Ayse Selen Yilmaz

Resistance Mechanisms for the Bruton's Tyrosine Kinase Inhibitor Ibrutinib

BACKGROUND Ibrutinib is an irreversible inhibitor of Brutons tyrosine kinase (BTK) and is effective in chronic lymphocytic leukemia (CLL). Resistance to irreversible kinase inhibitors and resistance associated with BTK inhibition have not been characterized. Although only a small proportion of patients have had a relapse during ibrutinib therapy, an understanding of resistance mechanisms is important. We evaluated patients with relapsed disease to identify mutations that may mediate ibrutinib resistance. METHODS We performed whole-exome sequencing at baseline and the time of relapse on samples from six patients with acquired resistance to ibrutinib therapy. We then performed functional analysis of identified mutations. In addition, we performed Ion Torrent sequencing for identified resistance mutations on samples from nine patients with prolonged lymphocytosis. RESULTS We identified a cysteine-to-serine mutation in BTK at the binding site of ibrutinib in five patients and identified three distinct mutations in PLCγ2 in two patients. Functional analysis showed that the C481S mutation of BTK results in a protein that is only reversibly inhibited by ibrutinib. The R665W and L845F mutations in PLCγ2 are both potentially gain-of-function mutations that lead to autonomous B-cell-receptor activity. These mutations were not found in any of the patients with prolonged lymphocytosis who were taking ibrutinib. CONCLUSIONS Resistance to the irreversible BTK inhibitor ibrutinib often involves mutation of a cysteine residue where ibrutinib binding occurs. This finding, combined with two additional mutations in PLCγ2 that are immediately downstream of BTK, underscores the importance of the B-cell-receptor pathway in the mechanism of action of ibrutinib in CLL. (Funded by the National Cancer Institute and others.).

Genomic Characterization of Non–Small-Cell Lung Cancer in African Americans by Targeted Massively Parallel Sequencing

PURPOSE Technologic advances have enabled the comprehensive analysis of genetic perturbations in non-small-cell lung cancer (NSCLC); however, African Americans have often been underrepresented in these studies. This ethnic group has higher lung cancer incidence and mortality rates, and some studies have suggested a lower incidence of epidermal growth factor receptor mutations. Herein, we report the most in-depth molecular profile of NSCLC in African Americans to date. METHODS A custom panel was designed to cover the coding regions of 81 NSCLC-related genes and 40 ancestry-informative markers. Clinical samples were sequenced on a massively parallel sequencing instrument, and anaplastic lymphoma kinase translocation was evaluated by fluorescent in situ hybridization. RESULTS The study cohort included 99 patients (61% males, 94% smokers) comprising 31 squamous and 68 nonsquamous cell carcinomas. We detected 227 nonsilent variants in the coding sequence, including 24 samples with nonoverlapping, classic driver alterations. The frequency of driver mutations was not significantly different from that of whites, and no association was found between genetic ancestry and the presence of somatic mutations. Copy number alteration analysis disclosed distinguishable amplifications in the 3q chromosome arm in squamous cell carcinomas and pointed toward a handful of targetable alterations. We also found frequent SMARCA4 mutations and protein loss, mostly in driver-negative tumors. CONCLUSION Our data suggest that African American ancestry may not be significantly different from European/white background for the presence of somatic driver mutations in NSCLC. Furthermore, we demonstrated that using a comprehensive genotyping approach could identify numerous targetable alterations, with potential impact on therapeutic decisions.

Somatic Mutation Spectrum of Non–Small-Cell Lung Cancer in African Americans: A Pooled Analysis

Introduction: The mutational profile of non–small-cell lung cancer (NSCLC) has become an important tool in tailoring therapy to patients, with clear differences according to the population of origin. African Americans (AAs) have higher lung cancer incidence and mortality than Caucasians, yet discrepant results have been reported regarding the frequency of somatic driver mutations. We hypothesized that NSCLC has a distinct mutational profile in this group. Methods: We collected NSCLC samples resected from self-reported AAs in five sites from Tennessee, Michigan, and Ohio. Gene mutations were assessed by either SNaPshot or next generation sequencing, and ALK translocations were evaluated by fluorescence in situ hybridization. Results: Two hundred sixty patients were included, mostly males (62.3%) and smokers (86.6%). Eighty-one samples (31.2%) were squamous cell carcinomas. The most frequently mutated genes were KRAS (15.4%), epidermal growth factor receptor (EGFR, 5.0%), PIK3CA (0.8%), BRAF, NRAS, ERBB2, and AKT1 (0.4% each). ALK translocations were detected in two nonsquamous tumors (1.7%), totaling 61 cases (23.5%) with driver oncogenic alterations. Among 179 nonsquamous samples, 54 (30.2%) presented a driver alteration. The frequency of driver alterations altogether was lower than that reported in Caucasians, whereas no difference was detected in either EGFR or KRAS mutations. Overall survival was longer among patients with EGFR mutations. Conclusions: We demonstrated that NSCLC from AAs has a different pattern of somatic driver mutations than from Caucasians. The majority of driver alterations in this group are yet to be described, which will require more comprehensive panels and assessment of noncanonical alterations.

Differential mutation frequencies in metastatic cutaneous squamous cell carcinomas versus primary tumors

Exome and targeted sequencing studies have identified potential driver mutations for a variety of tumor types. Cutaneous squamous cell carcinoma (cSCC) is one of the most highly mutated cancers but typically is associated with low rates of metastasis and high survival rates. Nevertheless, metastatic cSCC is a significant health threat; up to 8800 individuals die each year of this disease.

Impact of Pre-Analytical Variables on Cancer Targeted Gene Sequencing Efficiency.

Tumor specimens are often preserved as formalin-fixed paraffin-embedded (FFPE) tissue blocks, the most common clinical source for DNA sequencing. Herein, we evaluated the effect of pre-sequencing parameters to guide proper sample selection for targeted gene sequencing. Data from 113 FFPE lung tumor specimens were collected, and targeted gene sequencing was performed. Libraries were constructed using custom probes and were paired-end sequenced on a next generation sequencing platform. A PCR-based quality control (QC) assay was utilized to determine DNA quality, and a ratio was generated in comparison to control DNA. We observed that FFPE storage time, PCR/QC ratio, and DNA input in the library preparation were significantly correlated to most parameters of sequencing efficiency including depth of coverage, alignment rate, insert size, and read quality. A combined score using the three parameters was generated and proved highly accurate to predict sequencing metrics. We also showed wide read count variability within the genome, with worse coverage in regions of low GC content like in KRAS. Sample quality and GC content had independent effects on sequencing depth, and the worst results were observed in regions of low GC content in samples with poor quality. Our data confirm that FFPE samples are a reliable source for targeted gene sequencing in cancer, provided adequate sample quality controls are exercised. Tissue quality should be routinely assessed for pre-analytical factors, and sequencing depth may be limited in genomic regions of low GC content if suboptimal samples are utilized.

MiR-34a Regulates the Invasive Capacity of Canine Osteosarcoma Cell Lines

Background Osteosarcoma (OSA) is the most common bone tumor in children and dogs; however, no substantial improvement in clinical outcome has occurred in either species over the past 30 years. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and play a fundamental role in cancer. The purpose of this study was to investigate the potential contribution of miR-34a loss to the biology of canine OSA, a well-established spontaneous model of the human disease. Methodology and principal findings RT-qPCR demonstrated that miR-34a expression levels were significantly reduced in primary canine OSA tumors and canine OSA cell lines as compared to normal canine osteoblasts. In canine OSA cell lines stably transduced with empty vector or pre-miR-34a lentiviral constructs, overexpression of miR-34a inhibited cellular invasion and migration but had no effect on cell proliferation or cell cycle distribution. Transcriptional profiling of canine OSA8 cells possessing enforced miR-34a expression demonstrated dysregulation of numerous genes, including significant down-regulation of multiple putative targets of miR-34a. Moreover, gene ontology analysis of down-regulated miR-34a target genes showed enrichment of several biological processes related to cell invasion and motility. Lastly, we validated changes in miR-34a putative target gene expression, including decreased expression of KLF4, SEM3A, and VEGFA transcripts in canine OSA cells overexpressing miR-34a and identified KLF4 and VEGFA as direct target genes of miR-34a. Concordant with these data, primary canine OSA tumor tissues demonstrated increased expression levels of putative miR-34a target genes. Conclusions These data demonstrate that miR-34a contributes to invasion and migration in canine OSA cells and suggest that loss of miR-34a may promote a pattern of gene expression contributing to the metastatic phenotype in canine OSA.

Genetic variants in thyroid cancer distant metastases

Thyroid carcinoma is the most rapidly increasing solid tumor in the United States; although there has been a great emphasis on the analysis of primary tumors, predictors of both late-stage progression and response to therapy are more poorly defined due in part to the scarcity of progressive metastatic tissues. Therapeutic evidence of mixed responses in metastatic lesions and the limited available genomic data suggest that distant metastases in thyroid cancer are heterogeneous, driven by known oncogenes and other pathways that also might be therapeutic targets. We analyzed the genomes of a small number of rare surgically resected metastatic thyroid cancer lesions along with paired normal and primary tumor samples when available, in an effort to better characterize progressive distant metastases. The findings confirm the presence of mutations to known tumor drivers (BRAF and RAS) in metastatic samples. The results also identified the co-occurrence in predicted functional variants to the DNA damage repair (DDR) genes ATM and ERCC4 in metastatic lesions that did not show alterations of the MAPK pathway. We examined the exomes of 19 samples (including 5 paired normal tissues) from 11 follicular cellderived thyroid cancer patients with surgically resected distant metastases by custom exomeSeq (Supplementary Materials and Methods, see section on supplementary data given at the end of this article). Tumors with different histopathologies were included and were confirmed by an expert thyroid pathologist (PW). All patients were treated with TSH suppression, eight of them received I-131 therapy and none of them received chemotherapy or kinase inhibitors. Overall, 19,299 unique variants in the 682 genes and genomic regions were identified by an exomeSeq custom panel. We sought to identify rare, conserved, exonic variants that were likely to have functional effects. Thus, we focused on 1742 exonic variants in exons of sequenced genes. We excluded synonymous variants and variants with higher than 0.01 minor allelic frequency in the 1000 Genomes Project. We then filtered to include variants located in regions that were conserved through vertebrate evolution. These criteria produced a list of 349 variants in 199 genes. Unsupervised clustering using complete linkage and Euclidian distance identified clusters based on patients, suggesting that most variants were primarily associated with individuals rather than histology or tissue location (data not shown). We finally applied a filter to identify variants predicted or known to have a damaging (SIFT) or deleterious (PolyPhen) effect on the protein. Figure 1 summarizes the filtering strategy. In the primary tumors of patients with distant metastases, 33 variants in 31 genes were identified by comparing with normal samples. We focused on genes affected in more than one tumor sample and found three such genes: BCR, BRAF and MAP4 (Fig. 2). Two variants that affect BRAF in five primary tumor samples were identified. A Hürthle cell carcinoma (HCC) bore a BRAF T241M variant, although its functional significance is uncertain. The remaining samples with BRAF had mutations resulting in BRAF V600E and included two papillary thyroid cancers (PTC) primary tumors and a metastatic lesion, and one anaplastic thyroid cancer (ATC) metastatic lesion. We also identified four samples from four patients with an identical insertion resulting in a frameshift (chr22:23653975 Indel: TCCGG) in BCR including three primary tumors and one metastasis. The primary tumor samples were from follicular (FTC) and HCC, and a metastatic lesion from BRAF V600E PTC. Finally, three primary tumors had an identical mutation (R1112P) in MAP4. Two of the tumors are from patients with HCC, and the third is a PTC with a concurrent BRAFV600E mutation. BCR and MAP4 are functionally involved in cytoskeletal dynamics, although those roles and functional implications of the variants identified are not completely characterized. In the metastatic tissues, we identified thirty variants in twenty-eight genes after filtering unique vs normal tissues. Variants in three genes, ATM, BRAF and ERCC4,

Identification of medium-sized copy number alterations in whole-genome sequencing.

The genome-wide discoveries such as detection of copy number alterations (CNA) from high-throughput whole-genome sequencing data enabled new developments in personalized medicine. The CNAs have been reported to be associated with various diseases and cancers including acute myeloid leukemia. However, there are multiple challenges to the use of current CNA detection tools that lead to high false-positive rates and thus impede widespread use of such tools in cancer research. In this paper, we discuss these issues and propose possible solutions. First, since the entire genome cannot be mapped due to some regions lacking sequence uniqueness, current methods cannot be appropriately adjusted to handle these regions in the analyses. Thus, detection of medium-sized CNAs is also being directly affected by these mappability problems. The requirement for matching control samples is also an important limitation because acquiring matching controls might not be possible or might not be cost efficient. Here we present an approach that addresses these issues and detects medium-sized CNAs in cancer genomes by (1) masking unmappable regions during the initial CNA detection phase, (2) using pool of a few normal samples as control, and (3) employing median filtering to adjust CNA ratios to its surrounding coverage and eliminate false positives.

Transcriptional targeting of oncogene addiction in medullary thyroid cancer

Metastatic medullary thyroid cancer (MTC) is incurable and FDA-approved kinase inhibitors that include oncogenic RET as a target do not result in complete responses. Association studies of human MTCs and murine models suggest that the CDK/RB pathway may be an alternative target. The objective of this study was to determine if CDKs represent therapeutic targets for MTC and to define mechanisms of activity. Using human MTC cells that are either sensitive or resistant to vandetanib, we demonstrate that palbociclib (CDK4/6 inhibitor) is not cytotoxic to MTC cells but that they are highly sensitive to dinaciclib (CDK1/2/5/9 inhibitor) accompanied by reduced CDK9 and RET protein and mRNA levels. CDK9 protein was highly expressed in 83 of 83 human MTCs and array-comparative genomic hybridization had copy number gain in 11 of 30 tumors. RNA sequencing demonstrated that RNA polymerase II-dependent transcription was markedly reduced by dinaciclib. The CDK7 inhibitor THZ1 also demonstrated high potency and reduced RET and CDK9 levels. ChIP-sequencing using H3K27Ac antibody identified a superenhancer in intron 1 of RET. Finally, combined inhibition of dinaciclib with a RET kinase inhibitor was synergistic. In summary, we have identified what we believe is a novel mechanism of RET transcription regulation that potentially can be exploited to improve RET therapeutic targeting.

Discovery and characterization of the feline miRNAome

The domestic cat is an important human companion animal that can also serve as a relevant model for ~250 genetic diseases, many metabolic and degenerative conditions, and forms of cancer that are analogous to human disorders. MicroRNAs (miRNAs) play a crucial role in many biological processes and their dysregulation has a significant impact on important cellular pathways and is linked to a variety of diseases. While many species already have a well-defined and characterized miRNAome, miRNAs have not been carefully studied in cats. As a result, there are no feline miRNAs present in the reference miRNA databases, diminishing the usefulness of medical research on spontaneous disease in cats for applicability to both feline and human disease. This study was undertaken to define and characterize the cat miRNAome in normal feline tissues. High-throughput sequencing was performed on 12 different normal cat tissues. 271 candidate feline miRNA precursors, encoding a total of 475 mature sequences, were identified, including several novel cat-specific miRNAs. Several analyses were performed to characterize the discovered miRNAs, including tissue distribution of the precursors and mature sequences, genomic distribution of miRNA genes and identification of clusters, and isomiR characterization. Many of the miRNAs were regulated in a tissue/organ-specific manner.