Immunohistochemical Profile of Lung Tumors in Image Guided Biopsies

Background: Lung cancer is the leading cause of cancer related mortality in both men and women worldwide. Establishing the histological type and grade of pulmonary carcinoma is very important especially for the therapy and prognosis. Study design: cross-sectional descriptive study. The study analyses various histomorphological patterns of lung tumors in correlation with immunohistochemical profile. Methods: All the bronchoscopic and CT guided needle biopsy specimens (50 biopsy specimens) received in the Pathology department of Coimbatore medical college hospital over a period of one year were analysed. Both H&E and immunohistochemical sections were studied with panel of markersCK7, CK20, TTF-1, chromogranin, synaptophysin, CD45, vimentin, smooth muscle actin. Result: The most common histological type was squamous cell carcinoma (48%), followed by adenocarcinoma (28%) and small cell lung carcinoma (18%). Large cell neuroendocrine carcinoma and metastatic deposit constituted 2% each. Out of 50 cases, 24 cases were squamous cell carcinoma which showed positivity with HMWCK (20 cases) and P63 (22 cases) (p<0.001). Fourteen cases reported as adenocarcinoma showed positivity with CK7 (14 cases) and TTF-1 (13 cases) (p<0.001). All the nine cases of small cell carcinomas showed positivity with both TTF-1 and Ki 67. One case of large cell carcinoma with neuroendocrine features showed immunopositivity with neuroendocrine markers. Conclusion: Integration of conventional histomorphological diagnosis with panel of immunohistochemical markers allows more accurate identification of histological type, which has significant treatment implications.

www.pacificejournals.com/apalm eISSN: 2349-6983; pISSN: 2394-6466 history of primary tumor elsewhere in the body and radiological investigations were reviewed in all the cases.
Ethical clearance for the study was obtained from the Ethics Committee of Coimbatore Medical College, Coimbatore.
Histomorphological patterns and immunohistochemical profiles of the lung tumors were analysed. Because of the availability of very limited tissue in the lung biopsy specimens, panel of markers limited to the particular histological type diagnosed in H&E were used. Panel included markers of squamous differentiation-p63, HMWCK, cytokeratin specific for primary pulmonary origin-CK7, TTF-1, cytokeratin for primary gastrointestinal tract origin-CK20, markers of neuroendocrine differentiation-chromogranin, synaptophysin, proliferation antigen-Ki67, LCA and vimentin. Tumour cells were scored positive based on the pattern and intensity of staining in the neoplastic cells.
Statistical data analysis of various histomorphological patterns and percentage positivity of various immunohistochemical markers were studied and compared with those in the literature.

Result
In our study, it was observed that the peak incidence of lung malignancies occured in the age group of 51-60 years (46%) with a male preponderance (84%). 43 out of 50 cases could be diagnosed and subtyped precisely using routine Hematoxylin & Eosin stained sections and confirmed by IHC. Remaining seven cases were diagnosed as NSCLC and could not be subtyped as SCC or adenocarcinoma requiring the aid of IHC. With the IHC findings, four cases were concluded as SCC and two cases as adenoarcinomas.
Only one case showed inconclusive result with IHC and reported as NSCLC. ( Table 1) In our study, we observed 24 cases were squamous cell carcinoma (48%), 14 cases of adenocarcinoma (28%) and 9 cases of small cell lung carcinoma (18%). Large cell neuroendocrine carcinoma and metastatic deposit constituted 2% each. One case was reported as NSCLC alone. (2%). It was observed that among the 20 cases reported as squamous cell carcinomas and 4 cases as NSCLCs histologically, HMWCK was expressed in 20 cases with a sensitivity of 83.3%. P63 was expressed in 22 out of 24 cases with a sensitivity of 91.7%. ( Table 2) P63 was not expressed in any of the adenocarcinoma cases with 100% specificity (Table 3). It was observed that both HMWCK and p63 were equally good immunohistochemical markers for the diagnosis of squamous cell carcinomas.
All the 12 cases reported as adenocarcinomas in H&E stained sections and 2 cases as NSCLCs, showed immunopositivity with CK7, with a sensitivity of 100%. 13 out of 14 cases of adenocarcinomas showed nuclear immunoreactivity with TTF-1. TTF-1 was not expressed in any of the squamous cell carcinomas; thus the sensitivity of TTF-1 in this study was found to be 92.8% and specificity 100%. (Table 3). One case reported as NSCLC alone histologically, gave inconclusive results with IHC markers of both squamous and glandular differentiation.

Discussion
Lung cancer is the leading cause of cancer related morbidity and mortality. The primary intent of histopathological study is to classify lung tumors as primary pulmonary tumors or metastatic lesions. Primary lung carcinomas are classified as small cell lung carcinomas (SCLC) and nonsmall cell lung carcinomas (NSCLC). NSCLC accounts for 75-80% of all the lung carcinomas and further subtyped as squamous cell carcinoma and adenocarcinoma. [6] The objective of this study is to analyze the histomorphological patterns and immunohistochemical profile of lung tumors in bronchoscopic and needle biopsy specimens. Panel of immunohistochemical markers were used to confirm the histopathological diagnosis and correctly classify the lung tumors. It is a global observation that lung cancer has a higher incidence in males than in females with a male to female ratio of 2.7:1. [7] These tumors commonly affect individuals in the 6 th to 7 th decades of life. In our study also, males are more commonly affected (84%) and the peak incidence occurred in the age group of 51-60 years.
In our study, NSCLC accounted for 78%; Squamous cell carcinoma was the most common histological pattern (48%), followed by adenocarcinoma (28% HMWCK is usually expressed in SCC and does not show reactivity in adenocarcinomas and SCLCs. [8] P63 is also expressed only in SCCs. Both HMWK and P63 are useful to differentiate SCC from adenocarcinoma and poorly differentiated SCC from SCLC and large cell neuroendocrine carcinoma. [9] In our study, out of 20 cases reported as SCC and 4 cases as NSCLC in H&E, HMWCK was expressed in 20 cases (sensitivity 83.3%) ( fig.1) [9] Recent studies have proposed a panel of markers for pulmonary adenocarcinomas-CK-7, CK-20, TTF-1. Primary lung adenocarcinoma shows strong and diffuse positivity with CK-7. CK-20 is expressed in metastatic deposits from colonic carcinomas. CK-7 is used in combination with CK-20, in differentiating primary pulmonary carcinoma from metastatic colonic carcinoma. [11] 75% of primary pulmonary adenocarcinomas express TTF-1. [12,13] Napsin A is also a sensitive marker for adenocarcinoma that has a stronger intensity than TTF-1. [14] Y.Su, Y.Hsu et al., used a panel of markers CK-7, CK-20, TTF-1 to differentiate primary from metastatic lung adenocarcinomas. They observed that 73% of primary pulmonary adenocarcinomas expressed TTF-1, whereas all the metastatic adenocarcinomas lacked TTF-1 staining. They concluded in their study that TTF-1 has high sensitivity and specificity for primary pulmonary adenocarcinomas. CK-7 expression was present in 75% of pulmonary adenocarcinomas and none of the cases expressed CK-20. In their study they found combination of CK-7 + /CK-20along with TTF-1 immunoreactivity was highly specific for primary pulmonary adenocarcinoma. [15] In a study by R.Ocque, N.Tochigi et al., 2011, CK-7 expression was found in all the cases of adenocarcinomas (100%) and 86.2% of cases expressed TTF-1. They also observed in their study the immunoreactivity of TTF-1 in 9 out of 43 cases of SCCs with a sensitivity of 86% and specificity of 73%. [9] In our study, all the 12 cases reported as adenocarcinomas and 2 cases reported as NSCLC, showed positivity with CK7 immunostaining with a sensitivity of 100% and negative immunoreactivity with CK-20. (fig.3) TTF-1 immunoreactivity was positive in 13 out of 14 cases of adenocarcinomas. (fig4) TTF-1 was not expressed in any of the squamous cell carcinoma cases with a sensitivity of 92.8% and specificity 100%. ( p value <0.001) ( Table-3 ).
The results were consistent with the prior studies.
In our study, 9 cases were reported as SCLCs in H&E stained sections with crush artefacts. Crush artifact can also occur in carcinoids, lymphomas, poorly differentiated nonsmall cell carcinomas and lymphocytes of inflammation. [16] Mitotic activity is impossible to assess in small biopsy specimens with crush artifacts. In such cases, Ki67 labelling can be more reliable.
In a study by D. L. Aslan, H. E. Gulbahce et al., 2005 they found all the 34 cases of SCLC showed immunoreactivity with Ki-67 even in the crushed areas, with diffuse staining in more than 80% of all tumor cells. [17] Small cell carcinoma is positive for TTF-1 in 90% of cases. [18] In our study, all the 9 cases of SCLC showed positivity with both TTF-1 (100%) and Ki67 (100%) immunohistochemically. (fig 5&6) Immunostaining with Ki67 showed reactivity in more than 80% of the tumor cells. (fig5) Immunostaining with LCA (CD 45) showed negative results and hence lymphoma was ruled out.
In our study, one case of large cell carcinoma with neuroendocrine features was reported in H&E. Immunohistochemistry showed strong cytoplasmic positivity with neuroendocrine markers-chromogranin and synaptophysin. HMWCK and p63 expression, largely restricted to NSCLCs showed negative results. [19] CK-7 showed focal cytoplasmic positivity. Thus the possibility of poorly differentiated NSCLC was excluded. With the combined H&E and immunohistochemical profile, it was concluded as 'Large cell neuroendocrine carcinoma.' [20] Large cell carcinoma with neuroendocrine architecture but without immunoreactivity for neuroendocrine markers should be concluded as 'Large cell carcinoma with neuroendocrine architecture'. [20] With the clinical history, radiological findings, H&E and IHC reports with vimentin positivity, one case was concluded as metastatic high grade spindle cell sarcomatous deposit.
We observed in our study that 43 out of 50 cases could be diagnosed and subtyped precisely in H&E stained sections histomorphologically and confirmed by IHC.
Remaining seven cases were diagnosed as NSCLC alone and proceeded with IHC. With the IHC findings, four cases were concluded as SCC and two cases as adenoarcinomas.
Only one case showed inconclusive result with IHC and reported as NSCLC. (Table 1) This negative result may be due to reaction bias like specimen fixation, tissue processing and antigen retrival.
Integration of conventional histomorphological diagnosis with immunohistochemistry increases the refinement of diagnosis , so that a diagnosis of NSCLC can be avoided. Subclassification of NSCLC has significant treatment implications, especially for advanced stage tumors for which chemotherapy is being considered. [18] Conclusion From this study it is concluded that immunohistochemistry should be done in all the small lung biopsy specimens to confirm the histomorphological diagnosis as well as in cases where histological subtyping is difficult with H&E sections. Also, panel of markers can be restricted to the histological type because of the limited availability of tissues in bronchoscopic and CT-guided biopsy specimens.