Keith Killian

Molecular Profiling and Targeted Therapy for Advanced Thoracic Malignancies: A Biomarker-Derived, Multiarm, Multihistology Phase II Basket Trial

PURPOSE We conducted a basket clinical trial to assess the feasibility of such a design strategy and to independently evaluate the effects of multiple targeted agents against specific molecular aberrations in multiple histologic subtypes concurrently. PATIENTS AND METHODS We enrolled patients with advanced non-small-cell lung cancer (NSCLC), small-cell lung cancer, and thymic malignancies who underwent genomic characterization of oncogenic drivers. Patients were enrolled onto a not-otherwise-specified arm and treated with standard-of-care therapies or one of the following five biomarker-matched treatment groups: erlotinib for EGFR mutations; selumetinib for KRAS, NRAS, HRAS, or BRAF mutations; MK2206 for PIK3CA, AKT, or PTEN mutations; lapatinib for ERBB2 mutations or amplifications; and sunitinib for KIT or PDGFRA mutations or amplification. RESULTS Six hundred forty-seven patients were enrolled, and 88% had their tumors tested for at least one gene. EGFR mutation frequency was 22.1% in NSCLC, and erlotinib achieved a response rate of 60% (95% CI, 32.3% to 83.7%). KRAS mutation frequency was 24.9% in NSCLC, and selumetinib failed to achieve its primary end point, with a response rate of 11% (95% CI, 0% to 48%). Completion of accrual to all other arms was not feasible. In NSCLC, patients with EGFR mutations had the longest median survival (3.51 years; 95% CI, 2.89 to 5.5 years), followed by those with ALK rearrangements (2.94 years; 95% CI, 1.66 to 4.61 years), those with KRAS mutations (2.3 years; 95% CI, 2.3 to 2.17 years), those with other genetic abnormalities (2.17 years; 95% CI, 1.3 to 2.74 years), and those without an actionable mutation (1.85 years; 95% CI, 1.61 to 2.13 years). CONCLUSION This basket trial design was not feasible for many of the arms with rare mutations, but it allowed the study of the genetics of less common malignancies.

A specific missense mutation in GTF2I occurs at high frequency in thymic epithelial tumors

We analyzed 28 thymic epithelial tumors (TETs) using next-generation sequencing and identified a missense mutation (chromosome 7 c.74146970T>A) in GTF2I at high frequency in type A thymomas, a relatively indolent subtype. In a series of 274 TETs, we detected the GTF2I mutation in 82% of type A and 74% of type AB thymomas but rarely in the aggressive subtypes, where recurrent mutations of known cancer genes have been identified. Therefore, GTF2I mutation correlated with better survival. GTF2I β and δ isoforms were expressed in TETs, and both mutant isoforms were able to stimulate cell proliferation in vitro. Thymic carcinomas carried a higher number of mutations than thymomas (average of 43.5 and 18.4, respectively). Notably, we identified recurrent mutations of known cancer genes, including TP53, CYLD, CDKN2A, BAP1 and PBRM1, in thymic carcinomas. These findings will complement the diagnostic assessment of these tumors and also facilitate development of a molecular classification and assessment of prognosis and treatment strategies.

Copy number aberrations of BCL2 and CDKN2A/B identified by array-CGH in thymic epithelial tumors

The molecular pathology of thymic epithelial tumors (TETs) is largely unknown. Using array comparative genomic hybridization (CGH), we evaluated 59 TETs and identified recurrent patterns of copy number (CN) aberrations in different histotypes. GISTIC algorithm revealed the presence of 126 significant peaks of CN aberration, which included 13 cancer-related genes. Among these peaks, CN gain of BCL2 and CN loss of CDKN2A/B were the only genes in the respective regions of CN aberration and were associated with poor outcome. TET cell lines were sensitive to siRNA knockdown of the anti-apoptotic molecules BCL2 and MCL1. Gx15-070, a pan-BCL2 inhibitor, induced autophagy-dependent necroptosis in TET cells via a mechanism involving mTOR pathways, and inhibited TET xenograft growth. ABT263, an inhibitor of BCL2/BCL-XL/BCL-W, reduced proliferation in TET cells when administered in combination with sorafenib, a tyrosine kinase inhibitor able to downregulate MCL1. Immunohistochemistry on 132 TETs demonstrated that CN loss of CDKN2A correlated with lack of expression of its related protein p16INK4 and identified tumors with poor prognosis. The molecular markers BCL2 and CDKN2A may be of potential value in diagnosis and prognosis of TETs. Our study provides the first preclinical evidence that deregulated anti-apoptotic BCL2 family proteins may represent suitable targets for TET treatment.

Genomic aberrations in pediatric diffuse intrinsic pontine gliomas

Diagnostic biopsy is not routinely performed for children with diffuse intrinsic pontine glioma (DIPG). Consequently, our understanding of DIPG biology is hindered by limited tissue availability. We performed comparative genomic hybridization (CGH) on autopsy specimens to examine the feasibility of determining DNA genomic copy number aberrations on formalin-fixed, paraffin-embedded (FFPE) blocks. Histology on FFPE blocks obtained from autopsy of pediatric patients with DIPG was reviewed. Regions were marked for processing, and DNA was extracted from the tissue core, labeled by chemical coupling with Cy5, and hybridized to 105K oligonucleotide CGH arrays. After hybridization and washing, arrays were scanned, and data segmented and processed with Nexus software. Twenty-two samples from 13 subjects were obtained. Histologic variability was noted. CGH was successfully performed on 18 of 22 samples, representing 11 of 13 subjects. All demonstrated DNA copy number abnormalities. High copy number amplification of known oncogenes and homozygous deletions of known tumor suppressor genes were observed. Additional regions of high copy number amplification and homozygous deletion and geographical variations in the CGH patterns were found. CGH performed on FFPE tissue obtained from autopsy yields satisfactory results. This sample set from patients with DIPG was highly informative, with the majority of specimens showing ≥1 abnormality related to a known cancer gene. Aberrations in candidate drug targets were observed. This study establishes the feasibility of genomic DNA analysis from DIPG autopsy material, identifies several targets for which molecular targeted therapy exists, and suggests significant heterogeneity among patients with DIPG.

Primary neuroendocrine tumors of the kidney: Morphological and molecular alterations of an uncommon malignancy

Primary neuroendocrine (NE) tumors of the kidney (PNRTs) are rare and frequently mistaken for other renal and urothelial cancers. We evaluated morphological and molecular findings of 11 PNRTs classified according to the World Health Organization classification of lung NE tumors. Patients included 5 men and 6 women with a median age of 50 years. These tumors occurred in the left (5/11), right (3/11), and horseshoe (1/11) kidney. The histologic patterns were predominantly solid, trabecular, and pseudoglandular. Lymphovascular invasion and calcification were found in 3 and 1 cases, respectively. There were 2 atypical and 9 typical carcinoids. At the time of surgery, 2 patients with atypical carcinoids had hepatic metastasis, and 1 of the typical carcinoid patients had lymph node metastasis. All cases showed <1% proliferative rate, except 2 cases with hepatic metastasis, which showed 3% to 5% with MIB1/Ki-67 immunostaining. Immunostainings were frequently positive for synaptophysin, chromogranin, CD56, CD99, and neuron-specific enolase. Follow-up data (average 4 years) were available for 6 patients. Two patients with distant metastasis were alive with disease, and four patients with no metastasis were alive without disease. We evaluated the association of PNRT and loss of heterozygosity (LOH) on chromosome 3p21 and found LOH in 2 of 3 cases. However, the comparative genomic hybridization study (2/2) did not demonstrate significant chromosomal imbalances. We conclude that PNRTs are positive for NE markers and may have LOH on chromosome 3p21. PNRTs should be classified as NE tumors in other sites, and proliferative rate can be an indicator of aggressive behavior/metastasis.

Leukocyte DNA methylation and colorectal cancer among male smokers.

AIM To explore the association between methylation in leukocyte DNA and colorectal cancer (CRC) risk in male smokers using the α-tocopherol, β-carotene cancer prevention study. METHODS About 221 incident CRC cases, and 219 controls, frequency-matched on age and smoking intensity were included. DNA methylation of 1505 CpG sites selected from 807 genes were evaluated using Illumina GoldenGate Methylation Cancer Panel I in pre-diagnostic blood leukocytes of study subjects. Tertiles of methylation level classified according to the distribution in controls for each CpG site were used to analyze the association between methylation level and CRC risk with logistic regression. The time between blood draw to cancer diagnosis (classifying cases according to latency) was incorporated in further analyses using proportional odds regression. RESULTS We found that methylation changes of 31 CpG sites were associated with CRC risk at P < 0.01 level. Though none of these 31 sites remained statistically significant after Bonferroni correction, the most statistically significant CpG site associated with CRC risk achieved a P value of 1.0 × 10(-4). The CpG site is located in DSP gene, and the risk estimate was 1.52 (95% CI: 0.91-2.53) and 2.62 (95% CI: 1.65-4.17) for the second and third tertile comparing with the lowest tertile respectively. Taking the latency information into account strengthened some associations, suggesting that the methylation levels of corresponding sites might change over time with tumor progression. CONCLUSION The results suggest that the methylation level of some genes were associated with cancer susceptibility and some were related to tumor development over time. Further studies are warranted to confirm and refine our results.

Somatic VHL Mutation in a Patient With MEN1-Associated Metastatic Pancreatic Neuroendocrine Tumor Responding to Sunitinib Treatment: A Case Report

Multiple endocrine neoplasia type 1 (MEN1) and von Hippel-Lindau (VHL) are autosomal-dominant diseases caused by germline mutations in tumor-suppressor genes. A patient with a germline MEN1 mutation and a somatic VHL mutation in the tumor has not been reported. Herein, we report on a patient with MEN1 and a metastatic nonfunctioning pancreatic neuroendocrine tumor (PNET) with a somatic VHL mutation. This patient underwent a pancreaticoduodenectomy for a grade 2 PNET obstructing her pancreatic duct. The patient developed liver and regional lymph node metastases as well as growth of a PNET in the remnant pancreas. As part of a clinical trial for mutation-targeted therapy, a biopsy of the metastatic tumor was obtained. The clinical diagnosis, confirmed by OncoVAR-NET and molecular profiling analysis, revealed MEN1 with a germline deletion in exon 2 and a c.402 deletion C, p.Phe134LeufsX51. In addition, a somatic mutation in the VHL gene—a nonsense mutation, c.529A>T, p.Arg177Ter—was identified by hybrid capture sequencing. The mutations were confirmed by Sanger sequencing. Comparative genomic hybridization showed loss of heterozygosity in both the MEN1 and VHL genes. The patient was treated with sunitinib and had a partial response to treatment. This case illustrates not only that a second hit occurs in tumor suppressor genes but that somatic mutations are also possible in additional tumor suppressor genes. This suggests that targeted therapy selection should include analysis of somatic mutations even when the susceptibility gene is known.

Abstract 5012: DNA methylation profiles of endometrial cancer and benign endometrium suggest differences by mismatch repair status and possible early detection markers

Background: Increased risk for endometrial cancer, the most common gynecological malignancy in the United States, is related to obesity, nulliparity, and postmenopausal hormone use. Several molecular pathways and mechanisms are prominently involved in endometrial carcinogenesis including mismatch repair deficiency (MMR), abnormalities in the PTEN pathway and DNA methylation. We compared DNA methylation profiles in endometrial cancers to identify markers related to MMR deficiencies and contrasted results to normal endometrial tissue to identify potential biomarkers for early detection. Methods: We conducted methylation profiling on 148 paraffin embedded endometrial cancer tissues collected in the population-based Polish Endometrial Cancer Study and 23 normal tissues included within a study of benign endometrium at Magee Women9s Hospital. 1.0 mm tissue cores removed from target-rich areas of tissue blocks were used for DNA extraction and bisulfite treatment. The Illumina Golden Gate platform was used to assess CpG methylation at 1505 sites. Microsatellite instability (MSI) testing using three markers (BAT25, BAT26, and CAT25) was performed to identify MMR deficient cases. We conducted unsupervised hierarchical clustering and class comparison analyses to evaluate DNA methylation by MMR status among cancers and class comparison and pathway analyses to identify differences in methylation markers between normal and tumor tissue. Results: Unsupervised hierarchical clustering of methylation profiles in cancers showed two major clusters with significantly different proportions of MSI cases (61.4% versus 10.6%, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5012. doi:1538-7445.AM2012-5012

Abstract 4475: Association of endometrial cancer risk factors and DNA methylation in benign endometrial tissue

Background: Increased risk for endometrial cancer is related to factors such as use of unopposed estrogen and obesity, and decreased risk is associated with oral contraceptive use and smoking. However, little is known about the molecular mechanisms by which these factors influence cancer risk. DNA methylation of promoter regions in tumor suppressor genes has been linked to endometrial cancer, suggesting DNA methylation may mediate the effects of risk factors on normal endometrium, leading to increased risk for cancer. Accordingly, we explored relationships between risk factors for endometrial cancer and DNA methylation patterns assessed in normal endometrial tissue. Materials and Methods: Formalin fixed paraffin embedded endometrial tissues from 63 women aged 28-53 (median= 43) who underwent a hysterectomy for benign indications were studied. DNA isolated from 1.0-mm cores was bisulfite treated and methylation profiles were generated using Illumina9s GoldenGate array which includes 1505 CpG sites representing over 800 genes. Unsupervised hierarchical clustering analysis was used to identify groups of patients with similar methylation patterns and class comparison analysis was employed to identify differentially methylated genes based on predefined categories, representing endometrial cancer risk factors. Results: Global changes in DNA methylation, as measured by total or average methylation levels or clustering analysis were not associated with age. However, methylation levels of MT1A (p=0.0002) and CDH13 (p=0.0009) were increased and methylation levels of PKD2 (p=0.0009) were decreased among older women. Increased methylation of HS3ST2 (p=0.00006), MLF1 (p=0.0001), PLAUR (p=0.0004), MLH3 (p=0.0007), ISL1 (p=0.0007), and RASSF1 (p=0.0009) was related to women with a lifetime menstrual span of more than 30 years compared to those who reported having menstruated for less than 15 years. One gene (MMP3) was more methylated in women who had an older age at menarche (14+ years old) compared to those who had a younger age at menarche ( Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4475. doi:1538-7445.AM2012-4475

Abstract LB-314: Array comparative genomic hybridization of thymic epithelial tumors identifies loss of CDKN2A as a prognostic factor and BCL2 family members as targets for therapy

Background : Thymic epithelial tumors (TETs) are rare and the genomic aberrations relevant to TET biology are unknown. The goal of this study was to identify recurrent genomic aberrations in TETs. Materials and Methods : From a series of 132 TET resected patients we selected 59 formalin fixed paraffin embedded (FFPE) samples with more than 80% of cancer cells in order to perform high-resolution array-CGH. We used ULS labeling kit (Agilent) and 105A array (Agilent) for 20 and 180K array (Agilent) for 39 samples. For the immunohistochemistry we used a tissue micro-array built from FFPE samples of the 132 patients. The viability assay MTS (Promega) was used to evaluate cytotoxicity of Gx15–070 and ABT-263 (Selleck Chemicals) in T1889, TY82 and T1682 TET cell lines. Results : Chromosome arm level copy number (CN) aberrations were considered those chromosome arms with more than 80% of their length affected by either CN gain or loss. Rare non-recurrent chromosome arm-level CN aberrations were observed in type A thymomas, whereas types B3 and thymic carcinomas shared frequent gains of chromosomes 1q, 5 and 7, as well as losses of chromosomes 6 and 13q. In addition, thymic carcinomas presented frequent losses of chromosomes 16p and 17q. We applied the GISTIC algorithm to the whole set of CN aberrations in order to distinguish aberrations driving the cancer phenotype from the background level of random CN aberrations. 72 CN gain and 54 CN loss peaks were identified (q 2 ratio > 0.3) containing BCL2 locus and one high-level CN loss (log 2 ratio INK4 and p14 ARF . Patients with CDKN2A-negative immunohistochemistry results (85/119) had a poorer disease related survival (LogRank p=0.041). TET cell lines did not have CDKN2A CN loss or BCL2 CN gain, none of them expressed CDKN2A proteins and T1682 and TY82 expressed BCL2. All TET cell lines expressed multiple BCL2 anti-apoptotic family members including MCL1, and were resistant to ABT-263, a BCL2, BCL-XL and BCL-W inhibitor, but sensitive to Gx15–070, a pan BCL2 anti-apoptotic family member (IC50: range 35.8–99.6 nM). Conclusion : Our data indicate that TET histotypes differ by distribution of chromosome arm level CN aberrations. Loss of CDKN2A is prognostic in TETs, whereas Bcl-2 family members may be valuable targets for treatment of this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-314. doi:10.1158/1538-7445.AM2011-LB-314