Theresa A. Boyle

Fluorescence In Situ Hybridization, Immunohistochemistry, and Next-Generation Sequencing for Detection of EML4-ALK Rearrangement in Lung Cancer

BACKGROUND The U.S. Food and Drug Administration-approved method for detecting EML4-ALK rearrangement is fluorescence in situ hybridization (FISH); however, data supporting the use of immunohistochemistry (IHC) for that purpose are accumulating. Previous studies that compared FISH and IHC considered FISH the gold standard, but none compared data with the results of next-generation sequencing (NGS) analysis. MATERIALS AND METHODS We studied FISH and IHC (D5F3 antibody) systematically for EML4-ALK rearrangement in 51 lung adenocarcinoma patients, followed by NGS in case of discordance. RESULTS Of 51 patients, 4 were positive with FISH (7.8%), and 8 were positive with IHC (15.7%). Three were positive with both. NGS confirmed that four of the five patients who were positive with IHC and negative with FISH were positive for ALK. Two were treated by crizotinib, with progression-free survival of 18 and 6 months. Considering NGS as the most accurate test, the sensitivity and specificity were 42.9% and 97.7%, respectively, for FISH and 100% and 97.7%, respectively, for IHC. CONCLUSION The FISH-based method of detecting EML4-ALK rearrangement in lung cancer may miss a significant number of patients who could benefit from targeted ALK therapy. Screening for EML4-ALK rearrangement by IHC should be strongly considered, and NGS is recommended in borderline cases. Two patients who were negative with FISH and positive with IHC were treated with crizotinib and responded to therapy.

PD-L1 Expression in Lung Cancer

&NA; Immunotherapies targeted against programmed death ligand 1 (PD‐L1) and its receptor (PD‐1) have improved survival in a subset of patients with advanced lung cancer. PD‐L1 protein expression has emerged as a biomarker that predicts which patients are more likely to respond to immunotherapy. The understanding of PD‐L1 as a biomarker is complicated by the history of use of different immunohistochemistry platforms with different PD‐L1 antibodies, scoring systems, and positivity cut‐offs for immunotherapy clinical trials with different anti‐PD‐L1 and anti‐PD‐1 drugs. Herein, we summarize the brief history of PD‐L1 as a biomarker and describe the challenges remaining to harmonize PD‐L1 detection and interpretation for best patient care.

Protein Kinase C δ Is a Downstream Effector of Oncogenic K-ras in Lung Tumors

Oncogenic activation of K-ras occurs commonly in non-small cell lung cancer (NSCLC), but strategies to therapeutically target this pathway have been challenging to develop. Information about downstream effectors of K-ras remains incomplete, and tractable targets are yet to be defined. In this study, we investigated the role of protein kinase C δ (PKCδ) in K-ras-dependent lung tumorigenesis by using a mouse carcinogen model and human NSCLC cells. The incidence of urethane-induced lung tumors was decreased by 69% in PKCδ-deficient knockout (δKO) mice compared with wild-type (δWT) mice. δKO tumors are smaller and showed reduced proliferation. DNA sequencing indicated that all δWT tumors had activating mutations in KRAS, whereas only 69% of δKO tumors did, suggesting that PKCδ acts as a tumor promoter downstream of oncogenic K-ras while acting as a tumor suppressor in other oncogenic contexts. Similar results were obtained in a panel of NSCLC cell lines with oncogenic K-ras but which differ in their dependence on K-ras for survival. RNA interference-mediated attenuation of PKCδ inhibited anchorage-independent growth, invasion, migration, and tumorigenesis in K-ras-dependent cells. These effects were associated with suppression of mitogen-activated protein kinase pathway activation. In contrast, PKCδ attenuation enhanced anchorage-independent growth, invasion, and migration in NSCLC cells that were either K-ras-independent or that had WT KRAS. Unexpectedly, our studies indicate that the function of PKCδ in tumor cells depends on a specific oncogenic context, as loss of PKCδ in NSCLC cells suppressed transformed growth only in cells dependent on oncogenic K-ras for proliferation and survival.

Replication and further characterization of a Type 1 diabetes‐associated locus at the telomeric end of the major histocompatibility complex

Background:  We recently reported an association between Type 1 diabetes and the telomeric major histocompatibility complex (MHC) single nucleotide polymorphism (SNP) rs1233478. As further families have been analyzed in the Type 1 Diabetes Genetics Consortium (T1DGC), we tested replication of the association and, with more data, analyzed haplotypic associations.

Polypharmacology-based ceritinib repurposing using integrated functional proteomics

Targeted drugs are effective when they directly inhibit strong disease drivers, but only a small fraction of diseases feature defined actionable drivers. Alternatively, network-based approaches can uncover new therapeutic opportunities. Applying an integrated phenotypic screening, chemical and phosphoproteomics strategy, here we describe the anaplastic lymphoma kinase (ALK) inhibitor ceritinib as having activity across several ALK-negative lung cancer cell lines and identify new targets and network-wide signaling effects. Combining pharmacological inhibitors and RNA interference revealed a polypharmacology mechanism involving the noncanonical targets IGF1R, FAK1, RSK1 and RSK2. Mutating the downstream signaling hub YB1 protected cells from ceritinib. Consistent with YB1 signaling being known to cause taxol resistance, combination of ceritinib with paclitaxel displayed strong synergy, particularly in cells expressing high FAK autophosphorylation, which we show to be prevalent in lung cancer. Together, we present a systems chemical biology platform for elucidating multikinase inhibitor polypharmacology mechanisms, subsequent design of synergistic drug combinations, and identification of mechanistic biomarker candidates.

Summary of microsatellite instability test results from laboratories participating in proficiency surveys: proficiency survey results from 2005 to 2012.

CONTEXT The College of American Pathologists surveys are the largest laboratory peer comparison programs in the world. These programs allow laboratories to regularly evaluate their performance and improve the accuracy of the patient test results they provide. Proficiency testing is offered twice a year to laboratories performing microsatellite instability testing. These surveys are designed to emulate clinical practice, and some surveys have more challenging cases to encourage the refinement of laboratory practices. OBJECTIVE This report summarizes the results and trends in microsatellite instability proficiency testing from participating laboratories from the inception of the program in 2005 through 2012. DESIGN We compiled and analyzed data for 16 surveys of microsatellite instability proficiency testing during 2005 to 2012. RESULTS The number of laboratories participating in the microsatellite instability survey has more than doubled from 42 to 104 during the 8 years analyzed. An average of 95.4% of the laboratories correctly classified each of the survey test samples from the 2005A through 2012B proficiency challenges. In the 2011B survey, a lower percentage of laboratories (78.4%) correctly classified the specimen, possibly because of overlooking subtle changes of microsatellite instability and/or failing to enrich the tumor content of the specimen to meet the limit of detection of their assay. CONCLUSIONS In general, laboratories performed well in microsatellite instability testing. This testing will continue to be important in screening patients with colorectal and other cancers for Lynch syndrome and guiding the management of patients with sporadic colorectal cancer.

MET–GRB2 Signaling-Associated Complexes Correlate with Oncogenic MET Signaling and Sensitivity to MET Kinase Inhibitors

Purpose: Targeting MET in cancer is hampered by lack of diagnostics that accurately reflect high MET signaling and dependence. We hypothesized that assays reflecting MET signaling associated protein complexes could redefine tumors dependent on MET and could add additional precision beyond genomic assessments. Experimental Design: We used biochemical approaches, cellular viability studies, and proximity ligation assays to assess MET dependence. We examined MET signaling complexes in lung cancer patient specimens (N = 406) and patient-derived xenograft (PDX) models of solid tumors (N = 308). We evaluated response to crizotinib in a MET-amplified cohort of PDX models of lung cancer (N = 6) and provide a case report of a lung cancer patient harboring a Δexon14 MET splice variant. Results: We found the interaction of MET with the adaptor protein GRB2 is necessary for oncogenic survival signaling by MET. MET-GRB2 complexes were identified only within MET-amplified PDX models and patient specimens but exhibit substantial variability. Lack of MET-GRB2 complexes was associated with lack of response to MET TKI in cell lines and PDX models. Presence of MET-GRB2 complexes can further subtype tumors with Δexon14 MET splice variants. Presence of these complexes correlated with response to crizotinib in one patient with Δexon14 MET lacking MET gene amplification. Conclusions: Proximity assays measuring MET-GRB2 signaling complexes provide novel insights into MET-mediated signaling and could complement current clinical genomics-based assay platforms. Clin Cancer Res; 23(22); 7084–96. ©2017 AACR.

Congruence as a measurement of extended haplotype structure across the genome

BackgroundHistorically, extended haplotypes have been defined using only a few data points, such as alleles for several HLA genes in the MHC. High-density SNP data, and the increasing affordability of whole genome SNP typing, creates the opportunity to define higher resolution extended haplotypes. This drives the need for new tools that support quantification and visualization of extended haplotypes as defined by as many as 2000 SNPs. Confronted with high-density SNP data across the major histocompatibility complex (MHC) for 2,300 complete families, compiled by the Type 1 Diabetes Genetics Consortium (T1DGC), we developed software for studying extended haplotypes.MethodsThe software, called ExHap (Extended Haplotype), uses a similarity measurement we term congruence to identify and quantify long-range allele identity. Using ExHap, we analyzed congruence in both the T1DGC data and family-phased data from the International HapMap Project.ResultsCongruent chromosomes from the T1DGC data have between 96.5% and 99.9% allele identity over 1,818 SNPs spanning 2.64 megabases of the MHC (HLA-DRB1 to HLA-A). Thirty-three of 132 DQ-DR-B-A defined haplotype groups have > 50% congruent chromosomes in this region. For example, 92% of chromosomes within the DR3-B8-A1 haplotype are congruent from HLA-DRB1 to HLA-A (99.8% allele identity). We also applied ExHap to all 22 autosomes for both CEU and YRI cohorts from the International HapMap Project, identifying multiple candidate extended haplotypes.ConclusionsLong-range congruence is not unique to the MHC region. Patterns of allele identity on phased chromosomes provide a simple, straightforward approach to visually and quantitatively inspect complex long-range structural patterns in the genome. Such patterns aid the biologist in appreciating genetic similarities and differences across cohorts, and can lead to hypothesis generation for subsequent studies.

P2.09-17 A Call to Action: Rapid Collection of Post-Mortem Lung Cancer Tissue in the Community to Enable Lung Cancer Research

panel is a 31-gene NGS multiplex assay that synchronously detects multiple oncogene fusion transcripts from formalin-fixed paraffinembedded (FFPE) tissue derived RNA (Figure 1). Method: QIAseq analysis was undertaken in 33 samples (31 NSCLC samples, 2 commercial controls). 12/31 NSCLC samples were positive controls with a known fusion genotype identified by Quantide X NGS or FISH +/RTPCR. The remaining 19/31 fusion negative NSCLC controls included 6 samples with EGFR/KRAS/NRAS mutations. Analysis required a minimum 2x 5u Î M thick FFPE scrolls with >30% neoplastic cell content. Manual RNA extraction was undertaken in all samples except n1⁄46 (ExScale automation extraction). NGS fusion breakpoints, crossing and spanning reads were calculated in QIAseq fusion-detected samples. An additional validation cohort of 40 NSCLC samples, including 20 with unknown fusion status will optimise QIAseq thresholds for fusion detection. Result: 48 QIAseq sequencing experiments was undertaken in 33 samples with 2 sequencing runs in 12 samples. QIAseq analysis detected a corresponding NGS fusion breakpoint in 14/14 (100%) positive controls including EML4-ALK (n1⁄48), CLTC-ALK (n1⁄41), CD74ROS1 (n1⁄43), CCDC6-RET (n1⁄41) and 5-fusion control (n1⁄41). QIAseq analysis was negative in 17/19 (89.4%) negative controls samples including all KRAS (n1⁄44), NRAS (n1⁄41) and EGFR (n1⁄41) mutation samples. QIAseq detected novel fusion gene CD74 Exon 6-CAMK2A Exon 2 in n1⁄41 sample subsequently confirmed on Sanger sequencing. Two separate runs detected TPM3 Exon 8-S100A7A Exon 2 fusion in n1⁄41 sample not identified with Sanger sequencing. Both fusions have uncertain clinical significance. Validation cohort results will be presented. Conclusion: QIAseq detects NSCLC oncogenic fusions with high sensitivity and specificity. Future applications include optimising use of small biopsy specimens for synchronous gene rearrangement screening and identification of novel gene fusion targets.

Phase I/II study of nivolumab and ipilimumab combined with nintedanib in advanced NSCLC.

TPS9112Background: Combination immunotherapy with nivolumab and ipilimumab has proven clinical activity in NSCLC. There is growing evidence to suggest that the tumor microenvironment (TME) may inte...