1. Murine Leukemia Virus (MLV) Related Human Retroviruses and Blood Safety BPAC meeting, December 14, 2010 Indira Hewlett, Ph. D Chief, Laboratory of Molecular Virology DETTD/CBER/FDA

2. Issue FDA is seeking the advice of the BPAC on  1) donor deferral based on a history or diagnosis of Chronic Fatigue Syndrome (CFS) or Prostate Cancer (PC) and,  2) testing for the newly identified MLV related retroviruses in humans recognizing that disease causation has not been established.

3. Identification of MLV related retroviruses in humans 3 Prostate Cancer Tissue DNAVirochip Urisman A, et al. PLoS Pathogen 2006; 2(3): e25 Silverman R, et al. Nat. Rev. Urol 2010; Schlaberg R, et al. PNAS 2009; 106(38):16351-6 Lo S, et al. PNAS 2010; 107(36):15874-9 polytropic MLV xenotropic MLV

4. MLV/XMRV and Blood Safety  In 2009, a US study reported XMRV detection in blood of 67% (68/101) of CFS patients and 3.7% (8/218) of healthy controls using PCR.  XMRV and MLV-related human retroviruses have since been detected in the blood of 0-14% of healthy controls.  However, several studies have shown lack of MLV/XMRV detection in CFS, PC and blood donors.  Although disease association has been reported, causation has not yet been established.

5. July 2010 BPAC Informational Session Topics discussed :  Identification and detection (or lack of) in CFS, PC blood donors.  Assay validation study plans and assay development efforts.  AABB recommendation for use of donor education materials on CFS and indefinite deferral for donors who voluntarily disclose their CFS diagnosis.  Health Canada’s policy of indefinite deferral for PC and recent introduction of deferral for those who voluntarily disclose their CFS diagnosis.

6. Update on MLV/XMRV Disease Association – CFS Lo et al, PNAS, Aug. 2010  32/37 (86.5%) of CFS patients positive for polytropic MLV-related sequences by PCR on PBMC DNA  3/44 (6.8%) of US healthy volunteer blood donors positive by PCR on PBMC DNA. Hanson et al, 1 st Intl. Workshop on XMRV, Sept. 2010  MLV related sequences detected in PBMC DNA of 8/10 severe CFS, 3/10 recovered CFS cases and 1/10 healthy controls.  Virus isolated from plasma of 6 individuals including 5 CFS patients. Mikovits et al, 1 st Intl. workshop on XMRV, Sept. 2010  50 CFS patients in UK, PCR, culture and serology; > 60% positive for XMRV by culture and serology.

7. Lack of Disease Association – CFS AuthorCountryNo. of SamplesDetection MethodsReference Switzer B, et alUSA51 CFS cases 53 healthy controls 41 U.S. blood donors Nested PCR, WB, ELISA, IFA (Done in three labs) Retrovirology 2010; 7:57 Henrich TJ, et alUSA32 CFS 261 controls PBMC DNA; nested PCRJ Infect. Dis 2010; 202:1478-81 Van Kuppeveld FJM, et al Netherlands32 CFS pts 43 controls Nested PCR of gag gene. Real time PCR of integrase BMJ. 2010; 340: c1018. Groom HC, et alUK170 CFS patients 395 controls PCR or real time PCR Viral neutralization assay for XMRV and MLV. Retrovirology. 2010; 7:10 Hong P, et al.China65 CFS patients 85 blood donors RT PCR PBMCs and plasma Virology Journal. 2010; 7:224. Hohn O, et alGermany36 CFS 50 MS with fatigue 30 controls Gag & env ELISAs Nested PCR 1 st International Worskshop XMRV. 2010. Blomberg JSweden40 CFS plasma 50 CFS PBMCs 200 controls RT qPCR Integrase, Gag & env PCR Serology, virus culture 1 st International Worskshop XMRV. 2010

8.  Petros et al, 1 st Intl. Workshop on XMRV, Sept. 2010  3 PC tissues from 3 separate PC patients tested and found to be positive for XMRV/MLV sequences by PCR.  Fischer et al, 1 st Intl. Workshop on XMRV, Sept. 2010  355 PC tissue, 40 PBMC from PC patients and 70 healthy controls from Northern Europe tested by PCR;  2/355 (0.56%) tissues positive for XMRV, 1 wild type and 1 heterozygous for R462Q.  PBMC from 2 XMRV positive PC patients negative by PCR.  All healthy controls negative for XMRV sequences.  Danielson et al, JID, epub Oct. 2010  Prostatic tissue of 144 PC patients from southern US by PCR for the env gene; 32/144 (22%) positive for XMRV sequences.  Both normal and tumor tissue for 57 cases tested; 21/57 (36.8%) normal and 25/57 (43.9%) tumor tissue samples positive for XMRV. Disease association - PC

9. Lack of Disease Association – PC AuthorCountryNo. of SamplesDetection MethodsReference Aloia, et alUSA161 tumor derived samples (PCR); 596 tumor tissue samples (IHC) 452 benign prostatic tissue samples (IHC) Nested RT-PCR IHC Cancer Research 2010; Oct 21. [Epub ahead of print] D’Arcy F, et alIreland139 PCnested PCREuropean Urology 2008; 7(suppl):271 Hohn O, et alGermany589 PC tissue samples 146 PC serum samples Gag & env ELISAs Nested PCR Retrovirology 2009;6:92 Blomberg etalSweden400 PC patientsRT-qPCR, gag, env PCR, serology, virus culture 1 st International Worskshop XMRV. 2010.

10. Evaluation of Transfusion Risk  Transfusion transmission of MLV/XMRV theoretically possible due to presence in blood cells, evidence of cell-free virus (Lombardi et al, 2009).  Low but detectable transient viremia in rhesus macaques 4-14 days and seroconversion 11-14 days post intravenous inoculation (Qiu et al, 2010).  Sero-prevalence of 1.7% (5/300) in Japanese blood donors; 0.1% (3/2861) in US blood donors (Qiu et al, 2010) and 6.8% (3/44) positive by PCR on PBMC DNA (Lo et al, 2010).  However, no evidence of XMRV in 1) US study of 121 blood donors by PCR and serology (Switzer et al, 2010) and 2) different US study of 1435 HIV-ve and 44 HIV-1 +ve blood donors by TMA ( Gao et al, 2010).  No XMRV sequences in 105 plasma and 19 PBMC from Cameroon blood donors, 94 HIV-1 infected individuals from Uganda (Tang et al, 2010).

11. Potential Sources of Discrepancies  Study populations and geographic differences in prevalence  Case definition criteria and stage of illness  Specimen processing, sensitivity and specificity of test methods  Potential genetic variation of the virus  Other unknown factors Need for assay standardization and validation to evaluate blood safety and disease association.

12. Assay validation for blood donor studies  NHLBI Scientific Research Working Group comprised of scientists from govt., academia and blood organizations established to validate assays for transfusion transmission studies.  BSRI’s analytical and clinical reference panels evaluated by multiple labs including CDC, NIH, WPI, BSRI and 2 labs at FDA for testing of blood donors in future studies.  FDA currently developing NAT and serology panels for future lot release, if needed.

13. NIH sponsored Proposed Study on XMRV and Related Viruses in Chronic Fatigue Syndrome Laboratory sites CDC, NCI, WPI, others Sample preparation TBD time to extraction Clinical collection sites TBD access, expertise, geography Assays molecular, cultivation/co-cultivation Organizational Meeting, 4-5 October 2010 Labs: Harvey Alter (NIH), Bill Switzer (CDC), Judy Mikovits (WPI) Consultants: John Coffin (NCI), Simone Glynn (NHLBI), Suzanne Vernon (CFIDS), Nancy Klimas (U of Miami) Columbia Faculty: Bruce Levin, Mady Hornig, W. Ian Lipkin

14. NIH sponsored Proposed Study on XMRV and Related Viruses in Chronic Fatigue Syndrome Laboratory sites CDC, NCI, WPI, others Sample preparation TBD time to extraction

15. MLV/XMRV and Blood Donation  No direct evidence for transfusion transmission or association with a transfusion transmitted disease, therefore, FDA has not established donor policy specific to MLV/XMRV.  FDA regulations require donors to be in good health at time of donation; medical directors exercise judgment on whether CFS patients meet criteria.  In regard to PC, large survey in Sweden and Denmark (SCANDAT) showed no increase in disease cases in 888,843 blood transfusion recipients without a prior cancer diagnosis at the index transfusion.  Indefinite deferral of donors who voluntarily disclose CFS diagnosis in Australia, New Zealand, UK and parts of Canada.  In the US, AABB issued bulletin (No. 10-03) recommending use of donor education materials on CFS and indefinite deferral for donors who voluntarily disclose their CFS diagnosis.

16. Questions for the Committee 1. Do the scientific data support asking donors about a medical history and/or diagnosis of CFS as a basis for indefinite deferral? 2. Please discuss whether the scientific data support asking donors about a medical history and/or diagnosis of prostate cancer as a basis for indefinite deferral.

17. Questions for the committee – con’t 3. Please comment on the scientific evidence that would be needed to justify a policy of donor testing for infection with MLV-related human retroviruses. In particular, should donor testing be considered in the absence of confirmed disease causation ? 4.Assuming that testing is warranted, please comment on the potential utility of NAT and /or serologic testing of blood donations to ensure safety of the blood supply from transmission of MLV-related human retroviruses.

18. BPAC presentations The BPAC session today will include the following presentations on MLV/XMRV:  Review of current data on MLV/XMRV including highlights of the Ist International Workshop.  Research updates and current data on association of MLV/XMRV with CFS.  Data from animal infectivity studies.  Update from the Blood Scientific Research Working group on assay validation using MLV/XMRV panels.  Prospective and retrospective donor prevalence studies.  Assay development efforts on MLV-related retroviruses.