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D. Aisner



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    MO01 - Lung Cancer Biology - Techniques and Platforms (ID 90)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      MO01.03 - Transitioning To Next Gen Testing Of Lung Carcinoma (ID 2891)

      10:30 - 12:00  |  Author(s): D. Aisner

      • Abstract
      • Slides

      Background
      The feasibility of multigene testing in a clinical setting has been demonstrated by the Lung Cancer Mutation Consortium (LCMC) which has evaluated over 1000 cases from multiple institutions in a CLIA environment. The initial platforms used by the LCMC were SNaPshot and Ion Torrent, allele specific tests. More recently the sequencing by synthesis method (Illumina) used for whole genome sequencing has been scaled for sequencing of a limited number of targeted genes. In this study we compare the performance characteristics of Next Generation testing on the MiSeq platform with the older allele specific SNaPshot platform and evaluate the applicability of Miseq-based testing to a clinical, CLIA regulated setting.

      Methods
      Two Illumina kits, the TruSeq and TruSight evaluating 221 hotspots in 48 gene and 175 exons in 26 genes, respectively, were compared. To assess analytical sensitivity, cell lines with known mutations and SNPs were titered into liver DNA known to be wild-type for the selected mutations, at tumor cell concentrations ranging from 3% to 50%. In addition, 24 formalin-fixed paraffin-embedded lung tumors that had previously been evaluated by SNaPshot or direct sequencing were tested to compare sensitivities and specificities of methods. Paraffin embedded human tumor tissue samples were enriched for tumor cells by coring of paraffin block or macrodissection using a pneumatic cell collector. DNA was extracted by proteinase K digestion and column chromatography, end repaired and phosphorylated. Libraries were prepared from each sample by ligating index adapters that allow for mixing of samples and binding adapters that link DNA fragments to flow cell. Combined libraries were added to flow cells at an appropriate concentration, clusters generated, and sequencing reaction commenced. Results were evaluated by software developed by Illumina or locally at the University of Colorado.

      Results
      Spiking studies indicated that analytic sensitivity for Miseq at loading quantities of 100 to 300 ng (TruSeq) was ~5% for known KRAS and TP53 mutations and several synonymous polymorphisms in other covered genes, comparable to SNaPshot. For clinical samples, average depth of coverage was 5700 (+/- 2267). Unfiltered results using Illumina software supplied with the Miseq instrument showed an average of 88 heterozygous SNPs, 12 insertions and 17 deletions (uncurated for relevance). All of the mutations that were previously found by SNaPshot were also detected by Miseq TruSeq and TruSight protocols (100% concordance). Variants representing known polymorphisms, synonymous changes and variants identified in the context of low coverage were excluded. Data analysis using locally developed software indicated the presence of 1-9 SNPs in each sample that were not predicted by SNaPshot testing, attributable to the wider coverage of the Miseq platforms. None of the additional mutations represented treatable targets with currently available drugs.

      Conclusion
      Next-generation testing is feasible in a CLIA environment using the Miseq platform. However, rigorous software validation is necessary before this platform can be adopted by a busy clinical laboratory. Software limitations currently being addressed include long turnaround time, inadequate vetting of new and recurrent SNPs for clinical significance and limited software development resources.

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    MO10 - Molecular Pathology II (ID 127)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Pathology
    • Presentations: 2
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      MO10.10 - Detection of RET fusions by FISH in unselected NSCLC (ID 2434)

      16:15 - 17:45  |  Author(s): D. Aisner

      • Abstract
      • Presentation
      • Slides

      Background
      Activation of the RET gene by fusion has been described in 1-2% of unselected population of non-small-cell lung cancer (NSCLC) and there is early evidence suggesting that patients with RET activated tumors obtain clinical benefit from RET inhibitors. The major fusion partner is KIF5B, but CCDC6, NCOA4 and TRIM33 have also been reported. The prevalence of RET fusions in different lung cancer subtypes and clinicopathologic characteristics of remain unclear. In this study, we sought to identify RET rearrangements in NSCLC using FISH and to investigate the association with histology and clinical features.

      Methods
      A 3-target, 3-color FISH probe set [3’RET in red, 5’RET in green, 5’KIF5B in yellow] was developed to simultaneously detect (a) disruption between 3’ and 5’ RET and (b) specific fusion between 5’KIF5B-3’RET. This probe set was used to interrogate a cohort of Caucasian NSCLC patients using tumor microarray. Inclusion of specimens on the tissue microarray was independent of gender, age, smoking history, histology and any known molecular profile and was only based on patient informed consent and tissue availability.

      Results
      Among 348 evaluable NSCLC patients, 6 (1.7%) were found to be positive for RET rearrangement (RET+): 2 showed typical KIF5B:RET pattern, 2 showed patterns consistent with CCDC6: RET fusion; and 2 had split 3’-5’ without suggestion of the fusion partner identity. The histology was adenocarcinoma in 4, large cell carcinoma in 1 and squamous cell carcinoma in 1. All RET+ tumors were wild type for EGFR and negative for ALK and ROS1 rearrangements. The mean age of RET+ patients at the time of diagnosis was 62 years (49-74) and they were predominantly male (5) and former (4) or current smokers (1). The 10p11-q11 region displayed high level of genomic instability, with RET doublets, KIF5B and RET doublets, unbalanced KIF5B copy number gain, fusion KIF5B with 5’ and 3’RET, and abnormal separation between KIF5B and RET in 8.5%, 5.1%, 9.6%, 2.3%, and 2% of specimens, respectively. These atypical patterns will be further investigated by RT-PCR.

      Conclusion
      The customized 3-target, 3-color probe set successfully detected KIF5B:RET rearrangements and identified patterns suggestive of RET rearrangements with non-KIF5B partners in small subset of unselected NSCLC. Interestingly, only a minority of RET + patients were never smokers and 1/3 of them had non-adenocarcinoma histology. Despite the benefits of using enrichment strategies based on clinicopathologic variables for molecular testing of NSCLC in search for personalized therapy, these findings argue against using variables such as smoking status and histology for screening selection when the aim is to detect all potential RET+ patients.

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      MO10.12 - ROS1 Fusions Diagnosed by Break-Apart FISH in NSCLC (ID 968)

      16:15 - 17:45  |  Author(s): D. Aisner

      • Abstract
      • Presentation
      • Slides

      Background
      Chromosomal rearrangements which generate constitutively activated ROS1 receptor tyrosine kinase (6q22.1) have been found in several tumor types, including non-small cell lung cancers (NSCLC). In clinical trials, the oral kinase inhibitor crizotinib has shown promise in treating tumors with ROS1 rearrangements. Currently, fluorescence in situ hybridization (FISH) using dual-color, break-apart (BA) probes is used to detect ROS1 rearrangements in clinical samples; however, further optimization of this method is necessary to ensure patients are accurately diagnosed. This study explores BA FISH assay characteristics in NSCLC samples.

      Methods
      Tumor sections from 464 NSCLC patients were screened for ROS1 rearrangement using ROS1 BA FISH. Of these samples, 206 were co-screened for ALK rearrangement. The copy number of fused and isolated 3’/5’ signals, as well as the incidence of atypical patterns (doublet and clustered multiple fusions) was investigated. Cells were considered ROS1 positive (ROS1+) when ≥ 15% of nuclei displayed split 5’/3’ signals or single 3’ signals. Specific fusion transcripts in ROS1+ cases were identified by RT-PCR or inverse PCR.

      Results
      ROS1 rearrangements (ROS1+) were found in 21 patients (5%). The copy number of native ROS1 differed significantly between positive and negative tumors (mean of 1.5 versus 2.5, p<0.0001). The percent of cells with FISH patterns compatible with ROS1 rearrangement ranged from 30% to 100%, with a mean of 81%, in ROS1+ patients. The distribution of positive cells between scored regions within ROS1+ tumors was investigated for 13 cases and found to follow a normal distribution, ruling out intra-tumoral heterogeneity. Among ROS1+ specimens, 71% had a split signal pattern, 19% displayed a single 3’ pattern, and 10% had both a split and single 3’ pattern of positivity. For positive tumors, ROS1 fusion partners were identified as SDC4 (S2;R32 and S2;R34), EZR (E10;R34) and CD74 (C6;R32 and C6;R34). Atypical negative patterns such as fused doublets, clusters, 3’ doublets, 5’ doublets, and single 5’ signals were observed in 4%, 1%, 1%, <1%, and <1% of negative patients. ALK and ROS1 were scored simultaneously in the same cells in 206 patients, including 5 ROS1+ and 10 ALK+; no double positive cases were found. In ROS1 negative specimens, mean native ALK copy numbers were significantly higher than native ROS1 in ALK negative samples (3.2 versus 2.3, p<0.0001).

      Conclusion
      ROS1+ tumors were detected in 5% of patients in this large NSCLC cohort. Since these patients were subject to various selection strategies, this frequency cannot be transferred to an unselected NSCLC population. The low native ROS1 copy number in the rearranged cells and lack of evidence of intra-tumoral heterogeneity suggests ROS1 rearrangements occur early in tumorigenesis, consistent with their known oncogenic driver role. Data from this sample also show that, in FISH negative cases, ROS1 copy number was lower than native ALK. This suggests ROS1 may exist in a relatively more stable portion of the genome, potentially explaining why ROS1 rearrangements exist at a lower frequency than ALK rearrangements in NSCLC.

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    P1.02 - Poster Session 1 - Novel Cancer Genes and Pathways (ID 144)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.02-006 - Identification of targetable driver mutations in molecularly selected never smoker lung adenocarcinomas (ID 2970)

      09:30 - 16:30  |  Author(s): D. Aisner

      • Abstract

      Background
      Approximately 25% of lung cancers occur in lifelong never smokers. Although no dominant risk factor has been identified yet, the discover of molecular drivers potentially targetable with biological agents, makes lung cancer in never smokers a unique disease, candidate for a personalized therapy. Through the FISH test, we performed a screening for ALK, ROS1, and RET rearrangements, in a highly selected population of lung adenocarcinoma never smoker patients, previously demonstrated to be wild-type for EGFR and K-RAS mutations.

      Methods
      We collected archived histological material of 28 EGFR and K-RAS wild-type patients (pts), from a 200 never-smoker advanced lung adenocarcinomas database, to be analyzed for the presence of rearrangements in ALK, ROS1 and RET genes. All pts were treated at the Division of Medical Oncology of the S Maria della Misericordia Hospital in Perugia from October 2003 to February 2013. 20 specimens were included in a tissue microarray (TMA) analysis, whereas 8 were screened in separate subset, due to the scarce samples. FISH test was performed using a combination of commercial reagents and custom designed probes. Median overall survival (OS) of mutated pts compared to the pan-negative ones, was evaluated by Cox multivariate analysis.

      Results
      Clinicopathological characteristics: among the 28 patients, 27 were never smokers and 1 former light smoker, with a good performance status; 20 (72%) presented with a metastatic disease at diagnosis, 8 (28%) were locally advanced; median age was 56 years-old, with a predominance of female sex (18/28, 64%). All cases were invasive adenocarcinomas and classified into 18 (64%) solid predominant type, 1 (3.5%) mixed acinar/lepidic pattern, 1 (3.5%) papillary, no predominant subtype for 8 (28%) patients, because of unsufficient histological material available. Of the 28 never smoker cases, we identified 7 gene fusions (25%), including 2 pts ALK+ (7.1%), 3 pts ROS1+ (10.7%) and 2 RET+ cases (7.1%), one compatible with KIF5B:RET and other with CCDC6:RET fusion. Median OS for the entire cohort was 24.5 months (mo), 61.2 mo for mutated pts (any rearrangement) vs 24.1 mo for not-mutated, respectively (P = .292).

      Conclusion
      Molecularly selected never smoker lung adenorcinomas associates with a high incidence of driver genes mutations and further investigations to confirm our frequencies in larger cohorts are needed. In line with literature data, our findings suggest a different survival outcome among genotypes, and identification of specific subsets in this special population can lead to successful treatment with target therapies.