A Validation Study for the Use of ROS1 Immunohistochemistry in Screening for ROS1 Translocations

A validation study for the use of ROS1 immunohistochemistry in screening for ROS1 translocations in lung cancer.

Patrizia Viola MD1, Manisha Maurya PhD2, James Croud MSc1, Jana Gazdova BSc2, Nadia Suleman BSc1, Eric Lim FRCS (C-Th)3,4, Tom Newsom-Davis FRCP5, Nick Plowman MD6, Alexandra Rice FRCPath1,4, M Angeles Montero MD1,4, David Gonzalez de Castro PhD2, Sanjay Popat FRCP7, Andrew G Nicholson DM1,4.

1Department of Histopathology, Royal Brompton & Harefield NHS Foundation Trust, London, UK

2Centre for Molecular Pathology, Royal Marsden Hospital, Sutton, UK

3Department of Thoracic Surgery, Royal Brompton & Harefield NHS Foundation Trust, London, UK

4National Heart and Lung Institute, Imperial College, London, UK

5Department of Oncology, Chelsea and Westminster Hospital, London, UK

6Department of Oncology, St Bartholomew’s Hospital, London, UK

7Department of Medicine, Royal Marsden Hospital, London, UK

Address for correspondence: Prof Andrew G Nicholson, Department of Histopathology, Royal Brompton & Harefield NHS Foundation Trust, Sydney St, London, UK.

Tel: 0044 207 3518425; Fax: 0044 207 3518293; Email: [email protected]

Acknowledgements: This project was supported by the National Institute of Health Research Respiratory Disease Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust and Imperial College London. SP acknowledges NHS funding to the Royal Marsden Hospital/Institute of Cancer Research NIHR Biomedical Research Centre.

Key words: Lung cancer, ROS1 gene rearrangement; immunohistochemistry, FISH. ABSTRACT

Introduction: The presence of ROS1 gene rearrangements in lung cancers confers sensitivity to ROS kinase inhibitors, including crizotinib. However, they are rare abnormalities (~1% in non-small cell lung carcinoma), typically identified via FISH, and so screening using immunohistochemistry (IHC) would be both cost and time-efficient.

Methods: A cohort of lung tumours, negative for other common mutations related to targeted therapies, were screened to assess the sensitivity and specificity of IHC in detecting ROS1 gene rearrangements, enriched by four other cases first identified by FISH. A review of published data was also undertaken.

Results: IHC was 100% (95% CI 48-100) sensitive and 83% (95% CI 86-100) specific overall when an h-score of >100 was used. Patients with ROS1 gene rearrangements were younger and typically never smokers, with the tumours all adenocarcinomas with higher grade architectural features and focal signet ring morphology (2/5). Four patients treated with crizotinib showed partial response, with three also showing partial response to pemetrexed. Three of four patients remain alive at 13, 27 and 31 months respectively.

Conclusion: IHC can be used to screen for ROS1 gene rearrangements, with patients herein showing response to crizotinib. Patients with tumours positive with IHC but negative for FISH were also identified, which may have implications for treatment selection. INTRODUCTION

Lung cancer remains a common cancer with high mortality, patients typically presenting with advanced stage disease that, until recently, has been primarily treated with platinum-based chemotherapy. However, the past decade has seen a shift in how these patients are managed due to the advent of targeted, therapy that leads to extended survival if such mutations are present.1,2 The most frequently recognised actionable mutations are epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) translocations, and most laboratories have protocols for handling specimens to ensure timely testing for these abnormalities, with fluorescence in-situ hybridisation (FISH) currently the test of choice for ALK translocations.3 However FISH testing can be time consuming, is complex to interpret and is comparatively expensive, so validated immunohistochemistry (IHC) has been recommended as a quicker and more cost efficient method of screening for ALK translocations,3 not least as the incidence of ALK translocations is no more than 2% in non- small cell lung cancer (NSCLC)s.

Similarly, FISH testing has been used to decide on whether targeted therapy with crizotinib should be given when ROS1 gene rearrangements (c-ros oncogene 1, located at 6q22) 4,5 6 are identified7-11 but, with a rate of only 1-2% in adenocarcinomas, this methodology is an expensive proposition, especially in a screening setting. Using immunohistochemistry (IHC) as a screening tool for ROS1 rearrangements may be a feasible option and antibodies are now available which identify the ROS1 protein in tumour cells.12

Herein, we present our findings from a validation study designed to determine whether IHC could be used as a screening tool for ROS1 gene rearrangements, using a selected group of mutation-negative lung cancers enriched with known positive cases from our diagnostic archive. We also review published data to date on using IHC to screen for ROS1 translocations.

MATERIALS AND METHODS

Given the relative rarity of the translocation and the fact the most driver mutations occur in isolation, a sequential test cohort of 103 cases was selected from 170 patients recruited from our institution to phase 1 of the Cancer Research UK-Stratified Medicine Project (CRUK- SMP), who were identified as negative for EGFR, KRAS and/or BRAF mutations, as well as ALK translocations.

Cases that did not harbour any of the above molecular abnormalities were then screened using immunohistochemistry. Sections were dewaxed and heat-induced antigen retrieval was carried out using a Dako PT Link module (PT10126). Slides were placed in pre-heated (65°C) target retrieval solution (EnvisionTM FLEX high pH TRS (pH9), DM828, diluted 1/50 from concentrate). Staining was carried out at room temperature using Dako EnvisionTM FLEX reagents and a Dako Link 48 Autostainer (AS48030). Slides were incubated for 30 minutes with the ROS1 antibody (D4D6, Cell Signalling, 1 in 300 dilution), before signal amplification (15 minute incubation with FLEX+ rabbit Linker solution (K8009)). Cases were scored semi-quantitatively as negative, weakly, moderately or strongly positive, along with the percentage of positive cells and an h-score was then generated with a score of >100 was viewed as positive,13,14 this being a score of 0-3 of intensity, multiplied by the percentage of positive cells (range 0-300).

FISH analysis was performed directly on 2μm FFPE tissue sections mounted on charged slides. We used the Cytocell ROS1 dual colour break-apart FISH Probe (Cytocell Ltd, Cambridge, UK) comprising of a 206kb Texas Red labelled probe covering the distal part of ROS1 and FITC (green) labelled probes situated proximal to the ROS1 gene to evaluate ROS1 translocations. FFPE slides were deparaffinised, treated with protease, co-denatured with FISH probe in a Thermobrite slide processor (Abbott Laboratories, Illinois, USA), washed, counterstained with DAPI and analysed using the Zeiss AxioImager Z2 Fluorescence microscope (Carl Zeiss AG, Jena, Germany). Images were captured and analysed using Metasystems ISIS software (MetaSystems GmbH, Altlussheim, Germany). ROS1 FISH test results were reported above a threshold of 15% in FFPE NSCLC tissue specimens as in previous studies.15,16

Cases showing > or = to15% split signals were classified as positive, and the sensitivity and specificity of positive staining for ROS1 was generated. Agreement was reported as sensitivity and specificity with 95% confidence intervals using FISH as the reference. Statistical analysis was performed on Stata 13 (College Station, Texas). Specimens were FISH-tested without knowledge of ROS1 IHC status. This study was approved as a service development study by the local R&D committee (Ref number 1240).

RESULTS

From 170 patients recruited from our institution into CRUK-SMP phase 1, a total of 103 patients were wild type for EGFR, KRAS, BRAF, and ALK aberrations. (90 patients were wild type for all 4 genetic abnormalities, whilst 9 cases had failed analyses for one mutation, and 4 patients had failed analyses for two mutations. Of the 103 cases, 39 were adenocarcinomas, 39 squamous cell carcinomas, 5 small cell carcinoma, 2 adenosquamous carcinoma, 3 pleomorphic carcinomas,4 large cell carcinoma, 2 large cell neuroendocrine cell carcinoma, 3 NSCLC on biopsy and 6 carcinoids (Table 1).

All cases classified as tumours other than adenocarcinoma were negative using IHC and these were not considered for further analysis, apart from two adenosquamous carcinomas (Table 1). In the adenocarcinoma group, five cases showed moderately intense staining for ROS1 protein (Figure 1), with positivity in 90% of cells in two cases and positivity in 20%, 40%, and 50% of cells in the other three cases. A further five cases showed weak staining in less than 50% of cells (<5%, <5%, 10%, 20%, 30%). There were therefore two cases in total classified as positive due to an h-score of more than 100.

Only cases with sufficient tissue available, classified as adenocarcinoma (n=36) and adenosquamous carcinoma (n=2), were subjected to FISH testing (n=38). Of these 38 cases, one case of adenocarcinoma was positive (78% of tumour cells showing a rearrangement). FISH testing was negative in 34 cases (32 adenocarcinomas and 2 adenosquamous carcinomas), with scores of 1-8%, and three cases failed.

The one positive case on FISH testing (signal pattern characteristic of an unbalanced ROS1 translocation resulting in loss of the red probe) was also positive on IHC (>90% of cells, moderate staining) (Figure 2a). Given the low number of cases within the screened cohort showing ROS1 gene rearrangement using FISH, the archive was reviewed for cases that had been found to be positive using FISH as part of clinical management.

A further four cases were identified from the diagnostic archive that had tested positive for a ROS1 gene rearrangement using FISH (Figure 2b). Of these, one case showed strong staining in 90% of tumour cells, two cases showed moderate staining in 80% and 100% of cells and one case showed weak staining in 60% of cells and moderate staining in 30% of cells (h- scores of 270, 200, 160 and 120 respectively). Overall, when the screening cohort of adenocarcinomas and adenosquamous cell carcinomas was combined with these additional four cases, the specificity was 83% (95% CI 86-100) with a sensitivity of 100% (95% CI 48- 100). When positive, staining was cytoplasmic and variably granular, with one (case 4) showing focal sub-membranous accentuation to the staining (Figure 2).

Reviewing the five FISH-positive/IHC-positive cases, these were all adenocarcinomas with the resection being cribriform predominant, along with solid and micropapillary areas, and the two biopsy samples showing predominant solid or micropapillary architectures and two cytology samples comprising micropapillary clusters. Two cases had focal signet ring cell morphology (Figure 1). There were three males and two female, and the average age was 41 years (Table 2) compared to 68 years for the entire screened cohort and 66 years for the screened adenocarcinomas. Of the 5 cases, four of five cases were known to be never smokers and one was an ex-smoker of only 3 pack years.

In relation to outcome, four of the patients received either sequential treatment with crizotinib and conventional chemotherapy, and one patient underwent resection, with no recurrence to date. All patients showed partial response to crizotinib, when treated. One patient relapsed after 5 cycles, and switched to pemetrexed and cisplatin with partial response and remains with stable disease on pemetrexed maintenance therapy. One patient remains alive at 31 months having started with 5 cycles of pemetrexed before switching to crizotinib. One patient had a ROS1 gene rearrangement identified at the time of a brain relapse with subsequent partial response to crizotinib but relapsed after 6 cycles of crizotinib. He then switched to pemetrexed, again with partial response, but died 14 months after diagnosis. One patient was treated with pemetrexed for 5 cycles, followed by crizotinib with partial response, and remains alive 27 months after diagnosis on continued crizotinib therapy.

Several cases also showed positive staining of entrapped pneumocytes (Figure 4) and alveolar macrophages, making scoring problematic in some adenocarcinomas. DISCUSSION

This study confirms that immunohistochemistry for ROS1 protein within the cytoplasm of tumour cells can potentially be used as a screening tool for ROS1 gene rearrangements, with excellent efficacy using crizotinib in three of four patients with advanced disease, alive at 13, 27 and 31 months respectively. The fourth patient died of disease at 14 months. Sensitivity and specificity using an h-score of 100 were 100% and 83% respectively, in keeping with the majority of the published literature (Table 3).12,14,15,17-22

This study also highlights the importance of individual laboratory validation of a screening programme, given the variation in platforms, antibody dilutions and optimisation techniques, reflecting the real-life variation in laboratory facilities. Even our chosen thickness of slides for FISH varied from other studies (2 microns versus 3-4 microns), this having been internally validated as not affecting FISH results but saving tissue. Scoring systems are a further variation, although we chose an h-score of 100 as a cut-off as this was the choice of most groups using this methodology, plus gave us the best specificity. Nevertheless, despite these variations and the fact that specificities would be lower if the cut-off level was reduced, the fact that nearly all studies show a sensitivity of 100% suggests that that IHC for ROS1 protein can be used to identify potential cases for confirmation by FISH testing, reducing the cost of screening significantly with a low likelihood of cases being missed. This IHC utility is similar to that identified for ALK fusions with many centres now adopting ALK-IHC as a standard screening tool.3,14,23-27

Our data and the published literature also raise the question of how to manage those patients where there is a positive result for ROS1 with IHC whilst the FISH test is negative. This phenomenon is described in some patients with abnormalities in relation to the ALK gene where clinical response to crizotinib has still been found.28 To our knowledge, there have not been any patients treated for ROS1 targeted agents with discordant ROS1 assessment results (ROS1-IHC positive and ROS1-FISH negative and vice versa), although it can only be a matter of time before this happens, as ROS1 IHC assessment becomes more routinely implemented. An alternative option for such patients would be to undertake additional PCR- based testing or next generation sequencing (NGS)-based testing29 because, as in the case of ALK fusions, some tumours have been found to show IHC and PCR positivity for ROS rearrangements whilst FISH tests have proven negative.17 Perhaps surprisingly, the number of cases identified within the quadruple- or triple-negative group did not show the kind of increased frequency seen in some publications where the positive rate was raised to 6%,15 although our rate of 2.5% is comparable with other “enriched” cohorts.

In ROS1-IHC positive cases, both our own data and the literature suggest that nearly all cases are adenocarcinomas and that ROS1 gene rearrangements are frequently seen in patients with solid, micropapillary and cribriform histologic patterns, as well as tumours containing tumour cells with signet ring morphology.30 This is similar to patients with ALK gene rearrangements,23,31-33 although histological pattern does not seem to be a good enough positive or negative predictor to direct ROS1-IHC screening, and is unlikely to be sufficiently specific or sensitive in relation to ROS1 gene rearrangements. Nevertheless, it is notable that these three histologic patterns are the ones associated with poorer prognosis,34 although this does vary between published studies (Table 3). Patients also seem to be younger and never smokers, consistent with what might be expected with driver mutations.

Pathologists need to be aware of staining of native pneumocytes and macrophages as ROS1 protein can be expressed in full length form in the normal lung, an important difference when comparing to ALK screening.19 In this study, several cases showed weak to moderate positive staining and careful review of the samples is required to ensure false positive are avoided. Indeed, some true positive cases show differences in staining compared to that seen in background staining in that there sometimes is sub-membranous accentuation, described particularly in relation to the EZR-ROS1 fusion,19 but we were unable to look for specific fusion partners in this study. However, our numbers are small and whether these differences in the staining characteristics could be used to define which cases should be sent for FISH confirmation requires further study.

The main limitation of the study is that the number of cases proving positive for the ROS1 gene rearrangement was low, necessitating enrichment from the diagnostic archive to support the high level of sensitivity. However, only one prior publication has more than 10 cases, highlighting the rarity of this gene rearrangement and the need for cost-efficient screening.

In conclusion, with a high sensitivity rate and relatively high specificity rate, IHC screening to identify patients that might harbour ROS1 gene rearrangements is feasible and would be less expensive and time consuming than FISH testing, which could be reserved for a confirmatory second step. Given the relative rarity of ROS1 gene rearrangements, further refinement of the screening population should be considered, limiting it to adenocarcinoma histology and further enriching by assessing only those patients with tumours that are wild type for EGFR, ALK and other more routinely assessed molecular abnormalities. Patients presenting at a younger age and those who are never smokers are further options. LEGENDS:

Figure 1; a) A cell block from a transbronchial needle aspirate of lymph node shows a micropapillary architecture. b) Immunohistochemistry for ROS1 protein shows variably granular cytoplasmic staining of mainly moderate intensity in 80% of cells within micropapillary clusters. c) A biopsy shows an adenocarcinoma showing focal signet ring cell morphology. d) This case shows moderate staining in the 90% of cells for ROS1 protein, with focal sub-membranous accentuation of staining. e) A nodal biopsy shows replacement by an adenocarcinoma with mixed micropapillary and cribriform patterns. f) This case show strong staining in the 90% of cells for ROS1 protein.

Figure 2: a) Fluoresence in-situ hybridisation (FISH) testing of case 5 shows a signal pattern characteristic of an unbalanced ROS1 translocation resulting in loss of the red probe, b) Fluorescence in-situ hybridisation (FISH) testing shows 3’ (red) and 5’ (green) regions of the ROS1 gene separated by rearrangement.

Figure 3: a-c) Mediastinal lymph nodes (a-c) and tumour in left hilum (d-f) show persistent response to crizotinib over 2.5 years.

Figure 4: Tumour cells are negative for ROS1 protein, but there is positive staining of background pneumocytes. REFERENCES:

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