ROS1 Immunohistochemistry Research Articles

A stitch in time saves nine: external quality assessment rounds demonstrate improved quality of biomarker analysis in lung cancer.

Biomarker analysis has become routine practice in the treatment of non-small cell lung cancer (NSCLC). To ensure high quality testing, participation to external quality assessment (EQA) schemes is essential. This article provides a longitudinal overview of the EQA performance for EGFR, ALK, and ROS1 analyses in NSCLC between 2012 and 2015.The four scheme years were organized by the European Society of Pathology according to the ISO 17043 standard. Participants were asked to analyze the provided tissue using their routine procedures.Analysis scores improved for individual laboratories upon participation to more EQA schemes, except for ROS1 immunohistochemistry (IHC). For EGFR analysis, scheme error rates were 18.8%, 14.1% and 7.5% in 2013, 2014 and 2015 respectively. For ALK testing, error rates decreased between 2012 and 2015 by 5.2%, 3.2% and 11.8% for the fluorescence in situ hybridization (FISH), FISH digital, and IHC subschemes, respectively. In contrast, for ROS1 error rates increased between 2014 and 2015 for FISH and IHC by 3.2% and 9.3%. Technical failures decreased over the years for all three markers.Results show that EQA contributes to an ameliorated performance for most predictive biomarkers in NSCLC. Room for improvement is still present, especially for ROS1 analysis.

Acral Lentiginous Melanoma Harboring a ROS1 Gene Fusion With Clinical Response to Entrectinib.

ROS1 gene fusions demonstrate oncogenic activity, and patients with non-small-cell lung cancer (NSCLC) harboring a ROS1 fusion benefit from the use of a ROS1 inhibitor; however, clinical response to ROS1 inhibitors remains largely uncharacterized outside of NSCLC. ROS1 fusions have been identified in multiple tumor types but have not been reported in cutaneous melanoma. Tumors from 22 patients with acral lentiginous melanoma (ALM) were analyzed with targeted RNA sequencing to detect fusions in ROS1, NTRK1, NTRK2, NTRK3, and ALK genes. A patient harboring a ROS1 fusion was enrolled in a phase I basket trial of a ROS1/TRK/ALK inhibitor (entrectinib). An additional 78 tumors with different subtypes of melanoma were screened by ROS1 immunohistochemistry. Targeted sequencing identified a GOPC-ROS1 fusion in a patient with ALM. The patient underwent a dramatic and durable response to entrectinib, with a RECIST (version 1.1) partial response of -38% at 3 months and -55% at 11 months. The response is ongoing, and the patient has not developed any new lesions. No additional ROS1 fusions were identified by immunohistochemistry, resulting in a frequency of 3.0% in ALM and 1.3% in all melanomas. ROS1 fusions occur and can respond to targeted therapy in cutaneous melanoma; however, they may be specific to ALM subtype. This report expands knowledge of ROS1 inhibitor response outside of NSCLC and identifies new therapeutic options for a subset of patients with ALM.

Prospective Clinical Integration of an Amplicon-Based Next-Generation Sequencing Method to Select Advanced Non–Small-Cell Lung Cancer Patients for Genotype-Tailored Treatments

IntroductionA substantial fraction of non–small-cell lung cancers (NSCLCs) harbor targetable genetic alterations. In this study, we analyzed the feasibility and clinical utility of integrating a next-generation sequencing (NGS) panel into our routine lung cancer molecular subtyping algorithm. Patients and MethodsAfter routine pathologic and molecular subtyping, we implemented an amplicon-based gene panel for DNA analysis covering mutational hot spots in 22 cancer genes in consecutive advanced-stage NSCLCs. ResultsWe analyzed 109 tumors using NGS between December 2014 and January 2016. Fifty-six patients (51%) were treatment-naive and 82 (75%) had lung adenocarcinomas. In 89 cases (82%), we used samples derived from lung cancer diagnostic procedures. We obtained successful sequencing results in 95 cases (87%). As part of our routine lung cancer molecular subtyping protocol, single-gene testing for EGFR, ALK, and ROS1 was attempted in nonsquamous and 3 squamous-cell cancers (n = 92). Sixty-nine of 92 samples (75%) had sufficient tissue to complete ALK and ROS1 immunohistochemistry (IHC) and NGS. With the integration of the gene panel, 40 NSCLCs (37%) in the entire cohort and 30 NSCLCs (40%) fully tested for ALK and ROS1 IHC and NGS had actionable mutations. KRAS (24%) and EGFR (10%) were the most frequently mutated actionable genes. Ten patients (9%) received matched targeted therapies, 6 (5%) in clinical trials. ConclusionThe combination of IHC tests for ALK and ROS1 and amplicon-based NGS is applicable in routine clinical practice, enabling patient selection for genotype-tailored treatments.

MA07.07 Ceritinib in ROS1-Rearranged Non-Small-Cell Lung Cancer: An Update of Korean Nationwide Phase II Study

ROS1 rearrangement is a distinct molecular subset of non-small-cell lung cancer (NSCLC). We investigated the efficacy and safety of ceritinib in patients with ROS1-rearranged NSCLC. We enrolled 32 patients with advanced NSCLC who tested positive for ROS1 rearrangement by fluorescent in situ hybridization (FISH). ROS1 immunohistochemistry (IHC) and next-generation sequencing (NGS) was performed in available tumor samples. Ceritinib 750mg was administered once daily and the primary endpoint was objective response rate (ORR) by central independent radiologic review. The secondary endpoints included disease control rate (DCR), duration of response, progression-free survival (PFS), overall survival (OS), toxicity and concordance between FISH and IHC. ROS1 fusion partners were identified with the use of next-generation sequencing (NGS) in available tumor samples. Between June 7, 2013, and February 1, 2016, a total of 404 patients underwent ROS1 prescreening, and 32 ROS1+ (by FISH) patients were enrolled. All patients except two (who did not respond to ceritinib) were crizotinib naïve. The median age of all patients was 62 years, and there were 24 females (75%). The majority of patients (84%) were never smokers, and all had adenocarcinoma histology. The median number of previous treatments before study enrollment was 3 (range, 2-7) and 17 (53%) patients had received three or more lines of chemotherapy. At the time of the data cut-off (April 18, 2016), the median follow-up was 7.5 months, and 15 (47%) patients had discontinued treatment. Of the 32 patients enrolled, 28 patients were evaluable for response by independent radiologic review. ORR was 63% (95% CI, 45.7-79.3), with 1 complete response and 19 partial responses. The median duration of response was 10.0 months (range, 0.4+-18.4+). Among 11 tumors that were tested by NGS, we identified 7 ROS1 fusion partners including ROS1-CD74, ROS1-SLC34A2, and ROS1-EZR. The median progression-free survival was 19.3 months (95% CI, 7.2-not reached), with 17 (53%) patients still in follow-up for progression. The median overall survival was not reached at the time of the data cut-off. Of 5 patients with retrospectively confirmed brain metastases, intracranial disease control was reported in 4 patients (80%). Gastrointestinal adverse events (vomiting, nausea, diarrhea) mostly grade 1-2, were the most frequent adverse events (80%); these events were manageable. Ceritinib demonstrated potent clinical activity in patients with advanced, ROS1-rearranged NSCLC, who received at least one prior line of platinum-based chemotherapy. ROS1 rearrangement defines a second molecular subgroup of NSCLC for which ceritinib is highly active (ClinicalTrials.gov number, NCT01964157).

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.

To assess the prevalence of ROS1 rearrangements in a retrospective and prospective diagnostic Australian cohort and evaluate the effectiveness of immunohistochemical screening. A retrospective cohort of 278 early stage lung adenocarcinomas and an additional 104 prospective non-small-cell lung cancer (NSCLC) cases referred for routine molecular testing were evaluated. ROS1 immunohistochemistry (IHC) was performed (D4D6 clone, Cell Signaling Technology) on all cases as well as fluorescence in-situ hybridization (FISH) using the ZytoVision and Abbott Molecular ROS1 FISH probes, with ≥15% of cells with split signals considered positive for rearrangement. Eighty-eight cases (32%) from the retrospective cohort showed staining by ROS1 IHC, and one case (0.4%) showed ROS1 rearrangement by FISH. Nineteen of the prospective diagnostic cases showed ROS1 IHC staining, 12 (12%) cases of which were confirmed as ROS1 rearranged by FISH. There were no ROS1 rearranged cases that showed no expression of ROS1 with IHC. The ROS1 rearranged cases in the prospective cohort were all EGFR wild-type and anaplastic lymphoma kinase (ALK) rearrangement-negative. The sensitivity of ROS1 IHC in the retrospective cohort was 100% and specificity was 76%. ROS1 rearrangements are rare events in lung adenocarcinomas. Selection of cases for ROS1 FISH testing, by excluding EGFR/ALK-positive cases and use of IHC to screen for potentially positive cases, can be used to enrich for the likelihood of identifying a ROS1 rearranged lung cancer and prevent the need to undertake expensive and time-consuming FISH testing in all cases.

1205PD - Ceritinib in ROS1-rearranged non-small-cell lung cancer: a Korean nationwide phase II study

ROS1 rearrangement is a distinct molecular subset of non-small-cell lung cancer (NSCLC). We investigated the efficacy and safety of ceritinib in patients with ROS1-rearranged NSCLC. We enrolled 32 patients with advanced NSCLC who tested positive for ROS1 rearrangement by fluorescent in situ hybridization (FISH). ROS1 immunohistochemistry (IHC) and next-generation sequencing (NGS) were performed in available tumor samples. The primary endpoint was objective response rate (ORR) by central independent radiologic review. The secondary endpoints included disease control rate (DCR), duration of response, progression-free survival (PFS), overall survival (OS), toxicity and concordance between FISH, IHC and NGS. Between June 7, 2013, and February 1, 2016, a total of 404 patients underwent ROS1 prescreening, and 32 ROS1+ (by FISH) patients were enrolled. The median age of all patients was 62 years, and the majority of patients (84%) were never smokers, and all had adenocarcinoma histology. The median number of previous treatments was 3 (range, 2-7) and 17 (53%) patients had received three or more lines of chemotherapy. At the time of the data cut-off (April 18, 2016), the median follow-up was 7.5 months, and 15 (47%) patients had discontinued treatment. The ORR was 63% (95% CI, 45.7-79.3), with 1 complete response and 19 partial responses. The median duration of response was 10.0 months (range, 0.4 + -18.4+). Among 11 tumors that were tested by NGS, we identified 7 ROS1 fusion partners including ROS1-CD74, ROS1-SLC34A2, and ROS1-EZR. The median progression-free survival was 19.3 months (95% CI, 7.2-not reached), and the median overall survival was not reached at the time of the data cut-off. Of 5 patients with retrospectively confirmed brain metastases, intracranial disease control was reported in 4 patients (80%). Gastrointestinal adverse events, mostly grade 1-2, were the most frequent adverse events (80%); these events were manageable. Ceritinib demonstrated potent clinical activity in patients with advanced, ROS1-rearranged NSCLC, who received at least one prior line of platinum-based chemotherapy. ROS1 rearrangement defines a second molecular subgroup of NSCLC for which ceritinib is highly active.

Abstract 4344: Comparison of nCounter, immunohistochemistry, RT-PCR and FISH to detect ALK, ROS1 and RET rearrangements in advanced non-small cell lung cancer (NSCLC)

Abstract Background: Targetable rearrangements in anaplastic lymphoma kinase (ALK), ROS1, and RET genes are present in approximately 7% of patients (p) with advanced NSCLC. Current methods for detecting gene fusions are based on FISH (FDA-approved companion diagnostic test for ALK), immunohistochemistry (IHC) or RT-PCR. However, these tests have disadvantages in terms of sensitivity, cost and throughput, and often show discrepancies. The nCounter platform allows simultaneous detection with no enzymatic reaction, within 72 hours, of several fusion genes in formalin-fixed paraffin-embedded (FFPE) samples using a transcript-based method. Methods: A custom set of 5′and 3′ probes and fusion-specific probes to detect ALK, ROS1 and RET fusion transcripts was designed and evaluated. A panel of ALK-ROS1-RET positive cell lines (H2228, H3122 [EML4-ALK], SUDHL-1 [NPM-ALK], HCC78 [SLC34A2-ROS1], BaF3 pBABE [CD74-ROS1], LC2/ad [RET]) and negative cell lines (PC9, H1975 [EGFR mut], H460, H23 [KRAS mut]) was used to validate the technique. Then, a total of 70 FFPE samples was analyzed, 49 of them positive by FISH, IHC and/or qRT-PCR for ALK (n = 30), ROS1 (n = 17) and RET (n = 2). Total RNA was isolated from cell lines and FFPE and < 225 ng were used for hybridization. Raw counts were normalized using positive controls, negative controls and four house-keeping genes (GAPDH, GUSB, OAZ1 and POLR2A) as described in Lira et al. J Mol Diagn 2013. Positive and negative translocations were defined by two criteria: (1) a 3’/5’ ratio score of > 2.0 and ≤ 2.0 respectively or/and (2) a signal for a fusion-specific probe above background. Response to crizotinib by RECIST criteria was retrospectively collected in p with ALK-positive NSCLC by any technique. Results: nCounter sensitivity to detect fusion transcripts ALK, ROS1 and RET in cell lines was 100% using the two criteria (3’/5’ and direct probes) and specificity was also 100%. Among 20 ALK-FISH-positive p, ALK 3’-5’ scoring was positive in 18 (95%). One p was non-evaluable (NE) by ALK 3’-5’ scoring. Among 48 ALK-FISH-negative p, nCounter score was positive in 13 (27%). All p positive for ALK by nCounter were either positive or NE for ALK by IHC. A total of 17 p were treated with crizotinib, 16 of whom responded to treatment and were positive by nCounter. Regarding FISH, five p responding to crizotinib were negative and one was NE. Finally, one p not responding to crizotinib was positive by RT-PCR but negative by nCounter. Conclusion: The ALK/ROS1/RET nCounter-based assay is a highly sensitive screening assay that identifies ALK-FISH-negative/NE NSCLC patients who could benefit from treatment with ALK inhibitors. Citation Format: Cristina Teixido, Noemí Reguart, Ana Giménez-Capitán, Miguel Ángel Molina-Vila, Patricia Galván, Sonia Rodriguez, Laia Paré, Santiago Viteri, Vicente Peg, Zaira Yeste, Núria Viñolas, Rafael Rosell, Aleix Prat. Comparison of nCounter, immunohistochemistry, RT-PCR and FISH to detect ALK, ROS1 and RET rearrangements in advanced non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4344.

Rare Incidence of ROS1 Rearrangement in Cholangiocarcinoma.

PurposeThe recent discovery and characterization of an oncogenic ROS1 gene rearrangement has raised significant interest because small molecule inhibitors are effective in these tumors. The aim of this study was to determine frequency and clinicopathological features associated with ROS1 rearrangement in patients with cholangiocarcinoma (CCA).Materials and MethodsA total of 261 patients who underwent surgery for CCA between October 1997 and August 2013 were identified from an international, multi-institutional database. ROS1 rearrangement was evaluated by break-apart fluorescence in situ hybridization using tissue microarrays of these patients.ResultsOf 261 CCA evaluated, three cases (1.1%) showed ROS1 rearrangement by fluorescence in situ hybridization (FISH), all of which were derived from intrahepatic origin. ROS1 protein expression was observed in 38 samples (19.1%). Significantly larger tumor size was observed in ROS1 immunohistochemistry (IHC)–negative patients compared with ROS1 IHC–positive patients. ROS1 FISH–positive patients had a single tumor with a median size of 4 cm and well-to-moderate differentiation. Overall, there was no difference in terms of baseline characteristics, overall survival, and recurrence-free survival between ROS1-positive and -negative patients.ConclusionROS1 rearrangement was detected in 1.1% of CCA patients. Although rare, conduct of clinical trials using ROS1 inhibitors in these genetically unique patients is warranted.

ROS-1 rearrangements in non-small cell lung cancer (NSCLC): a new target for a small subset of patients but a giant leap in therapeutics

ROS1 is a membrane tyrosine kinase receptor of which encoding gene has recently been found rearranged in non-small cell lung cancer (NSCLC), leading to constitutive activation of the ROS1 kinase activity. The ROS1 gene rearrangement has been described in roughly 1 % of patients with NSCLC and such rearrangement needs to be assessed by a FISH break-apart assay (CD74-ROS1) with ROS1 immunohistochemistry as a screening tool in tumor histological samples. The clinical and histological features of patients with ROS1-rearranged NCSCL include adenocarcinoma histology with TTF-1 expression, predominantly young women patients, and never-smokers. Targeting ROS1 fusion proteins with a kinase inhibitor of ROS1 kinase domain, such as crizotinib, seems to be promising. Indeed, this potential inhibitor of ROS1 kinase shows evidence of clinical efficiency either in patients treated off-label, in the crizotinib phase I trial, or in retrospective series. New kinase inhibitors such as ceritinib or lorlatinib also showed promising activities either in vitro, in mice xenograft models, or in patients treated off-label.

Molecular characterization of inflammatory myofibroblastic tumors with frequent ALK and ROS1 gene fusions and rare novel RET rearrangement.

Approximately 50% of conventional inflammatory myofibroblastic tumors (IMTs) harbor ALK gene rearrangement and overexpress ALK. Recently, gene fusions involving other kinases have been implicated in the pathogenesis of IMT, including ROS1 and in 1 patient PDGFRB. However, it remains uncertain whether the emerging genotypes correlate with clinicopathologic characteristics of IMT. In this study, we expand the molecular investigation of IMT in a large cohort of different clinical presentations and analyze for potential genotype-phenotype associations. Criteria for inclusion in the study were typical morphology and tissue availability for molecular studies. The lack of ALK immunoreactivity was not an excluding factor. As overlapping gene fusions involving actionable kinases are emerging in both IMT and lung cancer, we set out to evaluate abnormalities in ALK, ROS1, PDGFRB, NTRK1, and RET by fluorescence in situ hybridization. In addition, next-generation paired-end RNA sequencing and FusionSeq algorithm was applied in 4 cases, which identified EML4-ALK fusions in 2 cases. Of the 62 IMTs (25 children and 37 adults), 35 (56%) showed ALK gene rearrangement. Of note, EML4-ALK inversion was noted in 7 (20%) cases, seen mainly in the lung and soft tissue of young children including 2 lesions from newborns. There were 6 (10%) ROS1-rearranged IMTs, all except 1 presenting in children, mainly in the lung and intra-abdominally and showed a distinctive fascicular growth of spindle cells with long cell processes, often positive for ROS1 immunohistochemistry. Two of the cases showed TFG-ROS1 fusions. Interestingly, 1 adult IMT revealed a RET gene rearrangement, a previously unreported finding. Our results show that 42/62 (68%) IMTs are characterized by kinase fusions, offering a rationale for targeted therapeutic strategies. Interestingly, 90% of fusion-negative IMTs were seen in adults, whereas >90% of pediatric IMT showed gene rearrangements. EML4-ALK inversion and ROS1 fusions emerge as common fusion abnormalities in IMT, closely recapitulating the pattern seen in lung cancer.

Frequent aerogenous spread with decreased E-cadherin expression of ROS1-rearranged lung cancer predicts poor disease-free survival.

ROS1 rearrangement has been found in a subset of lung cancer and ROS1-rearranged tumors are sensitive to ALK kinase inhibitors. This study sought to evaluate the clinicopathological implications and histomorphological characteristics of ROS1-rearranged tumors, especially micropapillary and aerogenous spread growth and to investigate the usefulness of ROS1 immunohistochemistry as a diagnostic test for ROS1 rearrangement. ROS1 rearrangement characterizations by fluorescence in situ hybridization and ROS1 protein and E-cadherin expression by immunohistochemistry were performed using 754 non-small cell lung cancer surgical specimens. ROS1 rearrangement was identified in 10 samples. Histologically, all 10 ROS1-rearranged tumors harbored an adenocarcinoma component. Significantly, we noted a high association between ROS1 rearrangement with a micropapillary component (p<0.001), aerogenous spread (p=0.002), and E-cadherin loss (p=0.049). Survival analysis showed that ROS1 rearrangement was significantly associated with a higher risk of tumor recurrence (p=0.024). The best criterion to detect ROS1-rearrangement by immunohistochemistry was an H-score of ≥100, with a sensitivity and specificity of 90% and 93.5%, respectively. ROS1-rearranged adenocarcinoma exhibited distinct morphological and clinicopathological features. Decreased membranous E-cadherin expression and aerogenous spread may be associated with worse disease-free survival. ROS1 immunohistochemistry correlated well with ROS1 gene rearrangement.

Expression of ROS1 predicts ROS1 gene rearrangement in inflammatory myofibroblastic tumors

Inflammatory myofibroblastic tumor is a distinctive, rarely metastasizing mesenchymal neoplasm composed of fascicles of spindle cells with a prominent inflammatory infiltrate. Roughly 50% of inflammatory myofibroblastic tumors harbor ALK receptor tyrosine kinase gene rearrangements. Such tumors are usually positive for ALK by immunohistochemistry. The molecular pathogenesis of ALK-negative inflammatory myofibroblastic tumors is largely unknown. A recent study identified rearrangements of ROS1 (another tyrosine kinase receptor) in a subset of ALK-negative inflammatory myofibroblastic tumors. Immunohistochemistry for ROS1 has been shown to correlate with ROS1 rearrangement in lung adenocarcinomas. The purpose of this study was to determine whether immunohistochemistry for ROS1 could predict ROS1 rearrangement in inflammatory myofibroblastic tumor. In total, 30 inflammatory myofibroblastic tumors were evaluated, including 21 ALK-positive tumors (10 confirmed to harbor ALK rearrangements, with TPM3, CLTC, RANPB2, and FN1 fusion partners) and 9 ALK-negative tumors (including 2 known to harbor ROS1 rearrangements). Immunohistochemistry was performed on whole tissue sections following pressure cooker antigen retrieval using a rabbit anti-ROS1 monoclonal antibody. The results were scored as ‘positive’ or ‘negative,‘ and the pattern of staining was recorded. Three ALK-negative inflammatory myofibroblastic tumors (including both tumors with known ROS1 rearrangements) showed immunoreactivity for ROS1, whereas all ALK-positive inflammatory myofibroblastic tumors were negative for ROS1. One ROS1-positive inflammatory myofibroblastic tumor (with YWHAE-ROS1 fusion) showed strong, diffuse cytoplasmic and nuclear staining; one case (with TFG-ROS1 fusion) showed weak, diffuse and dot-like cytoplasmic staining; and one case (fusion partner unknown) showed moderate, diffuse and dot-like cytoplasmic staining. Expression of ROS1 correlates with ROS1 gene rearrangement in inflammatory myofibroblastic tumor. These findings suggest that immunohistochemistry for ROS1 may be useful to support the diagnosis of a subset of inflammatory myofibroblastic tumors and may select some clinically aggressive cases for targeted therapy directed against ROS1.

20. ROS1 immunohistochemistry is not a worthwhile screening test in breast carcinoma

The FIG-ROS1 translocation was initially reported in a glioblastoma cell line, but has gained prominence in lung cancer where it has been reported in up to 2% of adenocarcinomas. ROS1 is a member of the tyrosine-kinase gene family and the FIG-ROS1 translocation results in a constitutively active tyrosine kinase which drives neoplasia. Crizotinib is a tyrosine kinase inhibitor which inactivates the ROS1 tyrosine kinase through crystallization, and has shown early promise in the treatment of lung carcinomas which harbor the FIG-ROS1 translocation. Recently a study using next generation sequencing technologies and openly available exome and transcriptome datasets suggested that up to 0.2% of breast carcinomas harbor the FIG-ROS1 translocation. It was further suggested that ROS1 expression may correlate with other features associated with poor outcome such as a high proliferative index. Immunohistochemistry has been found to be an effective screening method for ROS1 translocations in lung cancer, however few studies have been done to evaluate its applicability to breast cancer. We sought to investigate the role of screening immunohistochemistry for ROS1 as a potential marker of targeted therapy in a large cohort of breast carcinoma. We constructed a tissue microarray (TMA) including all patients with breast cancer identified from the database of the department of Anatomical Pathology Royal North Shore Hospital from 2009 to 2012. Screening immunohistochemistry for ROS1 (clone D4D6) was performed on the TMAs and for any cases with any potential staining, immunohistochemistry was repeated on whole sections. None of 631 patients (mean age 61, 99% female) consecutive breast carcinomas demonstrated any positive staining. We conclude that immunohistochemistry for ROS1 is not a worthwhile screening test in breast cancer.

ROS1 Immunohistochemistry Among Major Genotypes of Non–Small-Cell Lung Cancer

BackgroundROS1 gene fusions cause several cancers by constitutively activating the ROS1 tyrosine kinase receptor. ROS1-targeted inhibitor therapy improves survival in the approximately 1% to 2% of patients with lung adenocarcinoma with ROS1 gene fusions. Although fluorescence in situ hybridization (FISH) is the standard diagnostic procedure for detecting ROS1 rearrangements, we studied immunohistochemistry (IHC). Materials and MethodsROS1 IHC was performed on a selected cohort of 33 lung adenocarcinoma whole tissue specimens with alterations in the EGFR (n = 5), KRAS (n = 5), ERBB2 (HER2) (n = 3), ROS1 (n = 6), ALK (n = 5), and RET (n = 3) genes and pan-negative (n = 6) detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and FISH. ResultsIn the cohort of 33 specimens, both ROS1 gene fusion using RT-PCR and high ROS1 protein expression using IHC were detected in 6 specimens. Of these 6 specimens, 5 were also positive by FISH for ROS1 gene rearrangements. All 27 lung cancer specimens that were negative for ROS1 rearrangements by genetic testing had no to low ROS1 protein expression. ConclusionWe have optimized ROS1 IHC and scoring to provide high sensitivity and specificity for detecting ROS1 gene rearrangements in whole tissue. ROS1 IHC could be a practical and cost-effective method to screen for ROS1 gene rearrangements.

Ros1 Rearrangements and Copy Number Alterations in Nsclc Patients: High Frequency of Ros1 Deletions

ABSTRACT Aim: Recently, c-ros oncogene 1, receptor tyrosine kinase (ROS1, 6q22) has been identified as a driver oncogene in non-small-cell lung carcinomas (NSCLC). Patients carrying ROS1 translocations are candidates to targeted therapy with crizotinib®. We aimed to determine the prevalence, patterns and fusion partners in ROS1-rearranged NSCLC samples and to characterize ROS1 copy number alterations (CNAs) by fluorescence in situ hybridization (FISH). Methods: A total of 216 NSCLC patients (median age 64 years, 66% males, 51% smokers, 73% adenocarcinomas (ADC), 35% stage 4), were screened for ROS1 abnormalities using a break-apart FISH probe (Abbott Molecular). To characterize ROS1 partners, clones from Human 32K BAC Re-Array Library were used to develop CD74, SLC34A2 and EZR probes. Immunohistochemistry (IHC) with ROS1 D4D6 antibody (Cell Signalling Technology) was also performed in ROS1-translocated cases. Correlation with clinico-pathologic information was investigated. Results: Four samples were ROS1 translocated (1.9%): three showed deletion of the non-translocated allele and one had a 5'ROS1 deletion. One harbored a SLC34A2-ROS1 fusion and another a CD74-ROS1. No fusion partner was identified in the other two samples. All cases were positive for ROS1 IHC staining, three presenting a cytoplasmic pattern and one with membranous pattern. ROS1-translocated tumors were ADC, all EGFR, KRAS, and ALK wt. We observed an association with non-smoking habit (P=0.036). Regarding CNAs in ROS1 non-rearranged cases, 43.5% had gains (3-6 copies) and 26.4% had deletions. Conclusions: We confirm the low prevalence of ROS1-translocated lung ADC in a Spanish population. Moreover, our study reveals a significant frequency of ROS1 deletion as a potentially relevant aberration, also in ROS1-translocated patients. Disclosure: All authors have declared no conflicts of interest.

Immunohistochemical detection of ROS1 is useful for identifying ROS1 rearrangements in lung cancers

The recent discovery and characterization of an oncogenic ROS1 gene fusion in a subset of lung cancers has raised significant clinical interest because small molecule inhibitors may be effective to these tumors. As lung cancers with ROS1 rearrangements comprise only 1–3% of lung adenocarcinomas, patients with such tumors must be identified to gain optimal benefit from molecular therapy. Recently, immunohistochemical analyses using a novel anti-ROS1 rabbit monoclonal antibody (D4D6) have shown promise for accurate identification of ROS1-rearranged cancers. To validate this finding, we compared the immunostaining results of tissue microarrays (TMAs) containing 17 ROS1-rearranged and 253 ROS1-non-rearranged lung carcinomas. All 17 ROS1-rearranged cancers showed ROS1 immunoreactivity mostly in a diffuse and moderate-to-strong manner with an H-score range of 5–300 (median, 260). In contrast, 69% of ROS1-non-rearranged cancers lacked detectable immunoreactivity, whereas the remaining 31% showed reactivity mainly in a weak or focal manner. The H-score for the entire ROS1-non-rearranged group ranged from 0 to 240 (median, 0). The difference in H-score between the two cohorts was statistically significant, and the H-score cutoff (≥150) allowed optimal discrimination (94% sensitivity and 98% specificity). Similar but slightly less-specific performance was achieved using the extent of diffuse (≥75%) staining or ≥2+ staining intensity as cutoffs. CD74-ROS1 and EZR-ROS1 fusions were significantly associated with at least focal globular immunoreactivity and plasma membranous accentuation, respectively, and these patterns were specific to ROS1-rearranged cases. Although full-length ROS1 is expressed in some ROS1-non-rearranged cases, we showed that establishment of an optimal set of interpretative criteria makes ROS1 immunohistochemistry a valuable method to rapidly and accurately screen lung cancer patients for appropriate molecular therapy.

11. ROS1 gene rearrangements in non-small cell lung carcinoma - A new genetic target

<h3>Background</h3> Targeted therapies aimed at specific molecular genetic alterations are revolutionising cancer treatment, particularly in non-small cell lung cancer (NSCLC). Driver mutations involving translocation of the <i>ROS1</i> gene have recently been identified in NSCLC and show promise as a target for tyrosine kinase inhibitors. <h3>Aim</h3> To investigate the incidence and clinicopathological features of NSCLC harbouring <i>ROS1</i> rearrangements and the accuracy of IHC compared to FISH at identifying these tumours. Methods: We tested for <i>ROS1</i> translocations using a FISH break-apart probe and immunohistochemistry (IHC) in (1) tissue micro-arrays from a retrospective cohort of 308 early stage adenocarcinomas, (2) a prospective cohort of 23 NSCLC, selected on clinical grounds for mutation testing. <h3>Results</h3> Only 1 of 308 cases was positive by FISH in the retrospective cohort giving a 0.3% incidence of <i>ROS1</i> gene rearrangements. ROS1 IHC was positive in 7 (2.3%) of these cases, including the FISH+ case (sensitivity 100%, specificity 98%). In the prospective cohort, three cases with <i>ROS1</i> rearrangement were identified by FISH (all IHC positive). All 4 cases of ROS1 gene rearrangement identified by FISH occurred in adenocarcinomas that were <i>EGFR</i>-wild-type, ALST-negative, from non-smoking females (age range 33-81 years, mean 59). Two were of Asian ethnicity. <h3>Conclusions</h3> <i>ROS1</i> gene rearrangements occur in a very small percentage of lung adenocarcinomas and are mutually exclusive with EGFR and <i>ALK</i> alterations. Screening with IHC may be a suitable method of reducing the number of cases requiring FISH.

ROS1 Immunohistochemistry for Detection of ROS1-Rearranged Lung Adenocarcinomas

ROS1 gene rearrangements are reported in 1% to 2% of lung adenocarcinomas (ACAs) and are associated with response to the multitargeted tyrosine kinase inhibitor crizotinib. ROS1 rearrangements can be detected using fluorescence in situ hybridization (FISH); however, immunohistochemistry (IHC) for ROS1 protein is a promising alternate screening modality. In this study, we examine the correlation between ROS1 IHC and FISH and describe the clinicopathologic characteristics of ROS1-rearranged lung tumors. ROS1 IHC was performed using clone D4D6 on whole-tissue sections. In a validation cohort, IHC was compared with ROS1 break-apart FISH in 53 cases of lung ACA enriched for an absence of known genetic alterations and never-smoking status. In a screening cohort, we performed ROS1 IHC on 167 consecutive cases of lung ACA from a routine molecular diagnostic practice and confirmed positive results by FISH. In the validation cohort, 6 cases (11%) were both FISH and IHC positive. One FISH-negative case was strongly ROS1 IHC positive. All IHC-negative cases were FISH negative. In the screening cohort, 2 of 167 (1.2%) had strong, diffuse ROS1 protein expression; a rearrangement was confirmed by FISH in both. ROS1-translocated tumors were wild type for EGFR, KRAS, and ALK and commonly had solid growth with mucinous/cribriform features and psammomatous calcification. ROS1 protein expression in tumor cells is 100% sensitive and 92% specific for ROS1 rearrangements by FISH. ROS1 IHC is an effective screening tool for this rare but clinically important subset of lung ACAs.

Analysis of Receptor Tyrosine Kinase ROS1-Positive Tumors in Non–Small Cell Lung Cancer: Identification of a FIG-ROS1 Fusion

To deepen our understanding of mutant ROS1 expression, localization, and frequency in non-small cell lung cancer (NSCLC), we developed a highly specific and sensitive immunohistochemistry (IHC)-based assay that is useful for the detection of wild-type and mutant ROS1. We analyzed 556 tumors with the ROS1 D4D6 rabbit monoclonal antibody IHC assay to assess ROS1 expression levels and localization. A subset of tumors was analyzed by FISH to determine the percentage of these tumors harboring ROS1 translocations. Using specific and sensitive IHC assays, we analyzed the expression of anaplastic lymphoma kinase (ALK), EGFR L858R, and EGFR E746-A750del mutations in a subset of lung tumors, including those expressing ROS1. In our NSCLC cohort of Chinese patients, we identified 9 (1.6%) tumors expressing ROS1 and 22 (4.0%) tumors expressing ALK. FISH identified tumors with ALK or ROS1 rearrangements, and IHC alone was capable of detecting all cases with ALK and ROS1 rearrangements. ROS1 fusion partners were determined by reverse transcriptase PCR identifying CD74-ROS1, SLC34A2-ROS1, and FIG-ROS1 fusions. Some of the ALK and ROS1 rearranged tumors may also harbor coexisting EGFR mutations. NSCLC tumors with ROS1 rearrangements are uncommon in the Chinese population and represent a distinct entity of carcinomas. The ROS1 IHC assay described here is a valuable tool for identifying patients expressing mutant ROS1 and could be routinely applied in clinical practice to detect lung cancers that may be responsive to targeted therapies.