ROS1 Immunohistochemistry Research Articles

P38.16 Expression of ROS1 Gene Rearrangement in Pulmonary Inflammatory Myofibroblastic Tumor With Cerebral Metastasis: Case Report

Primary pulmonary inflammatory myofibroblastic tumor (IMT) is a rare benign neoplasm; the tumor has been characterized by the application of immunohistochemical techniques, molecular biology and cytogenetics, which are very precious for the diagnosis. The purpose of this case was to determine whether immunohistochemistry for ROS1 could predict ROS1 rearrangement in inflammatory myofibroblastic tumor. A 50-year-old Moroccan man with history of mitral stenosis and mechanical valve replacement; presented to our hospital with a complaint of fatigue; headaches and lower back pain for one month. Contrast-enhanced computed tomography of his chest revealed a heterogenous well-defined mass lesion with central irregular calcification with no mediastinal lymphadenopathy or satellite nodules; this mass involving the left seventh rib and vertebral body and left atrial cavity. CT brain showed four solid enhancing irregular shaped mass lesions with hypodense areas consistent with metastasis CT-guided biopsy of the chest lesion showed fibrosis with dystrophic calcification; and non-specific chronic inflammation. He underwent radical excision of the right frontal lesion, and histopathology was distinctive fascicular growth of spindle cells with with a prominent inflammatory infiltrate, showed weak, diffuse and dot-like cytoplasmic staining with anaplastic lymphoma kinase (ALK)-negative immunohistochemistry and Fluorescence in situ hybridization (FISH) analysis was performed on interphase nuclei on 5 micron thin sections of formalin-fixed paraffin-embedded tissue that was found to be positive for TFG-ROS1 fusion. Because surgical excision was not possible crizotinib therapy (250 mg, bid) was chosen for the existence of ROS1 fusion gene with brain radiotherapy boost to metastasis. Treatment with glucocorticoids (methylprednisolone 1 mg/kg) and radiotherapy (5 days/week for 3 weeks at 3 Gy/fraction, 45 Gy/15 days) was started for cerebral metastasis . After taking crizotinib for 3 months, the strong changes in the size of the pulmonary mass was showed at chest chest computed tomography with decreased infiltration around the adjacent structures. Inflammatory pseudotumour of the lung with brain metastasis is very uncommon; we must continued therapy with crizotinib and long-term follow-up is necessary, for detection of recurrence, metastasis and to understand the natural course, especially in patients with myofibroblastic differentiation ROS1 positive Crizotinib is effective for pulmonary IMT, and should be taken into consideration for the treatment of inoperable pulmonary IMT patients who lack ALK rearrangement.

Clinicopathological features and immunohistochemical utility of NTRK-, ALK-, and ROS1-rearranged papillary thyroid carcinomas and anaplastic thyroid carcinomas

NTRK1/3, ALK, and ROS1 translocations have been reported in a minor subset of papillary thyroid carcinomas (PTCs). We aimed to elucidate the prevalence and clinicopathological characteristics of these gene rearrangements and the utility of immunohistochemistry (IHC) in PTC and anaplastic thyroid carcinoma (ATC). We screened nonradiation-exposed cases of 307PTCs and 16 ATCs by IHC for pan-Trk, ALK, and ROS1, followed by fluorescence in situ hybridization (FISH). In the PTC group, IHC for pan-Trk, ALK, and ROS1 was positive in 18 cases (5.9%), 1 case (0.3%), and 12 cases (3.9%), respectively. Among the pan-Trk IHC-positive cases (n=18), 2 cases (11.1%; 0.7% of all PTCs) had NTRK1 or NTRK3 gene rearrangement with conventional PTC histology. The ALK IHC-positive case (n=1) was the follicular variant of PTC with consistent ALK gene rearrangement. ROS1 gene rearrangement was not detectable in the ROS1 IHC-positive PTCs (0/12) by FISH. Most (approximately 70%) of the pan-Trk or ROS1 IHC-positive/FISH-negative cases had BRAF gene mutation with conventional PTC morphology. In the ATC group, neither ALK nor ROS1 IHC was positive, whereas pan-Trk IHC was positive in 1 case (6.3%) in which NTRK1 gene rearrangement was confirmed by FISH. These results suggest that NTRK, ALK, and ROS1 rearrangements are rare molecular events in nonradiation-exposed Japanese patients with PTC and ATC. Although IHC is not an entirely specific surrogate for these abnormalities and does not serve as a stand-alone companion diagnosis, the combined use of IHC and molecular testing may be helpful for determining promising therapeutic strategies with tyrosine kinase inhibitors.

Correlating ROS1 Protein Expression With ROS1 Fusions, Amplifications, and Mutations

IntroductionIn this study, we sought to further characterize ROS1 protein expression in solid tumors with the complete spectrum of ROS1 genomic alterations. MethodsROS1 immunohistochemistry (IHC) was performed using the ROS1 (SP384) class I assay per manufacturer’s instructions on a variety of solid tumors (n = 32) with known ROS1 genomic alterations. Genomic alterations included fusions (n = 17), gene amplifications (n = 10), and short-variant mutations (n = 11). ResultsOf the 32 cases with ROS1 IHC results, 100% (11 of 11) with canonical ROS1 fusions were positive for ROS1 IHC. Among noncanonical ROS1 fusions, only two (of five) cases with SQSTM1-ROS1 and RDX-ROS1 fusions were positive for ROS1 IHC whereas PTPRK-ROS1 (two) and TTC28-ROS1 fusions were negative for ROS1 IHC. One sample with a canonical ROS1 fusion and co-occurring ROS1 resistance mutation (6094G>A, p.G2032R) was positive for ROS1 IHC. A total of 10% (one of 10) of ROS1 amplified tumors were positive for ROS1 IHC. None of the cases (zero of five) with ROS1 short-variant mutations were positive for ROS1 protein expression. ConclusionsThese findings suggest that if ROS1 IHC was used as a screening tool for ROS1 fusion, a subset of fusion-negative tumors will reveal positive IHC staining highlighting the value of reflexing to genomic profiling to confirm the presence of a targetable fusion-driver before the initiation of therapy. In addition, the ability of comprehensive genomic profiling to detect ROS1 resistance mutations will be important for clinical decision making.

Pseudosarcomatous myofibroblastic proliferations of the urinary bladder are neoplasms characterized by recurrent FN1-ALK fusions.

Pseudosarcomatous myofibroblastic proliferation is a descriptive term that designates a group of clinically indolent genitourinary lesions that most commonly arise in the urinary bladder. Given that pseudosarcomatous myofibroblastic proliferation may show morphologic overlap with inflammatory myofibroblastic tumor, the relationship, if any, between the two entities has been unclear. Moreover, pseudosarcomatous myofibroblastic proliferations are known to be positive for ALK immunohistochemistry in a subset of cases, although an inconsistent association with ALK rearrangement (ranging from 0 to 60%) has been reported. The objectives of this study were to determine the frequency of ALK rearrangement and to identify fusion partners using fluorescence in situ hybridization (FISH) and targeted RNA sequencing studies in a contemporary series of 30 pseudosarcomatous myofibroblastic proliferations of the urinary bladder, as well as to investigate ROS1 status by immunohistochemistry. ALK immunohistochemistry was positive in 70% (21/30) of pseudosarcomatous myofibroblastic proliferations; ROS1 immunohistochemistry was consistently negative (0/28). ALK rearrangements were detected by FISH in 86% (18/21) of cases, correlating with ALK immunohistochemical positivity in all but 3 cases. Of eight cases confirmed to be ALK rearranged by FISH, targeted RNA-sequencing detected FN1-ALK fusions in seven (88%) cases, which involved exons 20-26 of FN1 (5') and exon 18-19 of ALK (3'). In conclusion, ALK rearrangements are frequent in pseudosarcomatous myofibroblastic proliferations, typically involving exon 19, and FN1 appears to be a consistent fusion partner. Given the significant clinicopathologic differences between inflammatory myofibroblastic tumor and pseudosarcomatous myofibroblastic proliferation, our findings provide further support for classification of pseudosarcomatous myofibroblastic proliferation as a distinct clinicopathologic entity, and propose the alternate terminology "pseudosarcomatous myofibroblastic neoplasm of the genitourinary tract."

ROS1 immunohistochemistry as a screening tool for detection of ROS1 rearrangement in non-small cell lung cancer: Comparison between SP384 and D4D6 clones.

e21733 Background: ROS1 rearrangement occurs in approximately 1%-2% of non-small cell lung cancer, and it is predictive of treatment response to tyrosine kinase inhibitor. The gold standard method to detect ROS1 rearrangement is break-apart fluorescence in situ hybridization (FISH). ROS1 immunohistochemistry has been proposed as a cost-effective screening test for detection of ROS1 rearrangement. Most studies about the correlation between ROS1 FISH and immunohistochemistry derived from the D4D6 clone anti-ROS1 antibody. Recently, a novel anti-ROS1 antibody (SP384 clone) is available. However, data regarding the performance of this new anti-ROS1 antibody is still limited. In this study, we evaluated the performance of these two anti-ROS1 antibody as a screening tool to detect ROS1 rearrangement. Methods: Two hundred non-small cell lung cancers, including 18 ROS1 FISH-positive and 182 ROS1 FISH-negative samples, were studied. All specimens were stained with D4D6 clone and SP384 clone anti-ROS1 antibodies. The stained slides were scored by a pathologist using the H-score method, which was defined as the sum of products of multiplying intensity (0, 1, 2, and 3) by extent of each staining intensity (%). Receiver Operating Characteristics (ROC) curves were used to determine the optimal cut-off value that discriminates between ROS1-rearranged and non-rearranged tumors. Results: There was high correlation between the H-score of D4D6 clone and SP384 clone (Spearman's rho correlation coefficient = 0.862, P-value < 0.001). The SP384 clone showed a higher H-score than D4D6 clone (Wilcoxon signed rank test, P-value < 0.001). ROC analysis showed H-score of ≥ 150 was the optimal cut-off value for both D4D6 clone and SP384 clone to discriminate between ROS1-rearranged and non-rearranged tumors. Using this cut-off value, the D4D6 clone showed 100% sensitivity and 96.7% specificity; the SP384 clone showed 100% sensitivity and 95.1% specificity. The areas under ROC curve for D4D6 clone and SP384 clone were 0.992 and 0.990, respectively. Conclusions: The performance of D4D6 clone or SP384 clone is comparable. ROS1 IHC with either D4D6 clone or SP384 clone is a cost-effective screening tool for the presence of ROS1 rearrangements. [Table: see text]

Hepatic Angiosarcoma: Clinicopathologic Study With an Investigation of ROS1 Gene Rearrangements.

Primary hepatic angiosarcoma (PHA) is a rare disease entity with variable morphologic features. Recent findings regarding ROS1 gene rearrangements in PHA may lead to new targeted therapies. Thirteen cases (4 resected specimens and 9 biopsy samples) underwent histologic review and morphologic patterns were classified according to a previous study as 1) sinusoidal, 2) peliotic, 3) vasoformative, and 4) solid (epithelioid/spindled). ROS1 immunohistochemistry and investigation of the presence of a ROS1 fusion gene by reverse transcription-polymerase chain reaction were performed in available cases. Eight of 13 cases (62%) showed vasoformative patterns. Three cases (23%) were classified as sinusoidal and two (15%) as solid patterns. Mortality rate was 90% (9/10) except for three patients lost in follow up. Only one patient is still alive and has survived for 8 months with the disease. All cases tested did not have ROS1 expression (0/9) or a ROS1 fusion gene (0/4). We report 13 cases of PHA with 90% mortality. Vasoformative PHA is the most common histologic type. New findings on ROS1 fusion gene rearrangements could lead to the development of novel targeted therapeutics for PHA patients with dismal prognosis.

I.18-1 NTRK Fusion Proteins

We have a new approach to cancer therapy “the Agnostic Tumor” approach. Molecular mechanisms (DNA mutations, translocations, deletions, fusions, etc.) are now the ones responsible of the origin and behavior of most of the tumors, instead of the location of the tumor as we used to think. We are in the beginning of changing the focus of cancer therapy from a single organ to a molecular marker envision. One of the best examples are the NTRK fusion proteins, NTRK genes encode for the Trk-family proteins: TrkA, TrkB, and TrkC (encoded by NTRK1, NTTRK2, and NTRK3). Normally, the NTRK family plays a role in the development of the nervous system, however, NTRK fusions are also present in solid tumors as fusion proteins responsible for growth of cancer cells, and the presence of these oncogenes is associate with poor survival in lung cancers and other tumor types. It is not surprising that these oncogenes are present in several different tumors (They are actionable in at least 17 tumor types) including adults and children. These genomic alterations are becoming a great example why the tumor agnostic approach might be the new paradigm in fighting cancer. However, there are still many challenges ahead; first for example, the diagnostic of these genetic aberrations: we can do fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), reverse-transcriptase polymerase chain reaction (RT-PCR) and next generation sequencing (NGS) of DNA or RNA (or cfDNA in blood: liquid biopsies). Each of these approaches has strengths and weaknesses, but we also have to play this in the context of workups for other genetic abnormalities and keeping into consideration that tumor tissue specimen is very limited in many instances. Nowadays, for example, we do FISH for ALK and ROS-1 translocations, if we add three more FISH tests for each of the NTRK alterations (and maybe one more for RET fusions) will markedly increase the costs of workup in a lung cancer patient, and the gene fusion partner, that might become relevant, still might not be identified if we don’t have the right probe. RT-PCR is a great technique, but we will need a lot of primers (there are more than 60 NTRK fusions documented) to cover all the NTRK genetic abnormalities. Probably the solution is to develop better IHC? as we are doing for ALK translocations and hopefully get one day as IHC for ROS-1 and the other NTRK oncogenes so we can have a more cost-effective work up. A more practical approach might be to establish NGS in tissue or blood as the standard of care that can detect all these genetic alterations at once at the time of the diagnosis of the patient? Later we can do a more limited work up if we are looking for ALK and now NTRK resistant variants in patients that have failed treatment clinically. Nonetheless, for those who are not fans of NGS and don’t advocate this approach we can say that several NTRK 1-3 introns are not well covered by NGS. Then, we need to do whole genome gene sequencing to find them increasing the costs of the workup. These are some of the questions that remain open while we make all these diagnostic techniques more accurate and more cost effective. Some of these reasons are responsible to consider the treatment for NTRK challenging: how we can treat a patient if we can’t discover the genetic aberration? We are fortunate to have two agents: entrectenib and larotrectenib that have already been FDA approved for NTRK fusions with acceptable toxicities regardless of tumor type, patient age, and fusion type, and more data coming from other agents in development CEP-701, ARRRY-470 DS-6051b, TPX-0005. Probably LOXO 195 is the one that has been more studied for now and it’s able to rescue patients that develop resistance mutations during the treatment with larotrectenib or entrectinib.

P2.16-05 Population-Based ROS1 Testing in Lung Cancer: Creating Opportunity in a Publicly Funded System

ROS1 gene rearrangement is found in 1-2% of all non-small cell lung cancer (NSCLC) and is recommended as standard molecular diagnostic testing. This study models the most efficient ROS1 testing strategy to maximize detection of true positive cases (TP) while minimizing costs and turnaround time (TAT). A population-based ROS1 testing model was developed from a Canadian (Ontario) public healthcare system perspective examining the use of immunohistochemistry (IHC) and next generation sequencing (NGS) versus fluorescence in situ hybridization (FISH, gold standard), reflex versus molecular or clinical selection (never smokers), and blood- versus tissue-based testing. Model inputs were derived from existing literature and expert opinion. Direct testing costs and TAT were obtained from the Ontario public perspective (University Health Network, Cancer Care Ontario). The most cost-effective strategy for the outcomes of TAT and TP was reflex testing with IHC and subsequent FISH confirmation; this identified a high proportion of TP within a relatively short TAT. The most costly reflex strategy was NGS, with a greater proportion of missed TP (Table), and long TAT. Clinician selection of never smokers yielded the lowest proportion of TP. Population-based plasma ctDNA testing using commercial assays was the most costly strategy. One-way sensitivity analysis demonstrated that the TP outcome was most sensitive to the population prevalence of ROS1, while cost was most sensitive to the specificity of ROS1 IHC. Pathologist-initiated reflex testing using IHC with FISH confirmation provides the most cost-effective population-based testing strategy. Clinician-initiated testing significantly lengthens TAT. Selecting only never smokers for testing misses a larger proportion of TP patients who would benefit from targeted therapy despite potential cost savings.

Correlation of ROS1 Immunohistochemistry With ROS1 Fusion Status Determined by Fluorescence In Situ Hybridization.

The ability to determine ROS1 status has become mandatory for patients with lung adenocarcinoma, as many global authorities have approved crizotinib for patients with ROS1-positive lung adenocarcinoma. To present analytical correlation of the VENTANA ROS1 (SP384) Rabbit Monoclonal Primary Antibody (ROS1 [SP384] antibody) with ROS1 fluorescence in situ hybridization (FISH). The immunohistochemistry (IHC) and FISH analytical comparison was assessed by using 122 non-small cell lung cancer samples that had both FISH (46 positive and 76 negative cases) and IHC staining results available. In addition, reverse transcription-polymerase chain reaction (RT-PCR) as well as DNA and RNA next-generation sequencing (NGS) were used to further examine the ROS1 status in cases that were discrepant between FISH and IHC, based on staining in the cytoplasm of 2+ or above in more than 30% of total tumor cells considered as IHC positive. Here, we define the consensus status as the most frequent result across the 5 different methods (IHC, FISH, RT-PCR, RNA NGS, and DNA NGS) we used to determine ROS1 status in these cases. Of the IHC scoring methods examined, staining in the cytoplasm of 2+ or above in more than 30% of total tumor cells considered as IHC positive had the highest correlation with a FISH-positive status, reaching a positive percentage agreement of 97.8% and negative percentage agreement of 89.5%. A positive percentage agreement (100%) and negative percentage agreement (92.0%) was reached by comparing ROS1 (SP384) using a cutoff for staining in the cytoplasm of 2+ or above in more than 30% of total tumor cells to the consensus status. Herein, we present a standardized staining protocol for ROS1 (SP384) and data that support the high correlation between ROS1 status and ROS1 (SP384) antibody.

Comparison of in Situ and Extraction-Based Methods for the Detection of ROS1 Rearrangements in Solid Tumors

Clinical data confirmed that patients with ROS1 rearrangement are sensitive to specific inhibitors. Therefore, reliable detection of ROS1 rearrangements is essential. Several diagnostic techniques are currently available. However, previous studies were hampered by the low number of ROS1-positive samples. Thirty-five samples, including 32 ROS1 fluorescent in situ hybridization (FISH)-positive and three ROS1 FISH-negative samples were evaluated by ROS1 chromogenic in situ hybridization, ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) immunohistochemistry (IHC), an Agilent SureSelectXT HS custom panel, the Archer FusionPlex Comprehensive Thyroid and Lung panel, and a custom NanoString fusion panel. Some samples were additionally analyzed with the Illumina TruSight Tumor 170 assay. Eleven samples were ROS1 FISH positive by a break-apart signal pattern. In all 11 samples, a ROS1 fusion was confirmed by at least one other method. The other 21 samples tested ROS1 FISH positive by an isolated 3' green signal pattern. Ten of 21 samples could be confirmed by at least two other methods. The other 11 samples tested negative by ROS1 IHC and at least one other method, indicating a false-positive ROS1 FISH result. Our study found that all ROS1 FISH-positive samples with isolated 3' green signals should be confirmed by another method. When sufficient material is available, extraction-based parallel sequencing approaches for the verification of these cases might be preferable.

Assessment of a New ROS1 Immunohistochemistry Clone (SP384) for the Identification of ROS1 Rearrangements in Patients with Non–Small Cell Lung Carcinoma: the ROSING Study

IntroductionThe ROS1 gene rearrangement has become an important biomarker in NSCLC. The College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology testing guidelines support the use of ROS1 immunohistochemistry (IHC) as a screening test, followed by confirmation with fluorescence in situ hybridization (FISH) or a molecular test in all positive results. We have evaluated a novel anti-ROS1 IHC antibody (SP384) in a large multicenter series to obtain real-world data. MethodsA total of 43 ROS1 FISH–positive and 193 ROS1 FISH–negative NSCLC samples were studied. All specimens were screened by using two antibodies (clone D4D6 from Cell Signaling Technology and clone SP384 from Ventana Medical Systems), and the different interpretation criteria were compared with break-apart FISH (Vysis). FISH-positive samples were also analyzed with next-generation sequencing (Oncomine Dx Target Test Panel, Thermo Fisher Scientific). ResultsAn H-score of 150 or higher or the presence of at least 70% of tumor cells with an intensity of staining of 2+ or higher by the SP384 clone was the optimal cutoff value (both with 93% sensitivity and 100% specificity). The D4D6 clone showed similar results, with an H-score of at least 100 (91% sensitivity and 100% specificity). ROS1 expression in normal lung was more frequent with use of the SP384 clone (p < 0.0001). The ezrin gene (EZR)-ROS1 variant was associated with membranous staining and an isolated green signal FISH pattern (p = 0.001 and p = 0.017, respectively). ConclusionsThe new SP384 ROS1 IHC clone showed excellent sensitivity without compromising specificity, so it is another excellent analytical option for the proposed testing algorithm.

Abstract 3127: Multicenter study evaluating the ROS1 status in lung adenocarcinoma using the novel SP384 immunohistochemistry clone. Towards a new algorithm for ROS1 status assessment in routine

Abstract Background: The detection of a ROS1 rearrangement in advanced or metastatic lung adenocarcinoma (LUAD) lead to a targeted treatment with tyrosine kinase inhibitors with improved progression free survival (PFS) and overall survival (OS) of the patients. Thus, it is mandatory to screen systematically for the ROS1rearrangement in this patient population. ROS1 rearrangements can be detected using fluorescence in situ hybridization (FISH), however ROS1 immunohistochemistry (IHC) can be used as a screening test since is largely available, easy, rapid to perform, and cost-effective. However, some false positive and negative IHC results are observed when using the D4D6 clone leading to confirmatory ROS1 FISH in IHC positive samples. Patients and methods: We evaluated the sensitivity and specificity of anti-ROS1 SP384 (Ventana, Tucson, AZ) and D4D6 (Cell Signaling, Danvers, MA) antibodies in a multicenter population of 336 LUAD cases enriched for ROS1 FISH positive cases (n=51) provided from 6 French molecular pathology platforms. Three senior lung pathologists independently scored the SP384 slides as positive or negative around a cutoff of staining in &amp;gt;30% tumor cells at a ≥2+ intensity level, and for the D4D6 clone around a cutoff of ≥2+ intensity level in any tumor cells. Inter-reader precision between pathologists was assessed. Results were correlated to the PFS and the OS of patients treated with crizotinib. Results: Sensitivity and specificity rates were 100% (95%CI 93.2-100%) and 99.31% (95%CI 97.51-99.92%) for the SP384 clone, and 90.6% (95%CI 79.34-96.87%) and 99.65% (95%CI 98.05-99.99%) for the D4D6 clone, respectively. Inter-reader agreement was 97.5% (95%CI 94.8-99.7) for the SP384 clone and 86.3% (95%CI 91.3-95.6) for the D4D6 clone. Overall, when compared to ROS1 FISH analysis, the SP384 clone had an accuracy of 99.4%, while D4D6 clone of 98.2%. Using log-rank test, we observed that LUAD with positive ROS1 SP384 status had a longer PFS than those characterized by the D4D6 clone (P=0.021). Conclusions: Interpretation above a cutoff of &amp;gt;30% tumor cells with staining at a ≥2+ intensity level, the ROS1 SP384 clone demonstrates superior sensitivity to D4D6 clone, while preserving similar specificity rate for the detection of ROS1 rearrangements. The presented data provide evidence that the SP384 clone may be used for effective stratification prior to confirmation with orthogonal methods. Citation Format: Véronique Hofman, Isabelle Rouquette, Sandra Lassalle, Simon Heeke, Jean-Christophe Sabourin, Nicolas Piton, Julien Mazières, Jean-Michel Vignaud, Clémence Yguel, Anne Laure Lepage, Frédéric Bibeau, Elodie Long-Mira, Katia Zahaf, Hugues Begueret, Jonathan Benzaquen, Michel Poudenx, Charles-Hugo Marquette, Marius Ilie, Paul Hofman. Multicenter study evaluating the ROS1 status in lung adenocarcinoma using the novel SP384 immunohistochemistry clone. Towards a new algorithm for ROS1 status assessment in routine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3127.

Observational, multicenter, prospective study to assess the impact on patients' outcome of a systematic screening of oncogenic drivers in advanced cancer: The GETHI XX-16 study.

3082 Background: Identification of “agnostic” genetic drivers in cancer is foreseen as a major step forward in precision medicine.Unfortunately, “off label” use of targeted therapies is not widely available and many oncogenic alteration do not present the same behaviour accross all tumor types.We aimed to analyze the real impact on patients management of the implementation of a systematic screening of genetic alterations in centers of the Spanish Group for Rare Cancer (GETHI). Methods: We designed an observational, prospective and multicenter study to molecularly characterize any adult patient with advanced cancer.Formalin fixed paraffin-embedded samples were studied by TrkA-C,ROS1 and ALK immunohistochemistry followed by RT-PCR when positive to confirm gene fusions. Additonally, the Next Generation Sequencing paltform ArcherFusion Plex (able to detect point mutations and rearrangements in 53 cancer related genes) was implemented.Clinical data regarding treatment administered and outcome, were collected from patients identified as harboring drugable alterations. Results: Up to 26 hospitals all over the country got involved in the study. 341 tumoral tissues, representing 41 different histologies were collected. Molecular studies could be performed in 292 samples that led to the identification of 33 patients as harboring somatic oncogenic mutations. 21 were considered druggable and 5 got targeted therapy directed against the alteration identified (three glioblastoma patients with EGFR mutations received erlotinib, one prostate cancer with a BRAF fusion received trametinib and one lung cancer with ALK translocation, previously deemed as negative by standard screening, received crizotinib). One of the glioblastoma patients achieved a long lasting stabilitation and both the prostate and lung tumors presented dramatic partial responses. Conclusions: Though only few cases harboring drugable alteratons got specif treatment, 50% achieved a meanignful benefit. A wide access to molecular screening and targeted drugs could improve the outcome of cancer patients.

Histopathologic characteristics of advanced-stage ROS1-rearranged non-small cell lung cancers

Background: ROS1 rearrangement accounts for 1%–2% of non-small cell lung cancer (NSCLC) with a remarkable response to crizotinib. Although ROS1-rearranged tumors are known to have characteristic histologic features, only a few studies have investigated the histologic features of advanced-stage ROS1-rearranged tumors.Methods: We analyzed the histopathologic features of ROS1-rearranged tumors in advanced-stage NSCLC patients and assessed the ROS1 immunohistochemistry (IHC) staining patterns of ROS1-rearranged cases.Results: A total of 37 ROS1 fluorescence in situ hybridization (FISH)-positive cases and 64 ROS1 FISH-negative cases were analyzed, and all tumors were EGFR-, ALK-, and RET-negative. Solid pattern, round nuclei with macronucleoli, solid signet-ring cells, extracellular mucin, and a close relation with adjacent bronchioles were significantly associated with ROS1 rearrangement, and the solid signet-ring cell feature was exclusively identified in ROS1-rearranged tumors. ROS1 IHC showed a 97.3% sensitivity when weak to strong protein expression was considered positive.Conclusions: Our findings highlight distinct histologic features of ROS1-rearranged tumors, including their nuclear features. A thorough understanding of ROS1 rearrangement-related histologic features would be helpful to identify ROS1-rearranged tumors in advanced-stage NSCLC.

Multicenter Evaluation of a Novel ROS1 Immunohistochemistry Assay (SP384) for Detection of ROS1 Rearrangements in a Large Cohort of Lung Adenocarcinoma Patients

IntroductionThe detection of a ROS1 rearrangement in advanced and metastatic lung adenocarcinoma (LUAD) led to a targeted treatment with tyrosine kinase inhibitors with favorable progression-free survival and overall survival of the patients. Thus, it is mandatory to screen for the ROS1 rearrangement in all these patients. ROS1 rearrangements can be detected using break-apart fluorescence in situ hybridization (FISH), which is the gold standard; however, ROS1 immunohistochemistry (IHC) can be used as a screening test because it is widely available, easy and rapid to perform, and cost-effective. MethodsWe evaluated the diagnostic accuracy and interpathologist agreement of two anti-ROS1 IHC clones, SP384 (Ventana, Tucson, Arizona) and D4D6 (Cell Signaling, Danvers, Massachusetts), in a training cohort of 51 positive ROS1 FISH LUAD cases, and then in a large validation cohort of 714 consecutive cases of LUAD from six routine molecular pathology platforms. ResultsIn the two cohorts, the SP384 and D4D6 clones show variable sensitivity and specificity rates on the basis of two cutoff points greater than or equal to 1+ (all % tumor cells) and greater than or equal to 2+ (>30% stained tumor cells). In the validation cohort, the D4D6 yielded the best accuracy for the presence of a ROS1 rearrangement by FISH. Interpathologist agreement was moderate to good (interclass correlation 0.722–0.874) for the D4D6 clone and good to excellent (interclass correlation: 0.830–0.956) for the SP384 clone. ConclusionsROS1 IHC is an effective screening tool for the presence of ROS1 rearrangements. However, users must be acutely aware of the variable diagnostic performance of different anti-ROS1 antibodies before implementation into routine clinical practice.

Morphologic Overlap Between Inflammatory Myofibroblastic Tumor and IgG4-related Disease

Inflammatory myofibroblastic tumor (IMT), a locally aggressive neoplasm capable of metastasis, may show an immunoglobulin (Ig)G4-rich lymphoplasmacytic infiltrate. Prior reports suggest that storiform-fibrosis and obliterative phlebitis aid in the distinction of IMT from IgG4-related diseases. Herein, we highlight the morphologic overlap between the 2 diseases, and emphasize the importance of a multiplex fusion assay in the distinction of IgG4-related disease (IgG4-RD) from IMT. We identified 7 IMTs with morphologic and immunohistochemical features of IgG4-RD; 3 patients were originally diagnosed with IgG4-RD. Demographic, clinical and morphologic data was recorded. We also reevaluated 56 patients with IgG4-RD. We performed immunohistochemistry for IgG4, IgG, ALK, and ROS1. In situ hybridization for IgG4 and IgG was performed in selected cases. A multiplex next-generation sequencing-based RNA assay for gene fusions was performed to detect all known IMT-related gene fusions. All 7 IMTs showed a dense lymphoplasmacytic infiltrate and storiform-type fibrosis, with obliterative phlebitis noted in 3 cases. The neoplastic stromal cells constituted <5% of overall cellularity and stromal atypia was either absent or focal and mild. Elevated numbers of IgG4 positive cells and increased IgG4 to IgG ratio was identified in all cases. Four cases showed ALK related abnormalities: 3 fusions and one alternative transcription initiation; while 2 patients showed ROS1 and NTRK3 fusions. One tumor was negative for known IMT-related gene fusions. All 56 IgG4-RD cases were negative for ALK and ROS1 on immunohistochemistry; 6 cases were negative on the fusion assay. Highly inflamed IMTs are indistinguishable from IgG4-RD both histologically and on immunohistochemistry for IgG4. We advocate scrutinizing patients with presumptive single organ IgG4-RD for IMT and the diagnostic algorithm should include ALK and ROS1 immunohistochemistry and, in selected cases, a next-generation sequencing-based fusion assay that covers known IMT-associated gene fusions.

Determination of ROS1 positivity by immunohistochemistry in a multicentric cohort of 426 non-small-cell lung cancer cases in India

Introduction: ROS1 is a receptor tyrosine kinase of the insulin receptor family that acts as a driver oncogene in 1%–2% of non-small cell lung carcinoma (NSCLC) patients via a gene translocation between ROS1 and other genes, the most common one being CD74. Histologic and clinical features that are associated with ROS1 translocations include never smokers, younger age, and adenocarcinoma (AC) histology. The identification of ROS1 fusion is important because it responds to targeted therapies such as crizotinib that can greatly improve the survival of the patient. The goal of this study was to determine the percentage of population harboring ROS1 mutation in India. Materials and Methods: ROS immunohistochemistry (IHC) method was optimized using Cell Signalling Technology, ROS antibody (ROS1 clone D4D6 rabbit mAb), and appropriate positive and negative controls in a fully automated procedure using Ventana automated stainer. Approximately 2000 retrospective lung cancer cases from six different Apollo hospitals were identified. The formalin-fixed paraffin-embedded blocks and corresponding diagnostic and demographic data were retrieved. A total of 426 cases with confirmed diagnosis of NSCLC were then tested by IHC for ROS1. Results: Given the retrospective nature of the cancer cases and diagnostic data being used for the study, there was high attrition in the availability of the blocks and data. Among the 2000 cases examined, there were 604 cases that were initially reported as lung cancer in the hospital medical records and for whom corresponding blocks were available, but blocks for only 485 cases had sufficient tumor content, as judged by hematoxylin and eosin staining and microscopic examination, to enable testing for ROS1 staining. Of these 485 cases, 225 were confirmed AC cases. A single case stained positive for ROS1 among the 225 AC cases. The percentage is calculated to be 0.44% based on this single positive case. No positive case was observed in squamous cell carcinomas or other cancers of the lung such as neuroendocrine tumors, germ cell tumors, and adenoid cystic tumors. Conclusion: The percentage positivity for ROS1 in the study comprising 225 Indian NSCLC AC cases appeared similar to the 0.3%–1.6% range reported for smokers and lower than 0.9%–1.7% positivity reported in mixed patient populations.

P2.09-13 Correlation of ROS1 (SP384) Immunohistochemistry with ROS1 Rearrangement Determined by Fluorescence in Situ Hybridization

P2.09-13 Correlation of ROS1 (SP384) Immunohistochemistry with ROS1 Rearrangement Determined by Fluorescence in Situ Hybridization

Clinicopathological significance and diagnostic approach of ROS1 rearrangement in non-small cell lung cancer: a meta-analysis: ROS1 in non-small cell lung cancer.

The aim of this study was to investigate the rate of ROS1 rearrangement and concordance between ROS1 immunohistochemistry (IHC) and molecular tests in non-small cell lung cancer (NSCLC). The study included 10,898 NSCLC cases from 21 eligible studies. ROS1 rearrangement rates were evaluated in NSCLC by a meta-analysis, including subgroup analyses. In addition, we performed a concordance analysis and a diagnostic test accuracy review of ROS1 IHC in NSCLC. The estimated overall rate of ROS1 rearrangement and IHC positivity was 2.4% (95% confidence interval (CI) 1.5, 3.7). In the subgroup analysis, which was based on tumor subtype, the rate of ROS1 rearrangement and IHC positivity was 2.9% (95% CI 1.9, 4.5) and 0.6% (95% CI 0.3, 1.2) in adenocarcinoma and non-adenocarcinoma, respectively. The overall concordance rate between ROS1 IHC and molecular tests was 93.4% (95% CI 78.3, 98.2). In ROS1 IHC positive and negative cases, the concordance rates were 79.0% (95% CI 43.3, 94.9) and 97.0% (95% CI 83.3, 99.5), respectively. The pooled sensitivity and the specificity of ROS1 IHC were 0.90 (95% CI 0.70, 0.99) and 0.82 (95% CI 0.79, 0.84), respectively. The diagnostic odds ratio and the area under the curve of the summary receiver operating characteristic curve were 118.01 (95% CI 11.81, 1179.67) and 0.9417, respectively. The rates of ROS1 rearrangement differed by tumor histologic subtype in NSCLC. ROS1 IHC may be useful for the detection of ROS1 rearrangement in NSCLC. Detailed criteria for evaluating ROS1 IHC are needed before it can be applied in daily practice.

A genomic and clinicopathological study of non‐small‐cell lung cancers with discordant ROS1 gene status by fluorescence in‐situ hybridisation and immunohistochemical analysis

ROS1 immunohistochemistry (IHC) using D4D6 antibody is a useful tool for screening patients with non-small-cell lung cancer (NSCLC) who may be suitable for targeted therapy. Many studies and our data have identified cases that express the ROS1 protein strongly but are negative for ROS1 by fluorescence in-situ hybridisation (FISH). The present study investigated the driver mutation and clinicopathological characteristics of 26 discordant cases (ROS1 IHC-positive but FISH-negative) to find new clues for distinguishing real ROS1-rearranged cases. Tumours from 26 discordant cases were analysed for clinicopathological characteristics, mutations in EGFR, KRAS, ERBB2, BRAF and PIK3CA; fusions in ALK and RET; and amplifications in MET, ERBB2 and ROS1. ROS1-rearranged NSCLCs were significantly more likely to be found in younger patients and at an advanced stage; they showed cribriform features, extracellular mucus and psammoma bodies, whereas ROS1-discordant cases were found in older patients at a relatively early tumour-node-metastasis (TNM) stage and showed a lepidic growth pattern (all P < 0.001). Most ROS1-rearranged NSCLCs had no concurrent mutation, whereas 73% of discordant cases harboured genetic aberrations, including EGFR and ERBB2. Compared with general lung adenocarcinomas, ERBB-2 abnormality was disproportionately high in ROS1-discordant cases. Moreover, we optimised the scoring criteria for ROS1 IHC as 'H score > 150 and no concurrent mutations'; the specificity was then increased to 81.6%. Compared with ROS1-rearranged cases, ROS1-discordant patients showed distinct clinical and morphological features and often harboured another oncogenic driver alteration. The use of optimised screening criteria will increase the specificity of ROS1 antibody.