Kosari, Farhad

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

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 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

Abstract Tumor-associated hypoxia facilitates malignant progression, metastasis and poor prognosis in prostate cancer (CaP); however, effects of oxygen tension (pO2) in CaP are just beginning to be investigated. To establish mechanisms whereby hypoxia enhances malignant properties and survival of CaP and to identify pO2-regulated tumor-associated macromolecules, we propagated CaP cell lines LnCaP, VCaP and DU145 in hypoxia (pO2=2 kPa) and compared them with normoxically grown cells (pO2=20 kPa); hypoxic cells proliferated faster and secreted more vascular endothelial growth factor (p<0.05). Transcriptome studies revealed different gene expression in cells grown in hypoxia relative to those in normoxia. Interestingly, in hypoxic cells transcripts associated with cancer and urologic disease were overexpressed in comparison to normoxic cells (p<0.001) suggesting an association of low pO2 and aggressive features of CaP. Further, we analyzed the transcriptome in primary CaP cells isolated by laser-capture microdissection (LCM) and whole CaP tissue. We compared the data to those from CaP cells cultured at pO2=20 kPa or pO2=2 kPa. Notably, hypoxia increased transcript levels for pyruvate dehydrogenase kinase isozyme 1, nuclear prelamin A recognition factor, glucose phosphate isomerase, and glyceraldehyde-3-phosphate dehydrogenase in all three cell lines (p<0.05) to levels comparable to those found in primary bulk tissue and LCM isolated cells (p<0.005). This finding suggests that gene expression in hypoxically cultured cells is more akin to that in tumor cells in situ than are cells grown normoxically. By 2D-gel electrophoresis, we found that change in pO2 affected the proteome mostly quantitatively (i.e., by change in spot intensity). Our results suggest that hypoxia affects transcriptome and proteome in CaP cells. In addition, we screened 20 patient sera and 20 healthy control sera for spontaneous antibodies cross-reactive with VCaP cells and found that the sera reacted with numerous proteins, some previously reported to elicit an immune response in CaP patients (e.g., nucleoporin 62 and transitional endoplasmic reticulum ATPase). By this method we detected numerous novel autoantigens (under validation). Currently we are clarifying the pO2 effects on the relationship of transcriptome, proteome and tumor-associated antigens in CaP cells. Support: DOD PC094680 (CRG), Minnesota Partnership for Biotechnology and Medical Genomics, Mayo Clinic Prostate SPORE 5P50CA091956 (FK); Mrs. Adelyn L. Luther, Singer Island, Florida and Mayo Clinic Cancer Center (SV-P). 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-308. doi:10.1158/1538-7445.AM2011-LB-308

Abstract The objective of this study was to assess the relationship of the tumor protein levels of TOP2A and MIB-1 and ERG status with cancer-specific outcomes in men with high-risk prostate cancer treated by radical prostatectomy (RP). A 150-pair case-control study was designed from RP patients who developed systemic progression (SP) within 6 years of RP (cases) and men who were free of disease at least 8 years after RP (controls). The cases and controls were matched on conventional prognostic clinical parameters. TOP2A and MIB-1 levels were assessed by immunohistochemical methods, and ERG status was assessed by quantitative reverse transcription-PCR. The prognostic abilities of TOP2A and MIB-1 were significantly better in ERG(−) patients, and TOP2A was superior to MIB-1. In receiver operating characteristic analysis, the TOP2A and MIB-1 scores exhibited AUCs of 0.81 and 0.78 for ERG(−) patients, versus 0.67 and 0.68 for ERG(+) patients, respectively. Clinical parameters attained an AUC of 0.65 in ERG(−) patients and 0.54 in ERG(+) patients. When both markers were incorporated into a model for ERG(−) patients, the AUC increased to 0.83, with TOP2A showing a stronger association with SP than MIB-1. The time to SP was significantly associated with TOP2A; higher 5-year SP rates were observed in patients with higher TOP2A protein levels. In addition, although patient numbers are small, the response to adjuvant androgen deprivation therapy is associated with ERG status, showing more significant treatment effect in ERG(+) patients. Cancer Res; 70(22); 8994–9002. ©2010 AACR.

Purpose In men who are at high-risk of prostate cancer, progression and death from cancer after radical retropubic prostatectomy (RRP), limited prognostic information is provided by established prognostic features. The objective of this study was to develop a model predictive of outcome in this group of patients. Methods Candidate genes were identified from microarray expression data from 102 laser capture microdissected prostate tissue samples. Candidates were overexpressed in tumor compared with normal prostate and more frequently in Gleason patterns 4 and 5 than in 3. A case control study of 157 high-risk patients, matched on Gleason score and stage with systemic progression or death of prostate cancer as the end point, was used to evaluate the expression of candidate genes and build a multivariate model. Tumor was collected from the highest Gleason score in paraffin-embedded blocks and the gene expression was quantified by real-time reverse transcription polymerase chain reaction. Validation of the final model was performed on a separate case-control study of 57 high-risk patients who underwent RRP. Results A model incorporating gene expression of topoisomerase-2a, cadherin-10, the fusion status based on ERG, ETV1, and ETV4 expression, and the aneuploidy status resulted in a 0.81 area under the curve (AUC) in receiver operating characteristic statistical analysis for the identification of men with systemic progression and death from high grade prostate cancer. The AUC was 0.79 in the independent validation study. Conclusion The model can identify men with high-risk prostate cancer who may benefit from more intensive postoperative follow-up and adjuvant therapies.