Vasmatzis, George

Abstract Introduction: Pulmonary carcinoid tumors account for up to 5% of all lung malignancies in adults, and comprise 30% of all carcinoid malignancies. They are defined histologically as typical carcinoid (TC) and atypical carcinoid (AC) tumors, and are characterized by neuroendocrine differentiation and the potential to metastasize. Method: We characterized genomic alterations in pulmonary carcinoid tumors using whole genome and exome sequencing in addition to mRNA expression and SNP genotyping from specimens of normal lung, typical and atypical carcinoid, and SCLC. Results: Analysis of the sequencing data identified novel mutations in the pulmonary neuroendocrine tumor spectrum, including KRAS, ABCA9, GALNT10, HSFX1, PTHLH, RANBP2, BRIP1, FXR1, BACE2, NCOR2, DGKI, HERC1, HERC5 and GLI3. Pathway analysis found 45% of the cases to harbor a mutation in genes that are involved in protein-protein interaction with ubiquitin c (UBC) and may therefore play a role in ubiquitin protein degradation pathway. We also identified genes differentially expressed in corresponding regions of copy number variation, 52 of which were linked to ubiquitination pathways in 88% of the samples genotyped. Genes amplified in 15 to 23% of the cases included ITM2A, MAOA, EGR2, HAGH, TSC22D3, NBEA, TCEAL2, and TSPYL2 whereas deleted regions in 15% of the cases implicated CFD, GDI1, IFITM1 and IFITM2. Conclusion: These results suggest that in addition to previously identified pathways, dysregulation of the ubiquitin pathway may contribute to lung carcinoid tumorigenesis and could be exploited for therapeutic purposes. Citation Format: Michael K. Asiedu, Charles L. Thomas, Jie Dong, Prasidda Khadka, Zhifu Sun, Farhad Kosari, Jin Jen, Julian Molina, George Vasmatzis, Marie Christine Aubry, Ping Yang, Dennis Wigle. Genomic alterations modify ubiquitination pathways in pulmonary carcinoid tumors. [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 3895. doi:10.1158/1538-7445.AM2015-3895

We used a target-centric strategy to identify transporter proteins upregulated in pancreatic ductal adenocarcinoma (PDAC) as potential targets for a functional imaging probe to complement existing anatomical imaging approaches. We performed transcriptomic profiling (microarray and RNASeq) on histologically confirmed primary PDAC tumors and normal pancreas tissue from 33 patients, including five patients whose tumors were not visible on computed tomography. Target expression was confirmed with immunohistochemistry on tissue microarrays from 94 PDAC patients. The best imaging target identified was SLC6A14 (a neutral and basic amino acid transporter). SLC6A14 was overexpressed at the transcriptional level in all patients and expressed at the protein level in 95% of PDAC tumors. Very little is known about the role of SLC6A14 in PDAC and our results demonstrate that this target merits further investigation as a candidate transporter for functional imaging of PDAC.

Abstract Introduction: Identification of causal DNA copy number variations (CNVs) is critical to the understanding of cancer development and progression, and is important clinically for accurate prognosis and efficient treatment. T-cell lymphomas are replete with amplifications and deletions that result in over- and under-expression of genes. Array comparative genomic hybridization (aCGH) has been the technology of choice to detect CNVs but recently, it has been proposed that mate-pair Next Generation Sequencing (mpNGS) can produce similar results. Here, we report a comparison between the two techniques. Methods: We have developed an algorithm based on extreme value theory to identify CNVs across the genome at high resolution from mate-pair NGS data. The algorithm is based on plotting the distribution of read-counts in windows across the genome, and detecting deletions and gains by finding the windows that differ significantly from the distribution of the normal part of the genome. We compared this algorithm with aCGH data to detect CNVs. We ran five cases of T-cell lymphoma with both techniques and compared the results using concordance statistics. Results: With 50kb lower cutoff for a CNV, mpNGS detected 98% of the cumulative deleted genome and 94% of the amplified/gained genome that were called by aCGH. Most of the discordant CNVs that were detected by only aCGH were in regions of repetitive areas where mapping fails. Conversely, aCGH detected 75% of the cumulative deleted genome and 74% of the amplified/gained genome that were called by mpNGS. Conclusion: A detailed comparison of these techniques showed that both have pros and cons. In general, mpNGS can detect most CNVs detected by aCGH, as long as they are more than 50KB. Additionally, mpNGS offers confirmatory data, particularly for deletions, based on detection of aberrant mate pairs that map to the rejoined ends of deleted regional. Finally, the mate-pair technology can also detect balanced translocations and can also predict fusion genes. Citation Format: George Vasmatzis, Andrew L. Feldman, Sarah H. Johnson, Erik C. Thorland, Rafael Fonseca, Esteban Braggio, Troy J. Gliem. Copy number detection using genomics technologies: A comparison between aCGH and NGS. [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 3572. doi:10.1158/1538-7445.AM2014-3572

Abstract Background: Fusion genes, transcripts, and proteins are key drivers of hematopoietic oncogenesis and can serve as effective therapeutic targets. Among peripheral T-cell lymphomas (PTCLs), little is known about fusion genes except for those involving ALK. Discovery of fusion genes may provide therapeutic targets for PTCLs, which have poor outcomes with conventional therapy. We have developed methods for detecting candidate fusions using (1) mate-pair DNA sequencing (MPseq), an approach that spans the entire genome at a fraction of the cost of traditional whole-genome sequencing, and (2) RNA sequencing (RNAseq). Each technique has strengths, limitations, and false discovery rates. Here, we integrated results of both methods to discover and orthogonally validate expressed fusion genes in PTCLs. Methods: DNA and RNA were extracted from 49 frozen, OCT-embedded clinical PTCL specimens, including 25 anaplastic large cell lymphomas; 16 PTCLs, not otherwise specified; and 8 other PTCLs. MPseq and RNAseq were performed using Illumina library protocols and a HiSeq 2000. Detection of fusion genes and transcripts followed our published methods (Blood 2012;120:2280 and Nucleic Acids Res 2011;39:e100, respectively). Functional annotation of genes involved in fusions was performed using DAVID. Results: Of 2818 candidate chromosomal rearrangements identified by MPseq and 90 candidate fusion transcripts identified by RNAseq, 45 events were present in both datasets (mean/case, 0.9; range, 0-7). Fusions were inter-chromosomal in 16 and intra-chromosomal in 29. Annotation clusters with the highest enrichment scores related to SH2 domain-containing proteins and tyrosine kinases (including FER and MAP2K3). Seven genes were recurrently involved in fusions, including known fusion partners such as ALK and previously unreported fusion partners such as PPP1R8, a phosphatase inhibitor important for the activity of Polycomb group proteins such as EZH2. Detection of candidate events only by MPseq could be explained in part by breakpoints mapping to non-genic regions (79% of events). Detection only by RNAseq could be explained in part by unannotated transcripts spanning neighboring named genes (median intra-chromosomal distance, 0.09 MB vs. 12.7 MB for intra-chromosomal candidates identified by MPseq; p<0.0001, Wilcoxon test). Conclusions: Integrated DNA/RNA sequencing is an effective, clinically feasible tool for simultaneous discovery and orthogonal validation of expressed fusion genes. This approach yielded fusions in PTCLs involving genes with key functions in lymphocytes, including novel and recurrent fusions, some of which might be targetable with compounds currently in clinical trial. Candidate intra-chromosomal fusion transcripts not identified by MPseq may represent unrecognized isoforms or the results of novel read-through mechanisms, and merit further study. Citation Format: Andrew L. Feldman, George Vasmatzis, Yan W. Asmann, Sarah H. Johnson, Bruce W. Eckloff, Sumit Middha, Jaime I. Davila, Paul J. Kurtin, Brian K. Link, Stephen M. Ansell, James R. Cerhan. Integrated DNA/RNA sequencing for discovery and orthogonal validation of expressed fusion genes in peripheral T-cell lymphomas. [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 5585. doi:10.1158/1538-7445.AM2014-5585

Abstract ASCL1 is an important regulatory transcription factor in pulmonary neuroendocrine cell development, but its value as a biomarker of neuroendocrine differentiation in lung adenocarcinoma (AD) and as a potential prognostic biomarker remains unclear. We examined ASCL1expression in lung cancer samples of varied histologic subtype, clinical outcome, and smoking status and compared to expression of traditional neuroendocrine markers. ASCL1 mRNA expression was found almost exclusively in smokers with AD, in contrast to non-smokers. ASCL1 protein expression by immunohistochemistry (IHC) correlated best with synaptophysin compared to chromogranin and CD56/NCAM. Analysis of a compendium of 367 microarray-based gene expression profiles in stage I lung adenocarcinomas identified significantly higher expression levels of the RET oncogene in ASCL1 positive tumors (ASCL1+) compared to ASCL1- tumors (q-value < 10 - 9). High levels of RET expression in ASCL1+ but not in ASCL1- tumors was associated with significantly shorter overall survival in stage 1 (p = 0.007) and in all AD (p = 0.037). RET protein expression by IHC had an association with overall survival in the context of ASCL1 expression. In silico gene set analysis and in vitro experiments by ASCL1 shRNA in AD cells with high endogenous expression of ASCL1 and RET implicated ASCL1 as a potential upstream regulator of the RET oncogene. Also, silencing ASCL1 in AD cells markedly reduced cell growth and motility. These results suggest that ASCL1 and RET expression define a clinically relevant subgroup of approximately 10% of AD characterized by neuroendocrine differentiation. Citation Format: Farhad Kosari, Cristiane M. Ida, Marie Christine Aubry, Lin Yang, Irina V. Kovtun, Janet L. Schaefer Klein, Sandra C. Tomaszek, Stephen J. Murphy, Ping Yang, Dennis Wigle, George Vasmatzis. ASCL1 and RET expression define a clinically relevant subgroup of lung adenocarcinoma characterized by neuroendocrine differentiation. [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 5251. doi:10.1158/1538-7445.AM2014-5251

Purpose Distinguishing independent primary tumors from intrapulmonary metastases in non–small-cell carcinoma remains a clinical dilemma with significant clinical implications. Using next-generation DNA sequencing, we developed a chromosomal rearrangement–based approach to differentiate multiple primary tumors from metastasis. Methods Tumor specimens from patients with known independent primary tumors and metastatic lesions were used for lineage test development, which was then applied to multifocal tumors. Laser capture microdissection was performed separately for each tumor. Genomic DNA was isolated using direct in situ whole-genome amplification methodology, and next-generation sequencing was performed using an Illumina mate-pair library protocol. Sequence reads were mapped to the human genome, and primers spanning the fusion junctions were used for validation polymerase chain reaction. Results A total of 41 tumor samples were sequenced (33 adenocarcinomas [ADs] and eight squamous cell carcinomas [SQCCs]), with a range of three to 276 breakpoints per tumor identified. Lung tumors predicted to be independent primary tumors based on different histologic subtype did not share any genomic rearrangements. In patients with lung primary tumors and paired distant metastases, shared rearrangements were identified in all tumor pairs, emphasizing the patient specificity of identified breakpoints. Multifocal AD and SQCC samples were reviewed independently by two pulmonary pathologists. Concordance between histology and genomic data occurred in the majority of samples. Discrepant tumor samples were resolved by genome sequencing. Conclusion A diagnostic lineage test based on genomic rearrangements from mate-pair sequencing demonstrates promise for distinguishing independent primary from metastatic disease in lung cancer.

Abstract So far, therapeutic vaccination with allogeneic cancer cells has been only marginally effective. A reason for failure could stem from the fact that vaccine cells are usually cultured in air (pO2=20 kPa), while tumors in situ are often highly hypoxic. Hence, it is possible that vaccine cells expressed antigens differently than tumor cells in situ. We postulate that cells grown at low pO2 provide a better antigen match to tumors in situ and could be more effective vaccines. We tested the former hypothesis by comparing prostate cancer (CaP) cells propagated at pO2 = 2 kPa and 20 kPa. To identify potential tumor-associated antigens, we prepared CaP cell lysates, resolved them by 2D electrophoresis and immunoblotting using spontaneous antibodies from plasma derived from CaP patients and control subjects. Antibody-labeled spots were analyzed by MALDI-TOF mass spectrometry. Among identified molecules, we selected hypoxia-regulated HSP70 and hnRNP L and hypoxia-independent HSP60 and determined by ELISA the frequency of plasma samples reacting with these molecules two standard deviations above their reaction with normal sera. Frequency of HSP60-reactive plasma was 1/76 in healthy controls (HC; 1.3%), while it was 7/54 in CaP patients (13.0%; p<0.05). The corresponding values were 1/14 in colorectal cancer (7.1%), 1/10 in lung cancer (LC; 10.0%), 1/20 in renal cell carcinoma (5.0%) and 0/17 in rheumatoid arthritis (RA; 0%), not different from healthy controls. These data suggest that CaP patients develop humoral immunity to HSP60. Levels of autoantibodies to HSP70 in CaP patients (2/38, 5.3%) did not differ from HC (2/54, 3.7%) while they did differ from HC in LC (2/10, 20.0%) and RA (3/17, 17.7%). Similarly, frequency of autoantibodies to hnRNP L did no differ between HC (3/49, 6.1%) and CaP patients (2/38, 5.3%). Further, we evaluated the expression of hnRNP L protein in native CaP tissue by Western blot. In contrast to the levels of autoantibodies in plasma, hnRNP L antigen was present at twice the levels in CaP tissue (N=8) than in control benign prostate tissue (N=4; p<0.05). Overall, our results suggest that it is worthwhile to pursue evaluating of HSP60-specific autoantibodies as a CaP-associated marker. Currently, we are analyzing HSP60 and hnRNP L (hypoxia-regulated) protein and transcript expression by immunohistochemistry and PCR, respectively, in a large patient cohort. This approach could validate the use of hypoxia-sensitive molecules as effective prognostic biomarkers and help develop better therapy for CaP and, possibly, other tumors. Support: DOD PC094680 and PCF Creativity Award (both to CRG). Citation Format: Tangeng Ma, Abdelouahid Elkhattouti, Farhad Kosari, R. Jeffrey Karnes, John C. Cheville, George Vasmatzis, Stanimir Vuk-Pavlović, Christian R. Gomez. Hypoxia as a tool for identifying prostate cancer-associated antigens. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4056. doi:10.1158/1538-7445.AM2013-4056

Abstract Background: Peripheral T-cell lymphomas (PTCLs) represent a heterogeneous group of generally aggressive malignancies, with 5-year overall survival (OS) rates of ∼35%. Only ALK translocations, which define ALK-positive anaplastic large T-cell lymphoma (ALCL), have emerged as a genetic biomarker predicting favorable outcome following conventional chemotherapy (e.g. CHOP). Using next-generation sequencing, we discovered additional recurrent chromosomal translocations in PTCLs involving either the DUSP22 locus on 6p25.3 or TP63 on 3q28. The purpose of this study was to evaluate the prognostic utility of combined testing for both these events in PTCL patients. Methods: We examined the clinicopathologic features of 134 systemic PTCLs using WHO criteria, including 49 ALCLs, 23 angioimmunoblastic PTCLs, 50 PTCLs of “not otherwise specified” type, and 12 PTCLs of other subtypes. Immunohistochemistry was used to determine ALK expression, an established surrogate for ALK translocations in ALCL. Fluorescent in situ hybridization (FISH) was performed to detect translocations of DUSP22 or TP63 in all cases. OS was plotted using the Kaplan-Meier method. Comparisons were made using the log-rank test. Results: There were 83 males and 51 females, with a mean age of 58 years (range, 6 to 90 years). Median OS for all patients was 22 months (5-year OS, 36%) with a median follow-up of 24 months (range, 0.1 to 354 months). Translocations of DUSP22, TP63, and ALK were mutually exclusive. Patients with ALK-positive ALCL (n=17) had a median OS of 176 months (median follow-up, 68 months; 5-year OS, 69%). Despite being ALK-negative, cases with translocations of DUSP22 or TP63 shared pathologic features with ALCL. Median OS was not reached in patients with DUSP22 translocations (n=7; median follow-up, 88 months; 5-year OS, 100%). Patients with tumors harboring TP63 translocations (n=9) had a median OS of 13 months (all dead at 76 months; 5-year OS, 11%). Patients with none of these abnormalities (n=101) had a median OS of 17 months (median follow-up, 13 months; 5-year OS, 29%). Differences in OS among these groups were highly significant (p=0.0002). Conclusions: PTCLs with DUSP22 translocations had a favorable prognosis similar to ALK-positive ALCLs. In contrast, TP63 were associated with very poor prognosis. FISH for these biomarkers is a promising tool for risk stratification, particularly since both translocations occurred in cases with overlapping pathologic features. PTCLs with DUSP22 translocations may represent a genetically defined subset of ALK-negative ALCLs. Like patients with ALK-positive ALCLs, these patients may not derive additional benefit from intensified up-front strategies such as early transplantation. Patients with none of these abnormalities comprised 75% of patients studied and had an intermediate prognosis, indicating the need for additional biomarkers in this group to improve risk stratification. Citation Format: Edgardo R. Parrilla Castellar, George Vasmatzis, Ryan A. Knudson, Rhett P. Ketterling, Karen L. Grogg, William R. Macon, Mark E. Law, Stephen M. Ansell, Ahmet Dogan, Andrew L. Feldman. Genetic biomarkers for risk stratification of patients with peripheral T-cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3538. doi:10.1158/1538-7445.AM2013-3538