Johns Hopkins University, Baltimore, USA. National Cancer...

Presented at the 1st Intl. Workshop on XMRV7-8 September 2010, Bethesda USA

K.S. Sfanos1, A.L. Aloia2, J.L. Hicks1, W.B. Isaacs1, Q. Zheng1, F. Maldarelli2, A.M. De Marzo1, A. Rein2

1Johns Hopkins University, Baltimore, USA.2National Cancer Institute, HIV Drug Resistance Program, Frederick, USA.


National Cancer Institute

Johns Hopkins University

Dr. Alan Rein Dr. Amanda Aloia

Dr. Angelo De Marzo


* To date, no virus has been causally linked to prostate cancer

HSV1/2

1973 1977

CMV

1990

PCR Invented

1996

HIV, HHV8/KSHVHPV

2002

EBV, BKV, JCV

2006

XMRV

1983


RealReal--time duplex PCR time duplex PCR -- reagentsreagentsXMRV probeXMRV probeFAMFAM BHQ1BHQ1

CCR5 probeCCR5 probeHEXHEX BHQ1BHQ1

XMRV primer and probe set in diverse region of Pol

(XMRV)

(XMRV)

XMRV-F probe

XMRV-R100 bpproduct


RealReal--time duplex PCR time duplex PCR –– positive controlpositive control

CWR22Rv1 contains XMRV (Knouf et al. J. Virol. 2009) Reported at least 10 integrated XMRV copies / cell Near-identical to published sequences of XMRV

Approx. VP62 nucleotide region sequenced VP62 compared 22rv1(VP62/Sample)

3930 – 4915 (985bp)Gag‐Pol

Identical

245 – 1089 (844bp)Leader‐Gag, with XMRV‐specific deletion

G/A (Val/Ile) at 790

6433 – 7294 (861bp)Env

A/C (Thr/Pro) at 6534T/A (Leu/Gln) at 6646


RealReal--time duplex PCR time duplex PCR –– positive controlpositive control CWR22Rv1 genomic DNA (gDNA) was diluted into HeLa or

293T cell gDNA Tests with XMRV plasmid VP62 indicate that there are ~ 15

copies of XMRV per diploid genome in CWR22Rv1 gDNA 0.01ng of CWR22Rv1 gDNA = about 1.4 diploid cells Assay can detect ~20 copies of XMRV DNA (single cell),

even in a vast excess (≥ 100 ng) of uninfected cell gDNA

XMRV probeXMRV probeFAMFAM BHQ1BHQ1CWR22Rv1 gDNA in 100ng HeLa gDNA

CWR22Rv1 gDNA1ng (red), 0.1ng (green),0.01ng (blue), 0.001ng (orange/pink)


Amplification of CCR5 in the same well demonstrates the DNA quality

CCR5 probeCCR5 probeHEXHEX BHQ1BHQ1CWR22rv1 gDNA in 100ng HeLa gDNA

CWR22Rv1 gDNA1ng (red), 0.1ng (green), 0.01ng (blue), 0.001ng (orange/pink)

RealReal--time duplex PCR time duplex PCR –– positive controlpositive control


RealReal--time duplex PCR time duplex PCR –– negative controlnegative control An amount of 293T or HeLa gDNA that corresponded to

the amount of sample being tested. The same amount of bacterial DNA (CCR5 negative

control)

100ng HeLa gDNA (red), 100ng DH5α gDNA (green)XMRV probeXMRV probe CCR5 probeCCR5 probe


RealReal--time duplex PCR time duplex PCR –– prostate samplesprostate samples Screened DNA from 161 prostate tumors, including 7

that had been micro-dissected and 10 that were metastases. All DNA samples were derived from fresh or frozen tissues.

Between 25 ng and 1 μg of DNA was used per reaction. 54 samples were tested at 25 – 35 ng, 54 at 40 – 60 ng, and 53 at 100 ng or greater.

In all cases CCR5 was successfully amplified, confirming the quality of the DNA preparation, but there was no amplification from the XMRV primers in any of the cases

All samples were tested at least twice


RealReal--time duplex PCR time duplex PCR –– prostate samplesprostate samples

XMRV probeXMRV probeFAMFAM BHQ1BHQ1 CCR5 probeCCR5 probeHEXHEX BHQ1BHQ1

Typical sample data: 100 ng of DNA from 4 prostate tumors

* In all cases negative controls are negative, 0.01 ng of CWR22Rv1 gDNA is positive


XMRV XMRV ImmunohistochemistryImmunohistochemistry (IHC)(IHC) Utilized MLV30 and MLV70 antisera, corresponding to the

MLV proteins p30CA and gp70SU, which are cleavage products of the viral Gag and Env polyproteins, respectively

Mock VP62 (XMRV)

VP62 (XMRV)

Mock

α-MLV30 α-MLV70


XMRV IHC XMRV IHC -- controlscontrolsα-MLV30 α-MLV70

pcDNA3.1

293T cells, formalin fixed and paraffin embedded in identical manner to prostate tissues

VP62 XMRV


XMRV IHC XMRV IHC -- controlscontrols

CWR2

2Rv1

DU1

45PC

3

α-MLV30 α-MLV70


XMRV IHC XMRV IHC –– prostate tissuesprostate tissues 596 prostate tumors and 452 benign prostate tissue

specimens, prepared either as full tissue sections or as tissue microarrays

Many of the prostatic tissues evaluated included areas of acute and chronic inflammation, atrophy, benign prostatic hyperplasia and high grade prostaticintraepithelial neoplasia (PIN)

Tumor samples were enriched for high-grade (i.e., Gleason ≥ 7) cases

Each experiment included positive and negative controls, which always gave the expected results

No staining of prostate tissue samples was ever observed with either antiserum


Normal

Low Grade High Grade LN Metastases

XMRV IHC XMRV IHC –– prostate tissuesprostate tissues

α-MLV30

α-MLV30

+ Control


* Numbers shown are total number of cases / total number of TMA spots analyzed. Multiple TMA spots (typically at least 4) were analyzed per case.

Summary of samples testedSummary of samples tested

Close to 800 samples, no positive cases detected.

PCR

Microdissected prostate tumor 12

Prostate tumor metastasis 10

Prostate tumor 139

IHC MLV30 MLV70

Cases Spots Cases Spots

TMA prostate tumor* 433 1524 433 1524

TMA prostate benign* 437 1890 437 1890

TMA prostate tumor metastasis* 52 121 52 121

Full sections prostate tumor 38 111

Full sections prostate benign 5 15


SummarySummary Used a real-time PCR assay capable of detecting XMRV

sequences in DNA from a very small number of infected cells, even in the presence of a vast excess (more than 10,000-fold) of uninfected cell DNA

Performed IHC with two antisera, each specific for a different MLV protein, under conditions where the sera reproducibly stained XMRV-containing cells but not identically treated control cells

Examined close to 800 total cases examined by either PCR or IHC, no positive cases identified


How can our negative results be reconciled?

We did not select RNaseL R462Q homozygotes for analysis This association has not held up in subsequent

studies both in PCa and in chronic fatigue In examining close to 800 cases, we undoubtedly

included a substantial number of these individuals

XMRV was present in our samples, but we failed to detect it because the viral sequences were somewhat different from the published XMRV sequence Little variation in XMRV sequences has been

observed to date IHC assays used broadly-reactive antisera


How can our negative results be reconciled?

Infected cells may be present at such a low level in virus-positive tumors that the samples we tested were too small to contain infected cells Schlaberg et al. initially reported that ~ 1 cell in

660 was XMRV-positive Might explain negative results with tissue

microarrays, but we analyzed >100 tumors by IHC on standard slides, which generally contain more than 105 cells

Would not support a causal role for XMRV in prostate tumorigenesis


Acknowledgements

Johns Hopkins University

Jessica Hicks

Dr. Angelo De MarzoQizhi ZhengDr. William IsaacsHelen FedorMarta Gielzak

National Cancer InstituteDr. Alan ReinDr. Amanda Aloia

DNA Sample ContributorsKenneth Pienta (University of Michigan)Frank Ruscetti (NCI)Cathy Vocke (NCI)Peter Pinto (NCI)W. Marston Linehan (NCI)The Brady Urological Institute Prostate Specimen Repository (Johns Hopkins)

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