| Abstract|| |
Background: C-ros oncogene 1, receptor tyrosine kinase (ROS 1) proto-oncogene 1, receptor tyrosine kinase (ROS-1) fusions are potent oncogenic drivers and these re-arrangements promote signal transduction programs leading to uninhibited cell survival and proliferation identified in 1–2% of cases of nonsmall-cell lung cancer. Mesenchymal epithelial transition factor (MET) receptor tyrosine kinase and its ligand are predominantly involved in epithelial mesenchymal transition and tissue regeneration. The MET amplification and overexpression is oncogenic in 3–7% cases. The objectives of this study were to identify the frequency of ROS-1 and c-MET protein expression in adenocarcinoma lung and to correlate it with the clinicopathological parameters and to analyze the histomorphology of cases that harbor the characteristic mutations (c-MET and ROS-1). Materials and Methods: Study group comprised a prospective cases series of 90 cases of adenocarcinoma lung. ROS-1 protein expression was determined by immunohistochemistry using the D4D6 rabbit monoclonal antibody (Cell Signaling, Danvers, MA) and c-MET protein expressed was analyzed using the SP-44 clone (Ventana Medical Systems). Results: c-MET protein expression was identified in 33.33% cases (n = 30/90) with statistically significant thyroid transcription factor-1 (TTF-1) positivity. ROS-1 protein expression was detected in 3.33% cases (n-3/90), in biopsies from the respiratory tree with TTF-1 expression. Conclusion: This is the first study from the Indian subcontinent to identify the frequency of ROS-1 re-arrangements and MET amplification in the Indian population. The availability of targeted therapy that has a significant impact on survival makes it essential to detect these less frequent mutations.
Keywords: Immunohistochemistry, mesenchymal epithelial transition, nonsmall cell lung carcinoma, ROS, targeted therapy
|How to cite this article:|
Shukla S, Pandey RK, Kant S, Garg R, Husain N. ROS-1 re-arrangements and c-MET amplifications in adenocarcinoma lung: A tertiary care center study from North India. Indian J Pathol Microbiol 2019;62:433-6
|How to cite this URL:|
Shukla S, Pandey RK, Kant S, Garg R, Husain N. ROS-1 re-arrangements and c-MET amplifications in adenocarcinoma lung: A tertiary care center study from North India. Indian J Pathol Microbiol [serial online] 2019 [cited 2019 Aug 19];62:433-6. Available from: http://www.ijpmonline.org/text.asp?2019/62/3/433/263502
| Introduction|| |
In the recent years, advances in lung cancer are occurring at an accelerated rate and major discoveries have been made in the regulation of growth, differentiation, apoptosis and metastasis of lung cancer., These findings have dramatically influenced the treatment protocols with molecular testing for targeted therapy being considered as the standard of care. C-ros oncogene 1, receptor tyrosine kinase (ROS 1) re-arrangements were identified by Rikova et al. in the year 2007. ROS 1 fusions are potent oncogenic drivers and promote signal transduction programs leading to uninhibited cell survival and proliferation.,,, The current national comprehensive cancer network guidelines recommend the use of Crizotinib for cases that harbor ROS 1 re-arrangements. Mesenchymal epithelial transition factor (MET) receptor tyrosine kinase and its ligand are involved in epithelial mesenchymal transition and tissue regeneration. The MET amplification and over-expression is oncogenic in 3-7% cases., MET targeting inhibitors like Onartuzumab and Tivantinib have shown promising results in clinical trials leading to improved overall survival.
In view of the clinical requirement the need for evaluation of frequency of mutations in the Indian population, the current study was undertaken with the objectives to detect the frequency of ROS-1 and c-MET protein over-expression in the Indian population and to co-relate with the clinic-pathological parameters. Further the histomorphology of cases that harbor the characteristic mutations (c-MET and ROS-1) was analyzed and compared with mutation negative cases.
| Materials and Methods|| |
This was a tertiary care hospital based case series that included 90 cases of nonsmall-cell lung cancer (NSCLC) conducted over a period of 1 year (2017–2018). A detailed clinical and radiological assessment was done. Biopsies from both primary and metastatic sites of NSCLC adenocarcinoma, adenosquamous carcinoma, or unspecified carcinoma excluding the squamous cell carcinoma subtype were included in the study. The cases were classified on the basis of histopathology as per the recommendations of the 2015 World Health Organization (WHO) classification criteria for lung tumors. A basic panel of Immunohistochemistry (IHC) that included napsin, thyroid transcription factor-1 (TTF-1), markers for squamous and neuroendocrine differentiation was performed. Molecular testing for epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) re-arrangement was done in all the cases. The testing for EGFR mutations was done by real time polymerase chain reaction (PCR) using the EGFR RGQ PCR kit (Qiagen technologies, Manchester UK) that detects 28 somatic mutations that span over exons 18 to 21. The standard PCR protocol for formalin fixed paraffin embedded (FFPE) tissue was used. ALK testing was done using the Ventana anti-ALK (D5F3) clone. The immunostaining was performed on the benchmark XT system using the Optiview DAB IHC detection kit and Optiview Amplification kit.
c-MET and ROS-1 protein expression
c-MET and ROS-1 expression was assessed using IHC was performed in 90 cases. The FFPE tissue blocks were sectioned at 3–4 μm, using a microtome (Leica, Germany), mounted on tissue bond-coated slides (Biocare, USA). The immunostaining was performed on the benchmark XT system using the Optiview DAB IHC detection kit and Optiview Amplification kit. The prediluted Ventana anti-total c-MET (SP 44) rabbit monoclonal primary antibody was used for cMET detection. For the detection of ROS-1, the D4D6 rabbit monoclonal antibody (Cell Signalling, Danvers, MA) at a dilution of 1:25. Positive (kidney) and negative (by omitting primary antibody) controls were run with all batches. The staining was performed as per the manufacturer's instructions.
Assessment of staining for c-MET and ROS-1 protein expression
Cytoplasmic and/or membranous staining was evaluated and semiquantitative assessment was done estimating the staining intensity and percentage of tumor cells that were positive. The following scores were given: Score 0-No staining, Score 1- Faint staining (+) in any % cells, Score 2: Moderate staining (++) in >50% cells, Score 3: Strong granular staining (+++) in >50% cells. The samples with a score of 0 and 1 were considered negative, while cases with a score of 2 and 3 were considered positive.
Analysis was done in terms of the pattern of arrangement of the cells that included solid, micropapillary, acinar, lepidic, loose clusters, or dispersed cells along with the presence of mucin. The presence or absence of necrosis was assessed and the tumors were histologically graded into well, moderately, or poorly differentiated lesions.
The statistical analysis was done using the Statistical Package for the Social Sciences (SPSS) software version 16. The association between two categorical variables was assessed using the chi square test. A P value of <0.05 was considered as statistically significant.
| Results|| |
The age range varied from 35 years to 72 years with a mean age of 57.75 years. The male to female ratio was 2.3:1. The site of the biopsy was from the respiratory tree (endobronchial, intrathoracic, lung, or pleural) in 87 (96.67%) cases, while biopsy was performed from the metastatic sites in three (3.33%) cases. The most common clinical finding in the cases was the presence of lung mass with or without the presence of pleural effusion. The basic panel of IHC was done to categorize the tumor included napsin, TTF-1, and p-40 [Figure 1]. ALK protein expression was identified in 4.44% (n = 4/90) cases and EGFR mutations were identified in 45.56% (n = 41/90) cases.
|Figure 1: (a and b): Biopsy from an intrathoracic mass with a tumor arranged in acinar pattern with positive staining for napsin (c) and TTF-1 (d) (a = H and E ×50, b = H and E ×100, c = DAB ×100, d = DAB ×100)|
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c-MET protein expression was detected in 30 cases (33.33%) [Figure 2]. The M: F ratio was 1.75:1 with a mean age of 57.5 years. TTF-1 was positive in 16/30 cases (53.33%) which was statistically significant (P = 0.013) when compared with the c-MET negative group [Table 1]. The histomorphological features of the c-MET positive (n = 26) and the c-MET negative (n = 53) was analyzed. The most common pattern in the cases that expressed c-MET was singly dispersed cells or loose cohesive clusters (73.07%), followed by the acinar and solid pattern (61.54%). In the c-MET positive group, 57.69% cases were moderately differentiated with a nuclear grade of 2 in 61.45%. In the c-MET negative group, 73.58% cases had singly dispersed cells or loose cluster followed by a solid pattern in 56.60% cases. Necrosis was present in 58.49% cases that were c-MET negative. However, none of these parameters were statistically significant.
|Figure 2: (a and b): c-MET protein expression in tumor cells performed using the SP-44 clone (a and b = DAB ×100)|
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ROS-1 protein expression was detected in three cases (3.33%) [Figure 3]. The M: F ratio was 1:2 with a mean age of 51.67 years. TTF-1 was positive in all the three cases which was statistically significant (P = 0.006) when compared with the ROS-1 negative cases. All the three cases were biopsies from the respiratory tree (lung, pleural, and endobronchial) which was statistically significant (P = 0.04) when compared with the ROS-1 negative group [Table 1]. The histomorphology of ROS-1 positive (n = 3) was analyzed. In the three cases that had ROS-1 protein expression solid pattern, acinar pattern, and mucin was present in 2/3 cases (66.67%), followed by solid pattern in 57.89%, while necrosis was present in one case (33.33%). All the three ROS-1 positive cases were moderately differentiated.
|Figure 3: (a and b): ROS-1 protein expression in the tumor cells performed using the D4D6 clone (a and b = DAB ×100)|
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Coexpression of ALK and c-MET was present in two cases in this study. Coexpression of c-MET with presence of EGFR mutation was identified in one case. There was one case that had EGFR mutation in association with ROS-1 re-arrangement. All the three cases that harbored ROS-1 re-arrangement had c-MET expression.
| Discussion|| |
Activated MET receptor signaling leads to tumor angiogenesis, tumor cell invasion, and metastasis. MET targeting inhibitors have shown marked results in clinical trials leading to improved overall survival.,, In this study c-MET expression was detected in 33.33% cases. In the study conducted by Casadevall et al. 47.8% cases had c-MET protein expression, while Watermann et al. documented c-MET expression 21.5% cases using IHC in FFPE tissue., In the study conducted by Pyo et al., the authors stated the c-MET IHC positivity significantly correlated with poor overall survival. In this study, TTF-1 expression in the group that had c-MET protein expression was statistically significant when compared with the c-MET negative group (P = 0.002). This may indicate that NSCLC with c-MET protein expression arise from terminal respiratory unit (TRU). The histomorphological pattern analysis for c-MET protein expression indicates that there is no specific pattern or criteria that can be considered as surrogate features for c-MET protein expression.
ROS 1 re-arrangements promote signal transduction programs leading top uninhibited cell survival and proliferation. Detection of ROS 1 re-arrangement using IHC has being strongly recommended with a sensitivity of 100% and a specificity of 92%.,,, In this study ROS-1 protein expression was assessed using the D4D6 and was present in 3.33% cases. Wu et al. reported ROS- protein expression in 4.20% cases, Rimkunas et al. in 1.6% while Sholl et al. reported the frequency of 1.2%.,, These re-arrangements are associated with younger age, never smoking history, Asian ethnicity, and advanced stage. In this study, all the three cases that had ROS-1 protein expression were adenocarcinomas in which the biopsies were performed from the respiratory tree (P = 0.01) and had TTF-1 expression (P = 0.05). The histomorphological analysis of the ROS-1 positive versus the ROS-1 negative yielded inconclusive results as the number of positive cases analyzed was low.
In NSCLC, 5% of driver alterations are concurrent double or triple mutations. In this study, an attempt has been made to identify coexpression of multiple predictive and prognostic markers. However, the number of cases with multiple marker expression is too few to derive valid conclusions.
This is the first study from the Indian subcontinent to identify the frequency of ROS-1 re-arrangements and MET amplification in the Indian population. c-MET protein expression was identified in 33.33% cases with statistically significant TTF-1 positivity. ROS-1 protein expression was detected in 3.33% cases, in biopsies from the respiratory tree with TTF-1 expression. The availability of targeted therapy that has drastic impact on survival makes it essential to detect these less frequent mutations.
The authors would like to acknowledge financial assistance through Intramural research project grant (IEC 6/16), Dr. Ram Manohar Lohia Institute of Medical Sciences, Lucknow, India.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Salgia R. Mutation testing for directing upfront targeted therapy and post-progression combination therapy strategies in lung adenocarcinoma. Expert Rev Mol Diagn 2016;16:737-49.
Dietel M, Bubendorf L, Dingemans AM, Dooms C, Elmberger G, García RC, et al
. Diagnostic procedures for non-small-cell lung cancer (NSCLC): Recommendations of the European expert group. Thorax 2016;71:177-84.
Cagle PT, Allen TC. Lung cancer genotype-based therapy and predictive biomarkers: Present and future. Arch Pathol Lab Med 2012;136:1482-91.
Gainor JF, Shaw AT. Novel targets in non small cell lung cancer: ROS1 and RET fusion. Oncologist 2013;18:865-74.
Viola P, Maurya M, Croud J, Gazdova J, Suleman N, Lim E, et al
. A validation study for the use of ROS1 immunohistochemical staining in screening for ROS1 translocations in lung cancer. J Thorac Oncol 2016;11:1029-39.
Wu J, Lin Y, He X, Yang H, He P, Fu X, et al
. Comparison of detection methods and follow up study on the tyrosine kinase inhibitors therapy in non small cell lung cancer patients with ROS 1 fusion re-arrangement. BMC Cancer 2016;16:599-601.
Sholl LM, Sun H, Butaney M, Zhang C, Lee C, Jänne PA, et al
. ROS 1 immunohistochemistry for detection of ROS 1 re-arranged lung adenocarcinoma. Am J Surg Pathol 2013;37:1-19.
Aisner DL, Marshall CB. Molecular pathology of non small cell lung cancer-A practical guide. Am J Clin Pathol 2012;138:332-46.
Watermann I, Schmitt B, Stellmacher F, Müller J, Gaber R, Kugler Ch, et al
. Improved diagnostics targeting c-MET in non small cell lung cancer: Expression, amplification and activation? Diagn Pathol 2015;10;130-42.
Pyo JS, Kang G, Cho WJ, Choi SB. Clinicopathological significance and concordance analysis of c-MET immunohistochemistry in non small cell lung cancers. A meta analysis. Pathol Res Pract 2016;212:710-6.
Boland JM, Jang JS, Li J, Lee AM, Wampfler JA, Erickson-Johnson MR, et al
. MET and EGFR mutations identified in ALK rearranged pulmonary adenocarcinoma. J Thorac Oncol 2013;8:574-81.
Casadevall D, Gimeno J, Clavé S, Taus Á, Pijuan L, Arumí M, et al
. MET expression and copy number heterogeneity in non squamous non small cell lung cancer (NSCLC). Oncotarget 2015;6:16215-26.
Inamura K, Takeuchi K, Togashi Y, Hatano S, Ninomiya H, Motoi N, et al
. EML4-ALK lung cancers are characterized by rare other mutations, a TTF-1 cell lineage, an acinar histology, and young onset. Mod Pathol 2009;22:508-15.
Rimkunas VM, Crosby KE, Li D, Hu Y, Kelly ME, Gu TL, et al
. Analysis of receptor tyrosine kinase ROS1-positive tumors in non-small cell lung cancer: Identification of a FIG-ROS1 fusion. Clin Cancer Res 2012;18:4449-57.
Department of Pathology, Dr. Ram Manohar Lohia Institute of Medical Sciences, Vibhooti Khand, Gomti Nagar, Lucknow - 226 010, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]