Best Practices Recommendations for Diagnostic Immunohistochemistry in Lung Cancer

Abstract

Since the 2015 WHO classification was introduced into clinical practice, immunohistochemistry (IHC) has figured prominently in lung cancer diagnosis. In addition to distinction of small cell versus non-small cell carcinoma, patients' treatment of choice is directly linked to histologic subtypes of non-small cell carcinoma, which pertains to IHC results, particularly for poorly differentiated tumors. The use of IHC has improved diagnostic accuracy in the classification of lung carcinoma, but the interpretation of IHC results remains challenging in some instances. Also, pathologists must be aware of many interpretation pitfalls, and the use of IHC should be efficient to spare the tissue for molecular testing. The International Association for the Study of Lung Cancer Pathology Committee received questions on practical application and interpretation of IHC in lung cancer diagnosis. After discussions in several International Association for the Study of Lung Cancer Pathology Committee meetings, the issues and caveats were summarized in terms of 11 key questions covering common and important diagnostic situations in a daily clinical practice with some relevant challenging queries. The questions cover topics such as the best IHC markers for distinguishing NSCLC subtypes, differences in thyroid transcription factor 1 clones, and the utility of IHC in diagnosing uncommon subtypes of lung cancer and distinguishing primary from metastatic tumors. This article provides answers and explanations for the key questions about the use of IHC in diagnosis of lung carcinoma, representing viewpoints of experts in thoracic pathology that should assist the community in the appropriate use of IHC in diagnostic pathology.

Keywords: Immunohistochemistry; Lung cancer; Neuroendocrine markers; TTF1; p40.

Figure 1.

ALK positive adenocarcinoma of the lung (A). A vast majority of ALK positive lung cancers are also positive for TTF1 as this case (B). Another characteristic of ALK positive tumor include discordant expression between p63 (C) and p40 (D), which can be a pitfall when p63 is used alone as squamous cell carcinoma marker.

Figure 2.

A case with focal positivity of TTF1. Histologically, the tumor cells do not show clear morphologic differentiation (A). The displayed TTF1 staining (B) should be evaluated as positive, thus the tumor is diagnosed as “NSCC, favor adenocarcinoma”. Another case with unclear morphologic differentiation (C). As the definition of a positive reaction with p40 is defined as 50% or more positive staining of the tumor cells, the widely scattered but sparse positive reaction with p40 (D) should not be considered as a definite diagnosis of squamous cell carcinoma (D).

Figure 3.

TTF1 expression according to antibody clones in primary squamous cell carcinoma of the lung (A, H&E staining). Positive reactions with clone SPT24 staining (B) is contrasted to weak or negative with clone 8G7G3/1 (C).

Figure 4.

Cytokeratin expression of squamous cell carcinoma in a bronchial biopsy specimen (A). Nuclear staining with p40 (BC28 Ab) supports the diagnosis (B). Both cytokeratin 7 (OVTL12/30 Ab, C) and cytokeratin 5/6 (D5/16B4 Ab, D) are positive.

Figure 5.

Solid adenocarcinoma of the lung (A), which is diagnosed by numerous intracytoplasmic vacuoles of mucus within the cytoplasm of tumor cells with Alcian blue staining (B). Diffuse cytokeratin 7 (OV-TL12/30, C) expression and absence of TTF1 (8G7G3/1 Ab, D) expression are noted.

Figure 6.

A lesion of typical ground glass attenuation on CT image (A) was surgically removed and histologically shows adenocarcinoma (B). This tumor has diffuse synaptophysin expression (C). Despite the diffuse expression, the tumor should be diagnosed as adenocarcinoma, because the tumor does not have any morphological neuroendocrine features.

Figure 7.

Typical immunoprofile of carcinoid tumor. Note the homogenous distribution of tumor cells, strong staining for three neuroendocrine markers and low MIB1 immunoreactions.

Figure 8.

Typical immunoprofile of small cell lung carcinoma. Note the irregular distribution of tumor cells, strong staining for two neuroendocrine markers and dot-like positivity with chromogranin A. Also, MIB1 labeling is high.

Figure 9.

Transbronchial biopsies often have crushed tumor cells as shown (A and C). In this situation, cytology specimens, if available, can be more useful for diagnosis, and Ki-67 staining (B and D) can help with the differential diagnosis between carcinoid tumor (A and B) and small cell carcinoma (C and D).

Figure 10.

Typical H&E appearance of NUT carcinoma: undifferentiated, primitive, but monomorphic features with focal abrupt squamous differentiation (A). NUT immunohistochemistry shows diffuse nuclear labeling with characteristic speckled pattern (B).

Figure 11.

Immunostaining of cytology specimens. A) TTF1 positive adenocarcinoma in cell block specimen (by brown 3,3’-diamonobenzidine, Ventana Benchmark XT immunostainer), BE) immunostaining on Papanicolaou stained, ethanol fixed, non-cell block specimens (Leica Bond automated immunostainer); B) TTF1 positive adenocarcinoma (detection by red 3-amino-9 ethylcarbazole), C) p40 positive non-keratinizing squamous cell carcinoma, D) p40 positive benign hyperplastic basal cells underlying ciliated respiratory cells (bronchial brush cytology), E: CD56 positive small cell carcinoma with corresponding Papanicolaou stained specimen (F).

Figure 12.

An example of invasive mucinous adenocarcinoma of the lung demonstrating lepidic and acinar patterns (A), diffuse CK7 expression (B), focal CK20 expression (C), scattered foci with weak TTF1 (D) and/or Napsin-A (E) expressions, and weak to moderate CDX2 expression (F). Of note, the entrapped type II pneumocytes are reactive to CK7 (B), TTF1 (D) and Napsin-A (E).

Figure 13.

A pancreatic ductal adenocarcinoma metastatic to the lung exhibiting a lepidic pattern (A, at a low-power magnification & B, at a high-power magnification), focal CK7 expression (C), negative TTF1 (D), diffuse weak CDX2 expression (E), and loss of SMAD4 (F). Of note, strong nuclear expression of TTF1 in the entrapped pneumocytes may give a false positive impression. Also, loss of SMAD4 expression has been reported as useful marker for the diagnosis of pancreatic adenocarcinoma, but a significant proportion of invasive mucinous adenocarcinomas of the lung harbors this alteration.

Figure 14.

Apical granular reactions of polyclonal Napsin-A are shown in non-pulmonary carcinoma (A, metastasis of pancreatic duct carcinoma and C, appendiceal adenocarcinoma) in contrast to negative reaction with monoclonal Napsin-A (clone IP64, B, D).

Figure 15.

Useful antibodies and pitfalls for some of the more common differential diagnosis of metastatic lung cancer. PAX8 (monoclonal) positivity in metastatic serous adenocarcinoma from the uterine corpus (A). Focal ER positivity in primary lung adenocarcinoma (B). GATA3 positivity in metastatic adenocarcinoma from the breast (C). MGB1 expression in salivary-type adenocarcinoma (mucoepidermoid carcinoma) of the lung (D).

Figure 16.

TTF1 expression in non-pulmonary carcinoma. A-C: Metastasis of ovarian endometrioid carcinoma to the lung (A) expresses PAX8 (B) and TTF1 (clone SPT24, C). The diagnosis was confirmed with identical KRAS mutation (G12A) between lung and ovarian cancer. D-F: Lymph node metastasis of mammary invasive ductal carcinoma (D) displays dual expression of ER (E) and TTF1 (clone SPT24, F).