XMRV among Prostate Cancer Patients from the Southern United States and Analysis of Possible Correlates of Infection
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1 XMRV among Prostate Cancer Patients from the Southern United States and Analysis of Possible Correlates of Infection Bryan Danielson Baylor College of Medicine Department of Molecular Virology and Microbiology
2 Prostate cancer Significance of prostate cancer (PCA) Leading cancer in men in western countries 1,2 American males, % of incident cancer cases 2 11% of cancer-related deaths 2 Some reasons to suspect that PCA may be caused by viral infection Environmental factors may promote prostate cancer development 1 20% of cancers worldwide have a known viral etiology 3 20% of all cancers are caused by chronic inflammation 1 1 De Marzo et al. Nat Rev Cancer (4): Jemal et al. CA Cancer J Clin (2): zur Hausen. Virology Sep 15;392(1):1-10.
3 Viral etiology for prostate cancer? Germline mutations in RNASEL are linked to familial prostate cancer 1 RNase L: Innate type I interferon-inducible antiviral effector Urisman et al. PLoS Path Virochip DNA microarray XMRV discovered Carcinoma-associated stroma infected Infection correlated with R462Q Familial PCA RNASEL R462Q XMRV infection 1 Casey et al. Nature genetics. 2002
4 XMRV, RNASEL R462Q, and PCA Yes Urisman et al. PLoS Path Correlation with R462Q? No Schlaberg et al. PNAS Lombardi et al. Science (PBMCS) Arnold et al. Cancer PCA Gleason score Familial PCA RNASEL R462Q? XMRV infection
5 Goals of the study Further define the geographic distribution of XMRV in PCA in the United States Investigate the association with RNASEL R462Q Verify the correlation with Gleason score Search for a correlation with other clinical pathological parameters of prostate cancer
6 Study group 144 prostate cancer patients from Texas No preoperative treatment All underwent radical prostatectomies Tissue cores stored at -80 C Normal tissue Tumor tissue only Tumor tissue
7 Methods Detecting provirus: Nested PCR targeting a 650 bp region of the envelope gene Each specimen screened in triplicate using 650ng of DNA template A C B LTR gag-pro-pol env LTR R462Q genotyping: XMRV provirus Real-time PCR-based allelic discrimination assay Primer binding sites
8 Provirus screen results Criteria for XMRV positivity: Specimens were considered positive if one or more of the three PCR replicates amplified a DNA fragment of the expected size Patients were considered positive for XMRV if any corresponding specimen was positive Of 144 patients, 32 (22.2%) tested positive for XMRV
9 Proviral load Nested PCR sensitivity: 1 infected cell per 10 5 uninfected cells in one of three samples with 600 ng (~10 5 cells) of DNA template 1kb 500 bp Positive control Specimen number: Majority of XMRV-positive specimens tested positive in only one or two of three replicates Number of proviruses is reaching the limit of detection
10 Sequence analysis PCR products from 17 of 32 XMRV positive samples were sequenced * All patient-derived sequences cluster with XMRV Frequency of amino acid differences from reference strain, VP62: Overall: 0.14 % Highest: 1.4 % Lowest: 0 % Patient-derived sequences *VP62: Previously characterized clone of XMRV
11 Normal versus tumor tissue XMRV was detected in both normal and tumor tissue Distribution of viral DNA between tissue types in XMRV-positive patients: Normal tissue only Normal and tumor tissue Tumor tissue only Patients were statistically more likely to test positive for XMRV in both normal and tumor tissue rather than either alone. (kappa coefficient, κ = 0.62; scale: -1 to 1)
12 XMRV positivity and RNASEL R462Q RNASEL Genotype a Total RR RQ QQ XMRV + 16 (24.2%) 13 (21.3%) 3 (17.6%) 32 (22.2%) XMRV Total Percentages of XMRV-positive patients: At least one wild-type allele: Homozygous R462Q variant: 22.8% 17.6% Chi-Square, p = 0.82 a RNASEL genotypes: RR, homozygous wild-type; RQ, heterozygous; QQ, homozygous R462Q variant.
13 Gleason score Percentage of patients 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% XMRV +XMRV Gleason score No significant trend found (Fisher s exact, p = 0.29) Few patients with high Gleason scores
14 Additional clinical pathological parameters of PCA Parameter a Pos. Neg. Test p value XMRV+ XMRV- XMRV- XMRV+ ECE 11(25.6) 32 21(21.4) 77 Chi-Square 0.59 SVI 5(33.3) 10 27(21.4) 99 Fischer s exact 0.33 SMI 8(23.5) 26 24(22.4) 83 Chi-Square 0.89 No significant association found a Clinical parameters are: ECE, extracapsular extension; SVI, seminal vesicle invasion; SMI, surgical margin invasion
15 Factors facilitating PCR-based detection of XMRV Relatively high quantity of DNA template ng DNA 3.2% 650ng 22.2% Multiple genes targeted Nested gag primers (1/10 th sensitivity of env primers) no detection Nested pol primers (competing chromosomal sequence) no detection Nested env primers detection
16 Summary 144 PCA patients were screened for XMRV provirus and genotyped for RNASEL R462Q 22.2% of patients tested positive for XMRV Detection of XMRV did not correlate with RNASEL R462Q XMRV mainly found in both normal and tumor tissue Detection of XMRV did not correlate with clinical pathological parameters of PCA
17 Conclusions XMRV can be found in PCA patients from the southern United States XMRV can be found in normal tissue, suggesting that infection may precede cancer onset The presence of XMRV is not influenced by the RNASEL R462Q polymorphism Further study is required to determine whether XMRV infection correlates with clinical pathological parameters of PCA
18 Acknowledgements Baylor College of Medicine Molecular Virology & Microbiology Dr. Jason Kimata Rajesh Thippeshappa Monica Yu-Kimata Dr. Cosmina Gingaras Pathology, Dan L. Duncan Cancer Center Dr. Gustavo Ayala Mohammad Sayeeduddin Pediatrics Dr. Claudia Kozinetz
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