastic type, (3) null cell type, and (4) osteoclast-like type. Though rare, tumors composed exclusively of giant cells can be seen as primary lung carcinomas. Immunohistochemical analysis of these cases showed that some giant cells have pneumocytic derivation, others syncytiotrophoblastic phenotype, and some lack differentiation except for positive staining for keratin antibodies. These last cells characteristically show emperipolesis of neutrophils, which has led to naming these giant cells as emperipoletic or “null” cell type.

Based on the available findings, we can establish a link, not only between spindle cell neoplasms with or without morphologic differentiation toward adenocarcinoma or squamous cell carcinoma, but in addition, we can also properly categorize the giant cell component of these tumors (see Fig. 12.24 ) and clearly separate all carcinomas into essentially three main categories: adenocarcinoma, squamous cell carcinoma, and large cell carcinoma.

Berg and Churg, using GATA-3 IHC, recently studied differences in immunostaining between 13 spindle cell carcinomas and 19 desmoplastic mesotheliomas. Their results suggest that the desmoplastic mesotheliomas are typically strongly positive for nuclear GATA-3, whereas the carcinomas are mostly negative or rarely focal/weak with GATA-3. Larger studies will be needed to confirm this finding as noted previously.

Key Diagnostic Points: Non–Small Cell Lung Cancer With Spindle and/or Giant Cell Components

• Adenocarcinoma, spindle cell type:

  • Tumors with morphologic evidence of adenocarcinoma and IHC evidence of positive pneumocytic markers (TTF-1 and/or napsin A) in the spindle cell component ( Fig. 12.21A–D )Or

  • Tumors composed exclusively of spindle cells and/or pleomorphic elements with immunohistochemical evidence of positive pneumocytic markers (TTF-1 and/or napsin A).

• Adenocarcinoma, dedifferentiated type:

  • Tumors with morphologic evidence of adenocarcinoma but negative pneumocytic markers (TTF-1 and napsin A) in the spindle cell component (keratin positive spindle cells).

• Squamous cell carcinoma, spindle cell type:

  • Tumors with morphologic evidence of squamous cell carcinoma and IHC evidence of positive squamous markers (p40 and/or keratin 5/6) in the spindle cell component ( Fig. 12.22A–C )Or

  • Tumors composed entirely of spindle cells and/or pleomorphic elements positive for squamous markers (p40 and/or keratin 5/6).

• Squamous cell carcinoma, dedifferentiated type:

  • Tumors with morphologic evidence of squamous cell carcinoma but negative staining for squamous markers (p40 and/or keratin 5/6) in the spindle cell component.

• Large cell carcinoma:

  • Conventional, round/oval cell type: Tumors composed of round or oval cell components (keratin positive) and negative differentiation with pneumocytic or squamous markers ( Fig. 12.23A and B)Or

  • Spindle cell type: Tumors composed entirely of spindle cells (keratin positive) and negative differentiation with pneumocytic and squamous markers ( Fig.12.24A and B ).

• NSCLC with giant cells

  • Giant cells, pneumocytic type ( Fig. 12.25A and B ):

    • Giant cells positive for TTF-1 and/or napsin A.

    

  • Giant cells, syncytiotrophoblastic type ( Fig. 12.26A and B ):

    • Giants cells positive for human chorionic gonadotrophin (HCG).

    

  • Giant cells, null cell type ( Fig. 12.27 ):

    • Phagocytic giant cells positive for keratin and negative for pneumocytic markers and HCG.

    

  • Giant cells, osteoclast type ( Fig. 12.28 ):

    • Giant cells usually positive for CD68 and negative for pneumocytic markers, keratin, and HCG.

    

Regarding giant cell carcinomas Osteoclast type, Lindholm et al. reported three cases under the designation osteoclast-like giant cell–rich carcinomas of the lung. The authors identified that the giant cells showed positive staining for CD68, cathepsin K, and histone H3, while the carcinomatous component was only positive for keratin and TTF-1. Interestingly, molecular results in two cases show that both tumors were negative for EGFR.

Pulmonary Blastoma

The terminology employed to designate this specific tumor in the lung has varied over the last decades, and the different designations mirror the different histologic features of the neoplasm. Terms such as fetal adenocarcinoma, pulmonary embryoma, or variants of carcinosarcoma have been put forward. However, the term pulmonary blastoma was coined in 1961 by Spencer, who found histologic characteristics similar to renal nephroblastoma. Pulmonary blastoma should not be confused with pleuropulmonary blastoma, which is a tumor that generally occurs in the pediatric age group.

As stated earlier, this tumor is found in the adult population, with a median age of 35 years. Clinically, patients may present with different symptomatology depending on the location of the tumor. For those tumors that are centrally located, the patients may present with symptoms of bronchial obstruction such as cough, dyspnea, and hemoptysis, whereas those patients with a peripheral tumor may present with symptoms of weight loss and chest pain.

Histologically, we divide pulmonary blastoma into two different histopathologic growth patterns:

• 1.

  Monophasic pulmonary blastoma ( Fig. 12.29A–C ): this tumor is composed essentially of one component—an epithelial component, which is arranged in a glandular growth pattern with features that resemble the pulmonary development of the lung at 11 to 18 weeks of embryologic development. The tumor may show a prominent glandular, pseudopapillary, or solid growth pattern. The glands are composed of columnar cells with clear cytoplasm and peripherally displaced nuclei. The presence of “morules” associated with the glandular component is a common characteristic of these tumors and can be seen in about 85% of monophasic pulmonary blastomas. Necrosis, increased mitotic activity, or marked nuclear atypia are not commonly seen in these tumors, but they can occur. The presence of those latter features has led some authors to subdivide these tumors into low and high grade. However, it is very important to highlight that monophasic pulmonary blastoma may be associated with another malignancy that is not necessarily of mesenchymal origin, as has also been our experience. Characteristically, the use of histochemical stains such as PAS in these tumors shows that the neoplastic cells are rich in glycogen, while negative for intracellular mucin with D-PAS and/or mucicarmine.

  

• 2.

  Biphasic pulmonary blastoma ( Fig. 12.30A ): the epithelial component is also associated with another spindle cell component of mesenchymal origin. Important to mention is that the epithelial component in the biphasic tumors usually shows more nuclear atypia, mitotic activity, necrosis, and hemorrhage. The mesenchymal component may either lack differentiation or may show areas of cartilage, bone, or skeletal muscle differentiation.

  

Pulmonary Carcinosarcoma

Even though this tumor is often included with pulmonary blastoma among the biphasic tumors of the lung, the histologic features of this tumor are different from those of blastoma. In addition, it is likely that these two biphasic tumors represent two different clinicopathologic entities. Only a few series of carcinosarcomas of the lung have been presented in the literature, which attest to the infrequency of this tumor as primary lung neoplasm. Kika in 1908 is credited for the initial description of this tumor, and ever since, many opinions have been advanced on the nature of this neoplasm. Perhaps one of the stumbling blocks in the proper classification of this tumor has been in the way it has been defined; nevertheless, in our opinion, the only tumors that we classify as carcinosarcomas are those with unequivocal heterologous component in the conventional forms of angiosarcoma, chondrosarcoma, rhabdomyosarcoma, etc. The current nomenclature of the WHO has obscured these tumors as they are lumped among other nonrelated neoplasms such as spindle cell carcinomas. The tumors that will be emphasized in this section are only those with unequivocal heterologous elements.

The tumors appear to be more common in adult men in the seventh decade of life. The symptomatology may vary and will depend on the anatomic location of the tumor. For those tumors centrally located, it is likely the patients will present with symptoms of obstruction, which will include cough, dyspnea, and hemoptysis, whereas if the tumor is peripherally located, the symptomatology may be that of chest pain, as those tumors are also likely to reach a larger size than those centrally located. Important to mention is that some patients are completely asymptomatic.

The histologic features of carcinosarcomas are rather straightforward and should not pose a problem in interpretation if enough material is available for histologic evaluation. The problem is often with small biopsies in which only one component of the tumor is present. Therefore, in the majority of cases, the diagnosis is performed after complete resection of the tumor in which more generous sampling is available for interpretation.

Histologically, the most common epithelial component of pulmonary carcinosarcomas is squamous cell carcinoma. However, adenocarcinoma and adenosquamous carcinoma also have been found to be part of these tumors. On the other hand, the mesenchymal component of these tumors is in the form of rhabdomyosarcoma, chondrosarcoma, osteosarcoma, and angiosarcoma as the most common forms encountered in these neoplasms ( Fig. 12.31A–D ).

The IHC evaluation of these tumors is essentially left to unequivocally identify the mesenchymal component; thus, IHC will be requested based on the morphologic evaluation of the tumor. In cases of osteosarcoma or chondrosarcoma, the morphology of the tumor is sufficient to make the diagnosis on morphology alone. However, in cases of rhabdomyosarcoma or angiosarcoma, the use of vascular markers such as CD34, CD-31, ERG, and D2-40 should be of aid, whereas the use of muscle markers such as caldesmon, desmin, myoglobin, and Myo-D may be of aid in cases of muscle differentiation.

Immunohistochemical Features

The immunophenotype of neuroendocrine carcinomas is characteristically positive for neuroendocrine markers, chromogranin, synaptophysin, and CD56. However, these tumors may also show positive staining for other markers, including TTF-1, keratin 7, and napsin A.

We consider that there are subtle differences in expression of some of these markers. For instance, chromogranin is highly specific but less sensitive, whereas synaptophysin is more sensitive but less specific, similar to CD56. In addition, the expression of any one of these markers in low- and intermediate-grade neuroendocrine carcinoma is strong, whereas such expression in small cell carcinoma is rather weak. Also, the expression of TTF-1 appears to be stronger in small cell carcinoma than it is in lower-grade neuroendocrine carcinomas. TTF-1 and napsin A expression is variable in large cell neuroendocrine carcinoma, which as it has been defined, requires positive staining for a neuroendocrine marker to define the tumor as large cell neuroendocrine carcinoma. Nevertheless, not all low- or intermediate-grade tumors express every single neuroendocrine marker; therefore, it is necessary in many cases to perform a panel rather than just a single antibody.

One additional antibody that is currently in use to try to determine grading in neuroendocrine carcinomas, mainly in the low or intermediate grade, is Ki-67 nuclear labeling. Even though studies defining a nuclear labeling of less than 5% have been claimed to be useful in separating low from intermediate grade, in a small biopsy, one will be confronted with sample issues, and the nuclear labeling in a small biopsy may be absent, whereas in a complete resection such findings may not be the case. In cases in which a resection is done, one rarely needs the Ki-67 to determine grade, as one is able not only to count mitotic activity but also to identify the presence of necrosis, both features important in the grading system of these tumors.

Pulmonary Paraganglioma

The existence of primary pulmonary paragangliomas has been recorded only rarely in the literature. However, the mere existence of these tumors in the lung can pose a significant challenge in the separation of paraganglioma from neuroendocrine carcinoma (low or intermediate grade). Pulmonary paragangliomas can be functioning tumors, and the tumor has been reported predominantly in adult patients. Similar to neuroendocrine carcinomas, pulmonary paraganglioma may present as a central or peripheral tumor.

The histologic features of paraganglioma may in some cases be similar to the histologic characteristics of low- and intermediate-grade neuroendocrine carcinomas. Paraganglioma may show a nested well-organized growth pattern composed of rather small and intermediate-sized cells admixed with large, bizarre cells with macronuclei. Extensive areas of fibrocollagenous material or discrete fibrovascular stroma commonly separate the nests of cells. The tumor may show prominent oncocytic features. However, the presence of mitotic activity in paragangliomas is rare, which is in contrast to the presence of large atypical, bizarre cells with macronuclei. In some cases, sporadic mitoses may be present. However, necrosis and hemorrhage are not common in these tumors ( Fig. 12.36A–C ).

Immunohistochemically, paragangliomas share similar immunophenotype with neuroendocrine carcinoma in terms of positive staining for chromogranin, synaptophysin, and CD-56; however, paragangliomas are generally negative for keratin antibodies, napsin A, and TTF-1, which is a common way to separate paraganglioma from neuroendocrine carcinomas in a small biopsy. In a comparative study of 46 cases of neuroendocrine tumors (carcinoid and paraganglioma), the authors found that all paragangliomas (22 cases) were negative for TTF-1, pancytokeratin, and napsin A, while 12/22 (55%) of the tumors showed positive staining for GATA-3. The authors concluded that the use of pancytokeratin, TTF-1, and GATA-3 are useful markers to separate carcinoid from paraganglioma. Unlike neuroendocrine carcinomas, paragangliomas regularly have a component of S100 positive sustentacular cells.