Vasmatzis, George

Key Points ALK-negative ALCLs have chromosomal rearrangements of DUSP22 or TP63 in 30% and 8% of cases, respectively. DUSP22-rearranged cases have favorable outcomes similar to ALK-positive ALCLs, whereas other genetic subtypes have inferior outcomes.

AbstractThe development of adenocarcinoma of the lung is believed to proceed from in situ disease (adenocarcinoma in situ, AIS) to minimally invasive disease with prominent lepidic growth (minimally invasive adenocarcinoma, MIA), then to fully invasive adenocarcinoma (AD), but direct evidence for this model has been lacking. Because some lung adenocarcinomas show prominent lepidic growth (AD-L), we designed a study to address the lineage relationship between the lepidic (noninvasive) component (L) and the adjacent nonlepidic growth component representing invasive disease within individual tumors. Lineage relationships were evaluated by next-generation DNA sequencing to define large genomic rearrangements in microdissected tissue specimens collected by laser capture. We found a strong lineage relationship between the majority of adjacent lepidic and invasive components, supporting a putative AIS–AD transition. Notably, many rearrangements were detected in the less aggressive lepidic component, although the invasive component exhibited an overall higher rate of genomic rearrangement. Furthermore, a significant number of genomic rearrangements were present in histologically normal lung adjacent to tumor, but not in host germline DNA, suggesting field defects restricted to zonal regions near a tumor. Our results offer a perspective on the genetic pathogenesis underlying adenocarcinoma development and its clinical management. Cancer Res; 74(11); 3157–67. ©2014 AACR.

Abstract Summary: Mate pair library sequencing is an effective and economical method for detecting genomic structural variants and chromosomal abnormalities. Unfortunately, the mapping and alignment of mate-pair read pairs to a reference genome is a challenging and time-consuming process for most next-generation sequencing alignment programs. Large insert sizes, introduction of library preparation protocol artifacts (biotin junction reads, paired-end read contamination, chimeras, etc.) and presence of structural variant breakpoints within reads increase mapping and alignment complexity. We describe an algorithm that is up to 20 times faster and 25% more accurate than popular next-generation sequencing alignment programs when processing mate pair sequencing. Availability: http://bioinformaticstools.mayo.edu/research/bima/ Contact: vasm@mayo.edu or vasmatzis.george@mayo.edu Supplementary Information: Supplementary data are available at Bioinformatics online.

Chromosomal translocations leading to expression of abnormal fusion proteins play a major role in the pathogenesis of various hematologic malignancies. The recent development of high‐throughput, “deep” sequencing has allowed discovery of novel translocations leading to a rapid increase in understanding these diseases. Translocations involving the anaplastic lymphoma kinase (ALK) gene leading to ALK fusion proteins originally were discovered in anaplastic large cell lymphomas (ALCLs). Among ALCLs, NPM1‐ALK fusions are most common and lead to nuclear localization of the fusion protein. Here, we present a 50‐year‐old male with ALCL demonstrating cytoplasmic ALK immunoreactivity only, suggesting the presence of a non‐NPM1 fusion partner. We performed deep RNA sequencing of tumor tissue from this patient and identified a novel transcript fusing Exon 6 of TRAF1 to Exon 20 of ALK. The TRAF1‐ALK fusion transcript was confirmed at the mRNA level by Sanger sequencing and the fusion protein was visualized by Western blot. The discovery of this TRAF1‐ALK fusion expands the diversity of known ALK fusion partners and highlights the power of deep sequencing for fusion transcript discovery. © 2013 Wiley Periodicals, Inc.

We used high resolution mate‐pair sequencing (HRMPS) in 15 patients with primary myelofibrosis (PMF): eight with normal karyotype and seven with PMF‐characteristic cytogenetic abnormalities, including der(6)t(1;6)(q21–23;p21.3) (n = 4), der(7)t(1;7)(q10;p10) (n = 2), del(20)(q11.2q13.3) (n = 3), and complex karyotype (n = 1). We describe seven novel deletions/translocations in five patients (including two with normal karyotype) whose breakpoints were PCR‐validated and involved MACROD2, CACNA2D4, TET2, SGMS2, LRBA, SH3D19, INTS3, FOP (CHTOP), SCLT1, and PHF17. Deletions with breakpoints involving MACROD2 (lysine deacetylase; 20p12.1) were recurrent and found in two of the 15 study patients. A novel fusion transcript was found in one of the study patients (INTS3‐CHTOP), and also in an additional non‐study patient with PMF. In two patients with der(6)t(1;6)(q21–23;p21.3), we were able to map the precise translocation breakpoints, which involved KCNN3 and GUSBP2 in one case and HYDIN2 in another. This study demonstrates the utility of HRMPS in uncovering submicroscopic deletions/translocations/fusions, and precise mapping of breakpoints in those with overt cytogenetic abnormalities. The overall results confirm the genetic heterogeneity of PMF, given the low frequency of recurrent specific abnormalities, identified by this screening strategy. Currently, we are pursuing the pathogenetic relevance of some of the aforementioned findings. Am. J. Hematol. 88:741–746, 2013. © 2013 Wiley Periodicals, Inc.

Abstract Gleason score 7 (GS7) prostate cancer [tumors with both Gleason patterns 3 (GP3) and 4 (GP4)] portends a significantly more aggressive tumor than Gleason score 6 (GS6). It is, therefore, critical to understand the molecular relationship of adjacent GP3 and GP4 tumor cell populations and relate molecular abnormalities to disease progression. To decipher molecular relatedness, we used laser capture microdissection (LCM) and whole-genome amplification (WGA) to separately collect and amplify DNA from adjacent GP3 and GP4 cell populations from 14 cases of GS7 prostate cancer. We then carried out massively parallel mate-pair next generation sequencing (NGS) to examine the landscape of large chromosomal alterations. We identified four to 115 DNA breakpoints in GP3 and 17 to 480 in GP4. Our findings indicate that while GP3 and GP4 from the same tumor each possess unique breakpoints, they also share identical ones, indicating a common origin. Approximately 300 chromosomal breakpoints were localized to the regions affected in at least two tumors, whereas more than 3,000 were unique within the set of 14 tumors. TMPRSS2–ERG was the most recurrent rearrangement present in eight cases, in both GP3 and GP4. PTEN rearrangements were found in five of eight TMPRSS2–ERG fusion–positive cases in both GP3 and GP4. Hierarchical clustering analysis revealed that GP3 has greater breakpoint similarity to its partner GP4 compared with GP3 from different patients. We show evidence that LCM, WGA, and NGS of adjacent tumor regions provide an important tool in deciphering lineage relationships and discovering chromosomal alterations associated with tumor progression. Cancer Res; 73(11); 3275–84. ©2013 AACR.

Abstract Tumor-associated hypoxia has been associated with malignant progression, metastasis and resistance to therapy. We studied the role of oxygen tension (pO2) in modulating the properties of cultured human prostate cancer (CaP) cells. In air, where oxygen tension (pO2) is approximately 20 kPa, cultured CaP cells expressed lower levels of transcripts associated with cancer and urologic disease; reducing pO2 to 2 kPa made these levels comparable to those in surgical CaP specimens suggesting that hypoxically cultured cells, rather than normoxically cultured cells, express genes at levels akin to those in tumor cells in situ. In human primary CaP tissue, transcripts of hypoxia-associated genes DLG7, CCNB1 and HMMR were overexpressed and associated with Gleason score and disease prognosis; this implies the potential prognostic value of these transcripts. While hypoxia affected the CaP cell proteome mostly quantitatively, it did affect expression of tumor–associated antigens and suggested the potential therapeutic relevance of the hypoxic antigenic landscape. Overall, our results suggest that hypoxia modifies cellular properties of cultured CaP cells towards a phenotype more similar to tumor cells in situ. Introduction of pO2 as a variable can constitute a tool for the identification of more effective prognostic biomarkers and development of better therapy for CaP and, possibly, other tumors. Citation Format: Christian R. Gomez, Stanimir Vuk-Pavlovic, Farhad Kosari, Jan Marie Munz, Claire Schreiber, Gaylord Knutson, Cristine Charlesworth, R. Jeffrey Karnes, John Cheville, George Vasmatzis. Prognostic value of hypoxia-associated genes in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C43.

Abstract Tumor-associated hypoxia has been associated with malignant progression, metastasis, resistance to therapy, and poor clinical outcome in prostate cancer (CaP). Prompted by the evidence that hypoxia affects CaP, we studied global gene expression in 100 CaP tissues and 71 samples of adjacent benign tissues. RNA was extracted from cancer cells isolated by laser-capture microdissection (LCM) or without isolation (“bulk tissue”). We found 24 hypoxia-associated genes significantly overexpressed in CaP (p≤0.02), both in bulk tissue and LCM. Among hypoxia-associated genes, the disc large (drosophila) homolog-associated protein 5 (DLG7), hyaluronan-mediated motility receptor (HMMR) and cyclin B1 (CCNB1) were associated with Gleason score and systemic progression. Since the products of HMMR and CCNB1 have been recently identified as molecular markers of CaP progression, we postulated that DLG7 has prognostic value also. To test this hypothesis we measured transcript levels for DLG7 in a 150-pair case-control cohort. The cases (progression to systemic disease within five years of surgery) and controls (no progression within seven years) were matched for clinical and pathologic prognostic variables, including grade, stage, and preoperative serum levels of PSA. The overall prognostic ability of DLG7, as tested in receiver operating characteristic analysis was of 0.74 (95% CL, 0.68 to 0.8), independent of the for ERG status. Overall, our data indicate that DLG7, a hypoxia-controlled gene have a prognostic value in high-risk CaP. To better define the value of DLG7 as a molecular predictor of CaP progression, we are currently assessing DLG7 protein levels by immunohistochemistry. Then we will analyze the association of DLG7 levels with clinical parameters. Introduction of oxygen tension as a variable may constitute a tool for the identification of novel biomarkers for CaP. Present address CRG: University of Mississippi Cancer Institute, Jackson, MS, USA. Support: DOD PC094680 (CRG), PCF Creativity Award (CRG), Mayo Clinic Prostate SPORE 5P50CA091956 (SV-P); Mrs. Adelyn L. Luther, Singer Island, Florida and Mayo Clinic Cancer Center (SV-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 116. doi:1538-7445.AM2012-116

Abstract The frequent observations of heterogeneous tumor foci in prostate cancers indicate that the molecular changes induced by carcinogenic insult take place over wide areas within the diseased prostate. These ‘field effect’ alterations provide important clues regarding the initiation of prostate cancer (PCa) and suggest targets for cancer prevention or biomarkers for early detection. However, PCa field effect biomarkers that have passed independent validation are lacking largely because these alterations are subtle and difficult to distinguish from unrelated small changes in gene expression in the benign prostate. We postulated that shared expression alterations in PCa and in benign prostate tissue in prostate glands that contain prostate cancer (BPC) would have a higher potential for independent validation than alterations identified in BPC alone. Expression analyses was performed on PCa (n = 37) and unmatched BPC (n = 36) and contrasted with benign prostate glands (BP) from patients free of prostate cancer (n =28). These analyses identified gene alterations that were concomitantly present in both PCa and BPC. The majority of the selected markers were validated by quantitative RT-PCR in an independent set of BPC (n = 33) and BP (n =18). Validated markers included genes and non-exonic sequences not previously associated with PCa field effect. A statistical model based on CCNB1 and non-exonic NACA locus discriminated between BPC and BP in the RT-PCR set and in an external microarray dataset with accuracies of 0.84 and 0.90, respectively. Genes with predominant expression in prostate stroma were identified by expression profiling of stroma and epithelial cells collected by laser captured microdissection. Pathway analysis identified enriched GO categories in BPC stroma including PDGFR signaling. These results validate our approach for finding PCa field effect alterations and demonstrate a prostate cancer transcriptome fingerprint in the adjacent non-neoplastic cells. 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 3008. doi:1538-7445.AM2012-3008