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Table of Contents
EDITORIAL
Year : 2019  |  Volume : 2  |  Issue : 1  |  Page : 66-68

ROS1 rearrangement testing: Is immunohistochemistry changing the horizon?


Department of Medical Oncology, Tata Memorial Hospital, Mumbai, Maharashtra, India

Date of Web Publication9-Sep-2019

Correspondence Address:
Anuradha Choughule
Department of Medical Oncology, Tata Memorial Hospital, Parel, Mumbai - 400 012, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/CRST.CRST_32_19

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How to cite this article:
Choughule A, D'Souza H. ROS1 rearrangement testing: Is immunohistochemistry changing the horizon?. Cancer Res Stat Treat 2019;2:66-8

How to cite this URL:
Choughule A, D'Souza H. ROS1 rearrangement testing: Is immunohistochemistry changing the horizon?. Cancer Res Stat Treat [serial online] 2019 [cited 2019 Nov 13];2:66-8. Available from: http://www.crstonline.com/text.asp?2019/2/1/66/266454



ROS1 is a receptor tyrosine kinase of the insulin receptor family. It results from a translocation between ROS1 and other genes, the most common one being CD74.[1] ROS1 mutation leads to the activation of downstream oncogenic pathways (STAT3, PI3K/AKT/mTOR, and RAS-MAPK/ERK pathways), thereby controlling the cell proliferation, survival, and cell cycling and eventually resulting in cell transformation.[2] It is a driver oncogene in 1%–2% of non-small-cell-lung cancer (NSCLC) patients.[3]

The interest in ROS1 gene arrangement stems from the fact that crizotinib, an anaplastic lymphoma kinase (ALK) inhibitor which is a Food and Drug Administration-approved small-molecule oral therapy for ALK mutation-positive NSCLC patients, is also effective for patients with ROS1 mutations. The approval for crizotinib was based on the efficacy and safety data from the expansion cohort of the Phase I PROFILE 1001 trial, in which the response rate to crizotinib was noted to be as high as 72% and the median progression-free survival was 19.2 months in patients with advanced ROS1-rearranged NSCLC. The clinical features associated with the presence of ROS1 mutation include a non-smoking history, younger age, and non-squamous and non-neuroendocrine lung cancer histopathology; a profile similar to ALK-positive NSCLC.[4]

Like ALK, ROS1 testing is usually performed on a small biopsy specimen or a cytology sample.[5] The studies for ROS1 testing published between 2011 and 2015 with the number of patients ranging from 108 to 1478 used fluorescence in situ hybridization (FISH), reverse transcriptase polymerase chain reaction (RT-PCR), and immunohistochemistry (IHC) as the detection methods.[6] In the Phase 1 study by Shaw et al., 49 of the 50 eligible patients had ROS1 rearrangements detected by a Break Apart FISH Probe. In the remaining patient, the ROS1 rearrangement was detected using an RT-PCR assay.[4]

There are relatively fewer pitfalls with FISH as compared to IHC. False-positive FISH results may be due to the detection of non-functional ROS1 fusions. One of the possible reasons is that the genomic break does not occur at the expected point to generate an active fusion or there is a possibility of an intervening post-transcription and post-translation phenomenon that inactivates a fusion product. On rare occasions, a false-negative FISH report can occur when the ROS1 rearrangement involves genes that are closely located on the same chromosome.[7] A very sensitive RT-PCR-based method has been devised to detect the overexpression of 3′ regions of fusion transcripts involving tumor genes constitutionally repressed or expressed at very low levels.[8] However, this method cannot be easily applied to ROS1 since the gene is also expressed in normal and hyperplastic lung tissue. Multiplex RT-PCR is easy to perform, rapid, and relatively inexpensive but may be challenging using ribonucleic acid (RNA) extracted from formalin-fixed paraffin-embedded (FFPE) samples.

The current data suggest that IHC is a cost-effective screening tool with a very high negative predictive value for ROS1 rearrangement followed by FISH confirmation. Several studies indicate a good correlation between FISH and IHC.[9],[10],[11],[12] Other reports also suggest that ROS1 testing by IHC seems to be highly sensitive; ironically, a strict 3+ cutoff significantly reduces the sensitivity of IHC as well. Assessment of IHC staining intensity is subjective and prone to interobserver variability. The results are highly dependent on the cutoff used to determine IHC positivity. By increasing the cutoff from 1+ to 3+ intensity, specificity can be increased from 76% to 100%. Diffuse moderate-to-strong staining is more commonly associated with ROS1 rearrangement than focal or heterogeneous staining; therefore, FISH testing is recommended in all cases with any ROS1 IHC positivity.[7]

In a meta-analysis, the overall concordance rate between ROS1 IHC and molecular tests, i.e., FISH and DNA sequencing, was 93.4% (95% confidence interval [CI], 78.3–98.2). In ROS1 IHC-positive and negative cases, the concordance rates were 79% (95% CI, 43.3–94.9) and 97% (95% CI, 83.3–99.5), respectively. The pooled sensitivity and specificity of ROS1 IHC were 0.90 (95% CI, 0.70–0.99) and 0.82 (95% CI 0.79–0.84), respectively.[6]

Regarding IHC testing, the published studies to date have done testing with only one ROS1 antibody which is commercially available, i.e., clone D4D6. There has been a lot of variability in the detection systems and staining conditions across various studies, and ultimately, this translates to a substantial difference in approaches to antigen retrieval or signal amplification, and there is no consensus on these approaches. Multiple challenges arise in the ROS1 IHC, both in performance of the test and in the interpretation of the results. Unlike ALK IHC, the levels of mRNA and protein expression may be typically low and very occasionally absent. ROS1 IHC is more prone to false-positive staining. In contrast to ALK IHC, tumors lacking ROS1 rearrangement, including tumors harboring ALK fusion or epidermal growth factor receptor (EGFR) mutations, can still express ROS1 mRNA at levels comparable to those of tumors with ROS1 rearrangement. A large proportion of ROS1 non-rearranged mucinous adenocarcinomas also show reactivity, with a distinct granular pattern indicating false positivity. Although nonspecific expression tends to be weak and heterogeneous, EGFR-mutant tumors can exhibit 3+ROS1 IHC staining, which is a false positive. In addition, a nonspecific staining can occur in reactive pneumocytes and alveolar macrophages, particularly giant cells.[9],[10] In contrast to ALK, to date, there are no approved companion assays for ROS1-rearranged NSCLC.

In this issue of the journal, Dr. Jain et al. from Sapien Biosciences have reported the retrospective analysis of their experience with ROS1 detection by IHC.[13] Two thousand lung cancer cases were screened at six Apollo Hospitals across India. Six hundred and four cases of NSCLC had FFPE blocks available for analysis and 426 contained adequate tissue for IHC testing. D4D6 antibody was applied with 1:150 dilutions using the Ventana platform with positive and negative controls. Two hundred and twenty-five were confirmed adenocarcinoma cases. A single case stained positive for ROS1; thus, the percent positivity rate was 0.44% in adenocarcinoma patients and 0.23% among all NSCLC cases. No positive case was observed in other cancers of the lung such as squamous cell carcinoma, neuroendocrine tumors, and germ cell tumors. This study presents the incidence of ROS1 positivity in NSCLC cases in India and confirms lack of ROS1 positivity in squamous cell lung carcinoma cases.

The incidence of ROS1 positivity was lower than that reported in Western literature (0.9%–1.7%).[3] The authors at Sapien Biosciences have heavily relied on IHC testing rather than on FISH testing; the details about the standardization of antigen retrieval or signal amplification have not been given, except for the optimization of dilution of antibodies. Although the authors did include the basic demographic data such as age and sex of the patients and diagnostic details for the biopsy or surgical tissue sample, there are no data of smoking history included. The arguments against the low incidence could be the age of the blocks as a sample may contain only a limited amount of tumor tissue, and there is only one opportunity available to fix and process the tissue.

The updated molecular testing guidelines from the College of American Pathology (CAP)[14]/the International Association for the Study of Lung Cancer/Association for Molecular Pathology suggest that similar to ALK, ROS1 testing should be done for patients with adenocarcinoma and lung cancers of mixed histology with an adenocarcinoma component in non-smokers.[10] The National Comprehensive Cancer Network guidelines also recommend testing for ROS1 – along with EGFR, ALK, and programmed death-ligand 1 – at the time of diagnosis of metastatic NSCLC.[15] In view of testing algorithms, ROS1 testing by IHC is to be used as an initial screening test to identify cases followed by molecular tests as a confirmatory test. Hence, at present, IHC is to be used as a screening test, followed by confirmatory tests with FISH (break apart probe assay) or RT-PCR or next-generation sequencing as reflex tests in the identification of lung adenocarcinomas harboring ROS1 fusions.

To summarize, ROS1 gene arrangements are oncogenic drivers in a small proportion of NSCLCs. There is an unmet need to have accurate and cost-effective methods to select these patients for therapy. The current methods for the detection of ROS1 gene rearrangements are FISH, RT-PCR, and IHC, and each has its attendant advantages and disadvantages. Regardless of which testing method is used, it is key that routine testing for ROS1 in the clinical setting has to be thoroughly optimized and validated, with appropriate controls, and there should be awareness that test outcomes may be confounded by preanalytical issues in tissue handling and processing. Collective efforts from clinicians, researchers, and regulatory bodies are needed to standardize the diagnostic testing.



 
  References Top

1.
Matsuura S, Shinmura K, Kamo T, Igarashi H, Maruyama K, Tajima M, et al. CD74-ROS1 fusion transcripts in resected non-small cell lung carcinoma. Oncol Rep 2013;30:1675-80.  Back to cited text no. 1
    
2.
Davies KD, Doebele RC. Molecular pathways: ROS1 fusion proteins in cancer. Clin Cancer Res 2013;19:4040-5.  Back to cited text no. 2
    
3.
Gupta V, Godre N, Alam M. 1337P A meta-analysis on epidemiology of ROS1 rearrangement in Asian and non-Asian population. Ann Oncol 2017;28 (Suppl 5):476.  Back to cited text no. 3
    
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Shaw AT, Ou SH, Bang YJ, Camidge DR, Solomon BJ, Salgia R, et al. Crizotinib in ROS1-rearranged non-small-cell lung cancer. N Engl J Med 2014;371:1963-71.  Back to cited text no. 4
    
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Bubendorf L, Büttner R, Al-Dayel F, Dietel M, Elmberger G, Kerr K, et al. Testing for ROS1 in non-small cell lung cancer: A review with recommendations. Virchows Arch 2016;469:489-503.  Back to cited text no. 5
    
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8.
Davies KD, Le AT, Sheren J, Nijmeh H, Gowan K, Jones KL, et al. Comparison of molecular testing modalities for detection of ROS1 rearrangements in a cohort of positive patient samples. J Thorac Oncol 2018;13:1474-82.  Back to cited text no. 8
    
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Sholl LM, Sun H, Butaney M, Zhang C, Lee C, Jänne PA, et al. ROS1 immunohistochemistry for detection of ROS1-rearranged lung adenocarcinomas. Am J Surg Pathol 2013;37:1441-9.  Back to cited text no. 9
    
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Yoshida A, Tsuta K, Wakai S, Arai Y, Asamura H, Shibata T, et al. Immunohistochemical detection of ROS1 is useful for identifying ROS1 rearrangements in lung cancers. Mod Pathol 2014;27:711-20.  Back to cited text no. 10
    
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Shan L, Lian F, Guo L, Qiu T, Ling Y, Ying J, et al. Detection of ROS1 gene rearrangement in lung adenocarcinoma: Comparison of IHC, FISH and real-time RT-PCR. PLoS One 2015;10:e0120422.  Back to cited text no. 11
    
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Selinger CI, Li BT, Pavlakis N, Links M, Gill AJ, Lee A, et al. Screening for ROS1 gene rearrangements in non-small-cell lung cancers using immunohistochemistry with FISH confirmation is an effective method to identify this rare target. Histopathology 2017;70:402-11.  Back to cited text no. 12
    
13.
Jain J, Chinta D, Jayaraman UB, Pathak N, Kaur M, Chatterjee S, et al. Determination of ROS1 positivity by immunohistochemistry in a multicentric cohort of 426 non-small-cell lung cancer cases in India. Cancer Res Stat Treat 2019;2:16-20.  Back to cited text no. 13
  [Full text]  
14.
Lindeman NI, Cagle PT, Aisner DL, Arcila ME, Beasley MB, Bernicker EH, et al. Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors: Guideline from the college of American pathologists, the international association for the study of lung cancer, and the association for molecular pathology. Arch Pathol Lab Med 2018;142:321-46.  Back to cited text no. 14
    
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NCCN Guidelines NSCLC, Ver; 5. 2019. Available from: https://www.nccn.org/professionals/physician_gls/pdf/nscl.pdf. [Last accessed on 2019 Aug 10].  Back to cited text no. 15
    




 

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