ALK FISH Research Articles

Combinational Analysis of FISH and Immunohistochemistry Reveals Rare Genomic Eventsin ALK Fusion Patterns in NSCLC that Respondsto Crizotinib Treatment.

The purpose of this study was to explore the complicated rearrangement mechanisms underlying cases with atypical and negative anaplastic lymphoma receptor tyrosine kinase gene (ALK) fluorescence hybridization (FISH) and positive immunohistochemistry (IHC) results and to stress the importance of combinational assay of these two methods in current pathological diagnosis. A total of 3128 NSCLCs were screened for ALK fusions through both FISH analysis and IHC assays with Ventana-D5F3 antibody. Fourteen cases with atypical and negative FISH results with the current criteria and positive IHC results were analyzed with targeted next-generation sequencing (NGS). Of the 3128 cases tested, 228 (7.3%) and 214 (6.8%) were ALK positive by IHC and FISH, respectively. Fourteen cases with negative and atypical FISH results all demonstrated IHC positivity. Of 2991 cases, eight (0.27%) with negative FISH results demonstrated echinoderm microtubule associated protein like 4 gene (EML4)-ALK fusions revealed by targeted NGS, and the relative abundance of fusion ranged from 0.9% to 46.8%. Three of 2991 cases (0.1%) did not exhibit any type of ALK fusions. In addition, two patients showed an isolated 5' side signal and targeted NGS revealed two novel ALK partner genes, baculoviral IAP repeat containing 6 gene (BIRC6) and phosphatidylinositol binding clathrin assembly protein gene (PICALM). One patient showed an isolated and attenuated 3' red signal and demonstrated a novel translocation partner with CCAAT/enhancer binding protein zeta gene (CEBPZ). Of all the patients, four received crizotinib treatment and demonstrated partial responses at the end of follow-up. Our study showed that patients with negative and atypical ALK FISH patterns may have positive results for IHC testing and harbor the translocation partners of EML4 or other genes. Therefore, additional testing with NGS should be conducted to explore the molecular mechanisms underlying the complicated gene rearrangement events.

Molecular characterization of pulmonary sarcomatoid carcinoma: analysis of 33 cases

AbstractSeveral targetable genetic alterations have been found in lung cancer, predominantly in adenocarcinomas, which have led to important therapeutic advancements with the advent of targeted therapy. In contrast, the molecular features and presence of targetable genetic abnormalities in pulmonary sarcomatoid carcinomas are largely unknown. Thirty-three cases of pulmonary sarcomatoid carcinoma were tested for approximately 2800 mutations in 50 oncogenes and tumor-suppressor genes, including EGFR, KRAS, NRAS, TP53, BRAF, ERBB2, JAK3, AKT1, ATM, MET, KIT, and PIK3CA. ALK immunostaining was performed, and ALK FISH was performed on cases with any degree of staining. Twenty-four of the 33 cases (72%) had at least one genetic abnormality: 19 cases (58%) had TP53 mutations; 10 cases (30%) had KRAS mutations; AKT1, JAK3, BRAF, NRAS, and PIK3CA mutations were observed in 1 case each (3%). Six of the 19 cases (32%) with a mutation in TP53 had simultaneous mutations in KRAS (18%). The cases with alterations in JAK3, BRAF, and NRAS also had mutations in TP53. The case showing a mutation in PIK3CA had a mutation in KRAS. No EGFR mutations were observed. One case had ALK gene rearrangement. ALK rearrangement was observed in a single case of sarcomatoid carcinoma (3%), which has currently available targeted therapy. Four tumors had mutations in genes with experimental molecular-based therapy, including BRAF, NRAS, PIK3CA, and AKT1. Testing for targetable mutations should be considered for patients with pulmonary sarcomatoid carcinoma, as a subset may benefit from currently approved drugs or clinical trials of novel therapeutic options available for other types of lung cancer.

Quantitative assessment of cell block cellularity and correlation with molecular testing adequacy in lung cancer

Determination of fine-needle aspiration (FNA) material adequacy is essential prior to performing molecular testing (MT) in order to ensure good results and maximize resources. This study investigates several quantitative measures of cellularity in FNA samples of lung carcinoma, and correlates the results with MT adequacy. A blinded retrospective analysis of 20 non-small-cell lung carcinoma cases was conducted: 13 contained "sufficient" material for EGFR/KRAS sequencing and ALK FISH studies; and 7 contained "insufficient" material for these tests. Three 400x fields-of-view (FOVs) were analyzed from digitized cell block glass slides of these cases. Cellularity in these FOVs was quantified using three methods: (1) visual estimation by cytopathologist; (2) manually annotated contours (MACs); (3) software derived, manually adjusted contours (SDMACs) using a custom segmentation script with adjustable parameters. These methods were evaluated using the Mann-Whitney-Wilcoxon test, paired t test, and receiver operating characteristic/area under the curve (AUC) analysis. There were significant differences between the insufficient/sufficient groups for each estimation method (visual P < 0.05, MAC P < 0.05, SDMAC P < 0.01). Variation of mean values was highest in the visual estimation method. AUC values were visual estimation = 0.903, MAC = 0.903, and SDMAC = 0.958. Mean variation of the 3 FOV values was found to be significantly higher for visual estimation compared with the other methods. Quantitative analysis of cellularity in digitized cell block material is feasible using different methods. In this investigation, the SDMAC method provided the highest accuracy and lowest variability. This supports image analysis as an objective and quantitative tool to assess FNA sample adequacy for guiding supplemental MT.

Abstract 4666: Revolutionizing clinical care using prospective, institution-wide tumor sequencing

Abstract Identification of predictive and prognostic biomarkers is central to clinical oncology. Use of targeted next generation sequencing (NGS) is increasing in molecular diagnostics labs, however the feasibility and impact of its routine application across all tumor types is largely untested. We launched an institution-wide effort to generate targeted NGS data (Oncopanel) for invasive tumors of all consenting patients. We hypothesized that this approach could replace traditional targeted testing, generate robust data on copy number alterations and structural variants, and provide novel patient-specific observations to facilitate clinical trial enrollment. Illumina NGS was performed on libraries prepared with Agilent SureSelect custom-designed hybrid capture of 4430 exons from 275 genes plus selected introns of 30 genes. Results were analyzed by an internally-developed computational pipeline for mutations, small insertions/deletions, copy number variation and rearrangements. Variants were characterized according to predictive, prognostic, or diagnostic actionability. Data is available for the first 4291 cases sequenced. Oncopanel succeeded in 96% of specimens with adequate DNA. In a subset analysis performed on the first 1000 cases, assay success ranged from 83-100% according to tumor type; breast carcinoma was most prone to failure (p&amp;lt;0.0001). Median number of mutations per case was 8 (range 0-205) and was lowest in endocrine malignancies and highest in skin malignancies. Three percent of tumors were hypermutated with mutational signatures revealing distinct pathogenic underpinnings, including prior temozolomide therapy, microsatellite instability, and UV exposure. Compared to clinical testing, Oncopanel showed 100% accuracy for detection of KRAS and BRAF point mutations in colon adenocarcinoma, EGFR exon 19 deletion mutations in lung adenocarcinoma, and for EGFR amplification in glioblastoma. Oncopanel was 97.5% sensitive and 87.5% specific for 1p19q deletion as compared to aCGH or FISH and was 80% sensitive and 100% specific as compared to ALK FISH and additionally detected 2 ALK rearranged tumors for which FISH failed. In the overall cohort, 26% of patients had an actionable variant (most commonly KRAS) and 39% had alterations with implications for clinical trial enrollment (most commonly in PI3K/PTEN/AKT pathway). TP53 was the most commonly altered gene overall and the most likely to be co-mutated with oncogenic drivers. High level amplifications were most common for EGFR, MDM2, CDK4, ERBB2, MYC, and CCND1. Two-copy deletions were most common for CDKN2A/B, followed by TP53 and PTEN. In several cases, Oncopanel data uncovered alterations that informed diagnosis and treatment of difficult-to-classify tumors. These efforts demonstrate that high quality, high throughput NGS data can be generated prospectively on an institutional level, thereby informing disease course and therapeutic options at an unprecedented scale. Citation Format: Lynette M. Sholl, Elizabeth Garcia, Yonghui Jia, Matthew Ducar, Bernard Fendler, Priyanka Shivdasani, Frank C. Kuo, Azra H. Ligon, Barrett J. Rollins, Neal I. Lindeman, Laura E. MacConaill. Revolutionizing clinical care using prospective, institution-wide tumor sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4666. doi:10.1158/1538-7445.AM2015-4666

Localisation secondaire atypique au « sein » des cancers bronchiques

In the context of bronchial cancers, the most frequent sites for metastases to occur are the lung, bone, brain, liver and adrenal glands. However, metastasis to other sites does additionally occur and this might be influenced by the biological characteristics of the tumour. We report the case of a 54-year-old woman with a primary bronchial adenocarcinoma with an EML4-ALK translocation. During her treatment with crizotinib, the patient developed a lesion in her right breast. The initial pathological diagnosis was of an invasive ductal adenocarcinoma of the breast. However, an additional immuno-histochemical analysis revealed it to be a metastasis from her bronchial tumour. This case is an illustration that, in the context of a lung cancer with ALK rearrangement, synchronous or secondary lesions must be interpreted with caution. Specific biological analysis -ALK immunohistochemistry or FISH- must be performed to confirm a primary or metastatic origin for these lesions.

Screening for ALK in non-small cell lung carcinomas: 5A4 and D5F3 antibodies perform equally well, but combined use with FISH is recommended.

Immunohistochemistry (IHC) has become a promising method for pre-screening ALK-rearrangements in non-small cell lung carcinomas (NSCLC). Various ALK antibodies, detection systems and automated immunostainers are available. We therefore aimed to compare the performance of the monoclonal 5A4 (Novocastra, Leica) and D5F3 (Cell Signaling, Ventana) antibodies using two different immunostainers. Additionally we analyzed the accuracy of prospective ALK IHC-testing in routine diagnostics. Seventy-two NSCLC with available ALK FISH results and enriched for FISH-positive carcinomas were retrospectively analyzed. IHC was performed on BenchMarkXT (Ventana) using 5A4 and D5F3, respectively, and additionally with 5A4 on Bond-MAX (Leica). Data from our routine diagnostics on prospective ALK-testing with parallel IHC, using 5A4, and FISH were available from 303 NSCLC. All three IHC protocols showed congruent results. Only 1/25 FISH-positive NSCLC (4%) was false negative by IHC. For all three IHC protocols the sensitivity, specificity, positive (PPV) and negative predictive values (NPV) compared to FISH were 96%, 100%, 100% and 97.8%, respectively. In the prospective cohort 3/32 FISH-positive (9.4%) and 2/271 FISH-negative (0.7%) NSCLC were false negative and false positive by IHC, respectively. In routine diagnostics the sensitivity, specificity, PPV and NPV of IHC compared to FISH were 90.6%, 99.3%, 93.5% and 98.9%, respectively. 5A4 and D5F3 are equally well suited for detecting ALK-rearranged NSCLC. BenchMark and BOND-MAX immunostainers can be used for IHC with 5A4. True discrepancies between IHC and FISH results do exist and need to be addressed when implementing IHC in an ALK-testing algorithm.

Clinical implication of mass-spectrometry-based selected reaction monitoring (SRM) assay in non-small cell lung cancer (NSCLC) patients exhibiting ALK gene rearrangement.

e22145 Background: FISH is considered as a standard diagnostic method in detection of ALK gene rearrangement, but clinical response of crizotinib (ALK/Met inhibitor) was various in ALK FISH (+) pat...

Discrepancies between FISH and immunohistochemistry for assessment of the ALK status are associated with ALK ‘borderline’-positive rearrangements or a high copy number: a potential major issue for anti-ALK therapeutic strategies

BackgroundPatients with advanced lung adenocarcinomas expressing ALK rearrangements are highly responsive to crizotinib, a dual ALK/c-MET inhibitor. Immunohistochemistry (IHC) is an easy clinically and routinely applicable cost-effective assay for ALK, c-MET and ROS1 protein expression for potential treatment with crizotinib. The purpose of this study was to evaluate the percentage and the pattern of ALK-rearranged cells, the variation in the native ALK copy number, as well as ALK, c-MET and ROS1 protein expression, and their significance on outcome of crizotinib-treated lung adenocarcinoma patients. Patients and methodsConsecutive lung adenocarcinoma specimens (n = 176) ‘double-negative’ (wild-type EGFR and KRAS) were tested for ALK rearrangements/copy number alterations and for ALK, c-MET and ROS1 protein expression using automated standardized protocols. Preliminary data on the outcome of crizotinib-treated patients were recorded. ResultsFISH analysis identified 26/176 (15%) cases with ALK rearrangements. Seven cases had discordant results between the ALK FISH and IHC. Five cases with discordant FISH-positive/IHC-negative revealed FISH ‘borderline’ positivity (15%–20%). Three cases overexpressed c-MET and responded to crizotinib, and two cases with ALK-‘borderline’ rearranged cells only, not associated with c-MET expression, progressed under crizotinib. Two cases with discordant FISH-negative/IHC-positive revealed ALK gene amplification without associated c-MET or ROS1 protein expression. ConclusionsThe discrepancies observed between the IHC and FISH data revealed unexpected biological events, rather than technical issues, which potentially can have a strong impact on the therapeutic strategy with crizotinib.

The combination of FISH and IHC for ALK assessment in NSCLC is more powerful than either technique alone

Aim Examination of the proposition that detection of ALK overexpression by IHC in NSCLC may act as triage or replacement for FISH. Method Results on 221 NSCLCs referred for ALK FISH testing (Abbott-Vysis LSI ALK Breakapart FISH Probe) were reviewed, FISH hybridisation pattern classified and compared to ALK IHC (Clone D5F3). Results 10 cases (4.5%) had insufficient material for testing. Thirty-four cases were FISH +ve, four equivocal (1.8%) and 173 negative. Seventeen positive cases had classic ‘split’ signal; 14 had isolated 3’ red signals, two had combined pattern and two cases (both IHC +ve) had variant patterns with additional 5’ (green) signals (previously regarded as FISH–ve). Twenty-nine of these 34 cases were IHC +ve (85% IHC sensitivity), three were IHC equivocal, 1 was untested, and 1 IHC–ve. Two FISH equivocal cases (minimal split) were strongly IHC +ve. One of 173 FISH–ve cases was strongly IHC +ve. IHC on 196 cases scored 32 as positive, 14 equivocal (7%) and 150 negative. 25 IHC +ve cases were straightforward FISH +ve, six had variant/equivocal FISH patterns, one was FISH–ve (97% IHC specificity). Three of 14 IHC equivocal cases were FISH +ve, as was one IHC–ve case. Discussion Combination of IHC and FISH for detection of ALK mutation is more powerful than either test in isolation. Without availability of IHC, six cases with equivocal or variant FISH patterns would likely have been missed, however four times more cases are equivocal by IHC than by FISH. Occasional cases (2 in this cohort:

The relevance of external quality assessment for molecular testing for ALK positive non-small cell lung cancer: results from two pilot rounds show room for optimization.

Background and PurposeMolecular profiling should be performed on all advanced non-small cell lung cancer with non-squamous histology to allow treatment selection. Currently, this should include EGFR mutation testing and testing for ALK rearrangements. ROS1 is another emerging target. ALK rearrangement status is a critical biomarker to predict response to tyrosine kinase inhibitors such as crizotinib. To promote high quality testing in non-small cell lung cancer, the European Society of Pathology has introduced an external quality assessment scheme. This article summarizes the results of the first two pilot rounds organized in 2012–2013.Materials and MethodsTissue microarray slides consisting of cell-lines and resection specimens were distributed with the request for routine ALK testing using IHC or FISH. Participation in ALK FISH testing included the interpretation of four digital FISH images.ResultsData from 173 different laboratories was obtained. Results demonstrate decreased error rates in the second round for both ALK FISH and ALK IHC, although the error rates were still high and the need for external quality assessment in laboratories performing ALK testing is evident. Error rates obtained by FISH were lower than by IHC. The lowest error rates were observed for the interpretation of digital FISH images.ConclusionThere was a large variety in FISH enumeration practices. Based on the results from this study, recommendations for the methodology, analysis, interpretation and result reporting were issued. External quality assessment is a crucial element to improve the quality of molecular testing.

Quantification of ALK From Non-Small Cell Lung Cancer (NSCLC) FFPE Tissue by Targeted Mass Spectrometry: Outcomes/Health Services Research

Crizotinib has significant antitumor activity in ALK-rearranged (AR) NSCLC. The current diagnostic test for AR, break-apart FISH, is a low throughput assay and not ideal for the detection of low-frequency oncogenic drivers. We sought to develop a quantitative ALK mass spectrometric (MS) assay and correlate the findings with ALK FISH, IHC results, and clinical outcomes.

P1-3-4 - Alk Translocation in Sarcomatoid Carcinoma of Head and Neck and Treatment Effect of Crizotinib: a Case Series

Sarcomatoid carcinoma of the upper aerodigestive tract, a variant of squamous cell carcinoma, is a rare biphasic tumor consisting of epithelial and mesenchymal components. Herein, we report a case of 34-year-old woman who had a sarcomatoid carcinoma in maxillary sinus and had been treated with surgery, radiation therapy and multiple lines of palliative chemotherapy. Because ALK translocation is common in sarcoma, ALK FISH was performed on the tumor tissue and showed break-apart signal indicative of ALK translocation. Thus, ALK inhibitor, crizotinib was administered to the patient. Although dramatic response was not observed, clinical improvement and stable disease with minimal tumor shrinkage was noted and lasted for 4 months. To further identify the incidence of ALK rearrangement in sarcomatoid carcinoma of head and neck, ALK FISH was performed on tumor samples of 10 patients. Among them, two patients were positive for ALK FISH. To the best of our knowledge, this is the first case report demonstrating clinical benefit of crizotinib in in sarcomatoid carcinoma of head and neck with confirmed ALK translocation . Our results suggest that ALK FISH test may be suitable and encouraged for patients with sarcomatoid carcinoma of head and neck.

Abstract 4675: Detection of ALK, ROS1, and RET translocations in non-small cell lung cancer (NSCLC) patients by intragenic differential expression analysis

Abstract BACKGROUND: ALK, ROS1, and RET translocations are frequently detected in NSCLC patients. Crizotinib, a tyrosine kinase inhibitor (TKI), was approved by the FDA in 2011 to treat NSCLC in patients harboring ALK translocations as detected by an FDA-approved assay. However, the FDA-approved ALK FISH assay is technically challenging, with failures due to pre-analytic variables. Another approach, intragenic differential expression (IDE), detects translocations by comparing expression levels of the 5′ end with the 3′ end of target gene transcripts. In this study we developed and evaluated a rapid IDE assay to screen for ALK, ROS1, and RET translocations, independent of the fusion partner. METHODS: A total of 419 samples (408 randomly-selected NSCLC clinical samples, ALK positive and ROS1 positive cell lines (2 each), and 7 previously-tested RET-positive clinical samples) were used to develop and evaluate performance characteristics of the IDE assays. To determine IDE scores, levels of ALK, ROS1, and RET expression were first determined by quantitative RT-PCR measurement of the 5′- and 3′- ends of the respective transcripts. The differences in expression levels were calculated as ΔCt (Ct5′ - Ct3′). High ΔCt values indicate presumptive presence of gene translocations. 212/408 NSCLC samples were analyzed by ALK FISH and EML4-ALK RT-PCR, and 196/408 samples were analyzed by EML4-ALK RT-PCR. RESULTS: Thirty-one of the 408 (7.6%) clinical samples tested positive for ALK rearrangements by IDE. Among them, 20 were confirmed by FISH and/or EML4-ALK (true positive, 64.5%), while 11 were negative by FISH and/or EML4-ALK (false positive, 35.5%). One of 10 ALK FISH positive samples tested negative by both ALK IDE and EML4-ALK RT-PCR analysis (false negative), while one of 202 FISH-negative sample tested positive by both EML4-ALK and ALK IDE. ALK IDE exhibited 94.5% (189/200) concordance with ALK FISH and 96.0% (356/371) concordance with the EML4-ALK assay. For ROS1, both ROS1-positive cell lines and 4/408 (1.0%) NSCLC samples tested positive for ROS1 by IDE. Among the 4 IDE-positive NSCLC samples, 1 was confirmed by ROS1 FISH. For RET, all 7 known positives and 10/408 (2.5%) NSCLC samples tested positive by IDE. Three of six RET IDE positive NSCLC samples were confirmed by RET FISH. Overall, ALK, ROS1, and RET translocations were mutually exclusive in NSCLC patients. The lung IDE assay had a failure rate of 3.7%. CONCLUSION: These findings demonstrate the feasibility of using IDE to detect ALK, ROS1, and RET gene translocations. These assays may have potential as a screening tool to select patients for further confirmation by FISH for TKI-targeted therapy. The IDE concept can be applied to a wide range of somatic translocations. Citation Format: Shih-Min Cheng, Cindy Barlan, Feras Hantash, Heather R. Sanders, Patricia H. Chan, Vladimira Sulcova, Marc A. Sanidad, Kevin Qu, Joann C. Kelly, Fatih Z. Boyar, Anthony D. Sferruzza, Frederic M. Waldman. Detection of ALK, ROS1, and RET translocations in non-small cell lung cancer (NSCLC) patients by intragenic differential expression analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4675. doi:10.1158/1538-7445.AM2014-4675

Abstract 2841: Characterization of ALK fusions in circulating tumor cells (CTCs) of NSCLC

Abstract Background: Many NSCLC patients have insufficient tumor sample or have high co-morbidities preventing access to tissue biopsies for FISH or IHC to determine ALK fusion and subsequent eligibility to Crizotinib therapy. We developed a gene/protein assay for the ALK fusion and examined CTCs and CTC subpopulation incidence and molecular characterization in newly diagnosed NSCLC patients. Methods: 10 samples from newly diagnosed NSCLC patients prior to therapy were recruited and blood specimens were collected and shipped to Epic Sciences. 2/10 had known ALK rearrangement through tissue analysis. All nucleated cells were plated onto glass slides and subjected to IF staining and CTC identification by fluorescent scanners and algorithmic analysis. CTCs, defined as classic (CK+ CD45- w/intact DAPI nuclei and morphologically distinct), apoptotic (CK+, CD45-, non-intact nuclei) and CK- (CK-, CD45-, intact and distinct) were identified. Samples were characterized with ALK IF to assess expression. Patients with known ALK rearrangements in tissue also had their CTCs assessed with ALK FISH. Results: Assays for the ALK protein and ALK gene rearrangement were developed and specificity confirmed utilizing H2228 (ALK positive) &amp; A549 (ALK negative) cells spiked into donor blood and run through the Epic Assay. Expression of ALK fusions was visualized in CTCs of 1/2 patients with known ALK rearrangements and 0/8 patients with no ALK rearrangements. ALK fusion protein expression was seen in classic, apoptotic, small and CK- CTCs. Conclusion: ALK fusion protein and ALK rearrangement assessment with FISH utilizing the Epic CTC platform demonstrates sensitivity and specificity to identification of patients eligible to Crizotinib. The identification of ALK in CTC subpopulations identifies unique tumor cell morphology and suggests evidence of epithelial plasticity. Further clinical studies are ensuing to determine the sensitivity and specificity of ALK in CTCs and the relevance of CTC subpopulations related to Crizotinib therapy and NSCLC progression. Citation Format: David Lu, Rachel Krupa, Natalee Bales, Jessica Louw, Dena Marrinucci, Ryan Dittamore. Characterization of ALK fusions in circulating tumor cells (CTCs) of NSCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2841. doi:10.1158/1538-7445.AM2014-2841

Abstract 4699: Analytical validation of solid tumor fusion gene detection in a comprehensive NGS-based clinical cancer genomic test

Abstract Background: As the number of clinically actionable cancer genes grows and the size of most diagnostic biopsies decreases, next-generation sequencing (NGS) becomes increasingly attractive as a diagnostic tool, as it can detect all classes of genomic alterations in all cancer genes in a single test. However, for NGS to achieve its full utility in the clinic, robust analytical validation and performance comparisons against established detection methodologies are required for each class of targetable genomic alteration. Methods: Previously, we reported on the development and validation of an NGS-based diagnostic test for accurate detection of clinically-relevant genomic alterations across all exons of 287 cancer genes in routine FFPE specimens (Frampton, et. al, Nat. Biotech. Oct 2013). Here, we present systematic validation of fusion gene detection in the test, enabled by hybrid-selection and deep sequencing of commonly rearranged introns in 19 (or, in an alternative version, 31) genes. We created reference samples reflecting key determinants of detection accuracy for gene fusions, including extent of stromal admixture and variety of partner genes: We obtained 5 solid tumor fusion-bearing cell-lines (2 ALK, 1 RET, 1 ROS1, 1 TMPRSS2) and mixed these into 23 variably sized pools, such that each fusion was represented at 100%, 50%, 33%, 25%, and 20% simulated cellular fraction at least once. Gene fusions were called if a well-mapped cluster of 5 chimeric reads or greater was observed in a targeted intron. We then verified that the observed performance translated to FFPE clinical samples by examining test concordance in 43 tumors (20+/23-) characterized for ALK rearrangement by FISH. Finally, we evaluated the test broadly by assessing detection prevalence of the three most common gene fusions (ALK, ROS1, RET) in 724 lung adenocarcinoma FFPE patient samples, including needle biopsies. Results: Of 32 tested gene fusion instances in the 28 cell-lines samples, all were successfully detected (sensitivity 100%, 95% CI 89%-100%), with no false positive calls. Robust performance translated to FFPE: of the 20 ALK FISH+ specimens, 18 were unequivocally (+) by NGS, with remaining 2 specimens showing sub-threshold evidence of the events. 22/23 FISH- specimens were NGS-, with the remaining specimen an apparent false (-) by FISH. Clinical lung adenocarcinoma samples showed 5% ALK, 3% RET, and 2% ROS1 rearrangement rate respectively, in line with published data. Conclusions: We present rigorous validation and performance benchmarks for efficient targeted fusion gene detection for solid tumors in an NGS-based test for use in clinical oncology. Given the ability of NGS to detect a much broader range of genomic alterations than currently available technologies, particularly on limited tissue, this type of testing can be a direct component of patient care and potentially expand targeted treatment options. Citation Format: Roman Yelensky, Amy Donahue, Geoff Otto, Michelle Nahas, Jie He, Frank Juhn, Sean Downing, Garrett M. Frampton, Juliann Chmielecki, Jeffrey S. Ross, Maureen Zakowski, Marc Ladanyi, Vincent A. Miller, Philip J. Stephens, Doron Lipson. Analytical validation of solid tumor fusion gene detection in a comprehensive NGS-based clinical cancer genomic test. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4699. doi:10.1158/1538-7445.AM2014-4699

Accurate identification of ALK positive lung carcinoma patients: novel FDA-cleared automated fluorescence in situ hybridization scanning system and ultrasensitive immunohistochemistry.

BackgroundBased on the excellent results of the clinical trials with ALK-inhibitors, the importance of accurately identifying ALK positive lung cancer has never been greater. However, there are increasing number of recent publications addressing discordances between FISH and IHC. The controversy is further fuelled by the different regulatory approvals. This situation prompted us to investigate two ALK IHC antibodies (using a novel ultrasensitive detection-amplification kit) and an automated ALK FISH scanning system (FDA-cleared) in a series of non-small cell lung cancer tumor samples.MethodsForty-seven ALK FISH-positive and 56 ALK FISH-negative NSCLC samples were studied. All specimens were screened for ALK expression by two IHC antibodies (clone 5A4 from Novocastra and clone D5F3 from Ventana) and for ALK rearrangement by FISH (Vysis ALK FISH break-apart kit), which was automatically captured and scored by using Bioview's automated scanning system.ResultsAll positive cases with the IHC antibodies were FISH-positive. There was only one IHC-negative case with both antibodies which showed a FISH-positive result. The overall sensitivity and specificity of the IHC in comparison with FISH were 98% and 100%, respectively.ConclusionsThe specificity of these ultrasensitive IHC assays may obviate the need for FISH confirmation in positive IHC cases. However, the likelihood of false negative IHC results strengthens the case for FISH testing, at least in some situations.

137IN - Importance of External Quality Assessement in Immuno-Oncology

ABSTRACT The important role of immunohistochemistry (IHC) is well known. IHC will recognize antigens and, consequently, identify and classify specific cells within a cell population whose morphology is heterogenous or apparently homogenous. Since many years external quality assessment (EQA) schemes for IHC are organized by NORDIQC and UK NEAQS. Since some years, IHC can be used for treatment selection in advanced NSCLC. In 2012, the European Society of Pathology (ESP) proposed an EQA scheme to promote high quality biomarker testing in NSCLC for EGFR mutation analysis and ALK rearrangement detection (IHC, FISH). From 2014 on, ROS1 testing is also included. The scheme aims to assess and improve the current status of testing in NSCLC, to provide education and remedial measures, to permit inter-laboratory comparison and to allow validation of test methods by distributing validated material harboring well-defined aberrations. In total, 173 different laboratories participated in the pilot rounds. In the first round, 29 laboratories submitted results for ALK IHC. In the second round, 58 laboratories submitted results for ALK IHC. If carefully clinically validated according to ISO 15189, ALK IHC may be considered as a screening method to select specimens for ALK FISH testing. In the context that at the time of the pilot studies no interlaboratory comparison validation studies were available, ALK IHC performance from our study shows that IHC testing is well implemented. Not surprisingly however, the error rates for IHC were greater than for FISH. Our study demonstrates improvement of ALK testing after only two rounds. It is expected that larger datasets, spanning a larger number of EQA participations will demonstrate a statistically significant improvement in performance. On scheme level, the decreased error rates in the second round demonstrated improvement for both ALK FISH and ALK IHC. Error rates for ALK FISH TMA and ALK IHC were still high (>5%), which stresses the need for continued education through EQA. Based on the results from this study, recommendations for the methodology, analysis, interpretation and result reporting were issued. External quality assessment is a crucial element to improve the quality of molecular testing. Disclosure: All authors have declared no conflicts of interest.

Large Cell Neuroendocrine Carcinomas (Lcnec) of the Lung: Pathologic Features, Treatment and Outcomes

ABSTRACT Aim: LCNEC is a rare thoracic malignancy, for which pathologic classification and optimal therapies are debated. We report the largest series of stage IV LCNECs, evaluating clinicopathologic features, treatment and survival. Methods: Pts with stage IV LCNEC evaluated at MSKCC from 2006-2013 were identified. Clinicopathologic and treatment data were collected. Radiologic RECIST evaluation was performed for pts with existing diagnostic imaging. Pts with available tissue underwent pathology re-review to confirm diagnosis, and potentially identify features that could impact outcomes. Results: 50 pts were identified (median age: 66 years; 62% male; 88% former/current smokers). Common sites of metastasis: lymph nodes (n = 29); brain (n = 22); liver (n = 13). 34% of pts had diagnostic molecular testing with PCR-based fragment length analysis, mass spectrometry based assay for point mutation genotyping, and ALK FISH testing: 24% had KRAS mutations (mtns) (n = 4/17; G12D, n = 2; G12C, n = 1; Q61H, n = 1). No EGFR mtns or ALK rearrangements were noted. 33 pts had archived tumor at MSKCC for central pathology re-review. The diagnosis of LCNEC was confirmed in all but 2 cases, which were reclassified as SCLC and combined SCLC/LCNEC (both brain metastases), respectively. Treatment data was available on 39 pts: 77% (n = 30) received first-line platinum(plt)/etoposide. 23% (n = 9) received other regimens: plt/taxane (n = 4), plt/pemetrexed (n = 1), plt/pemetrexed/bevacizumab(bev) (n = 1), temozolomide (n = 1)/bev (n = 1), clinical trial (n = 1). Objective response rate was 32% with plt/etoposide by RECIST (95% CI: 16-52%). Median OS was 12.2mos (range: 9-19.3mos) for all pts. Karnofsky performance status 250 were poor prognostic factors for OS on multivariate analysis (p Conclusions: Pts with stage IV LCNEC have low response to plt/etoposide treatment and poor OS. KRAS mutations are commonly observed. Prospective studies are needed to investigate optimum therapeutic strategies. Disclosure: All authors have declared no conflicts of interest.

Immunohistochemistry as a screening tool forALKrearrangement in NSCLC: evaluation of five different ALK antibody clones andALKFISH

ALK FISH analysis is used as the reference standard to demonstrate ALK rearrangements, which qualify patients with pulmonary adenocarcinomas for therapy with ALK inhibitors. The aim of this study was to find screening ALK antibody clones with the best positive and best negative percentage agreement with ALK FISH. Three hundred and three pulmonary adenocarcinomas were evaluated with ALK FISH and stained with five ALK antibody clones (5A4; D5F3; ALK1; ALK01; SP8) with standardized detection systems. D5F3 was additionally assessed using the OptiView enhanced detection and amplification system. ALK FISH found 14 cases (4.6%) that harboured ALK rearrangements. These stained at all intensities for D5F3 and 5A4. To identify rearranged cases among stained cases, we subsequently analysed all immunohistochemically positive cases with ALK FISH. D5F3 with OptiView exclusively stained rearranged cases with strong intensity, without a single false-positive or false-negative case. The number of subsequent ALK FISH analyses required would have decreased from 303 to 14 cases (-95.4%), reducing significantly the time, work and costs without any loss of diagnostic quality and accuracy.

Success and failure rates of tumor genotyping techniques in routine pathological samples with non-small-cell lung cancer

IntroductionIdentification of some somatic molecular alterations in non-small-cell lung cancer (NSCLC) has become evidence-based practice. The success and failure rate of using commercially available tumor genotyping techniques in routine day-to-day NSCLC pathology samples is not well described. We sought to evaluate the success and failure rate of EGFR mutation, KRAS mutation, and ALK FISH in a cohort of lung cancers subjected to routine clinical tumor genotype. MethodsClinicopathologic data, tumor genotype success and failure rates were retrospectively compiled and analyzed from 381 patient-tumor samples. ResultsFrom these 381 patients with lung cancer, the mean age was 65 years, 61.2% were women, 75.9% were white, 27.8% were never smokers, 73.8% had advanced NSCLC and 86.1% had adenocarcinoma histology. The tumor tissue was obtained from surgical specimens in 48.8%, core needle biopsies in 17.9%, and as cell blocks from aspirates or fluid in 33.3% of cases. Anatomic sites for tissue collection included lung (49.3%), lymph nodes (22.3%), pleura (11.8%), bone (6.0%), brain (6.0%), among others. The overall success rate for EGFR mutation analysis was 94.2%, for KRAS mutation 91.6% and for ALK FISH 91.6%. The highest failure rates were observed when the tissue was obtained from image-guided percutaneous transthoracic core-needle biopsies (31.8%, 27.3%, and 35.3% for EGFR, KRAS, and ALK tests, respectively) and bone specimens (23.1%, 15.4%, and 23.1%, respectively). In specimens obtained from bone, the failure rates were significantly higher for biopsies than resection specimens (40% vs. 0%, p=0.024 for EGFR) and for decalcified compared to non-decalcified samples (60% vs. 5.5%, p=0.021 for EGFR). ConclusionsTumor genotype techniques are feasible in most samples, outside small image-guided percutaneous transthoracic core-needle biopsies and bone samples from core biopsies with decalcification, and therefore expansion of routine tumor genotype into the care of patients with NSCLC may not require special tissue acquisition or manipulation.