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July 26-27, 2010: Blood Products Advisory Committee Meeting - Xenotropic Murine Leukemia Virus-Related Virus (XMRV) – Informational Presentation

BLOOD PRODUCTS ADVISORY COMMITTEE MEETING
98 th Meeting, July 26-27, 2010
Hilton Washington D.C./North
620 Perry Parkway, Gaithersburg, MD

Topic: Xenotropic Murine Leukemia Virus-Related Virus (XMRV) – Informational Presentation

Issue: FDA is updating the committee on XMRV, the newly identified human retrovirus (a gammaretrovirus), and FDA’s collaborative efforts with other public health agencies and blood establishments to determine whether XMRV poses a safety concern for the blood supply.

Background:

Xenotropic Murine Leukemia Virus-Related Virus (XMRV) is a newly identified retrovirus and the first gammaretrovirus of the Retroviridae family detected in humans. It is unrelated to HIV but highly similar to mouse retroviruses. Gammaretroviruses are widespread in vertebrates, but do not establish infection readily in immunocompetent hosts. Gammaretroviruses in animals are implicated in a wide range of diseases including cancers, immunodeficiency, and neurological disorders. XMRV is a simple retrovirus with gag, pol and env genes and unlike HIV or HTLV, has no accessory or regulatory genes. The genome is a dimer of linear, positive-sense, single stranded RNA about 8,300 nucleotides long.

XMRV was identified using a viral detection DNA microarray composed of oligonucleotides corresponding to the most conserved sequences of all known viruses (1). Using this array, gammaretroviral sequences were identified in samples from 7/11 prostate cancer (PC) patients that were homozygous (QQ) for the R462 mutation for RNAse L which is an important molecule in the innate antiviral response (2). A survey of an additional 86 PC tumors by RT-PCR specific for XMRV showed that 40% of QQ cases were positive for XMRV compared with 1.5% of heterozygous RQ and homozygous wild type RR cases. Subsequently an analysis of 334 consecutive prostate resection specimens using a quantitative PCR assay and immunohistochemistry showed that 6% were positive for XMRV DNA and 23% for protein expression. Taken together, these findings suggest an association of XMRV with prostate cancer (3).

A subsequent study showed that XMRV could also be detected in patients with Chronic Fatigue Syndrome (CFS), a disease of unknown etiology, known to affect several million people worldwide (4). DNA from peripheral blood mononuclear cells (PBMCs) of CFS patients and healthy controls was tested for the presence of XMRV sequences. XMRV sequences were found in 68 out of 101 CFS patients (67%), compared with 8 of 218 (3.7%) healthy US controls. The viral gene sequences identified in CFS patients clustered with sequences from PC and both sequences were virtually identical. Further investigation using activated CFS patient PBMC co-cultured with susceptible PC cells (LNCaP) showed that virus could be transmitted by cells and supernatant, as indicated by protein expression and transmission electron microscopy, suggesting that the virus being detected by protein expression was infectious. Virus could also be transmitted from 10/12 CFS patient plasma samples. Taken together, these studies suggested that both cell associated and cell-free transmission of XMRV is possible. Finally, antibodies to XMRV were detected in 9/18 CFS patients using a test based on the envelope of a closely related virus, spleen focus forming virus (SFFV).

Preliminary data from a limited study of XMRV in an animal model (rhesus macaques) showed disseminated infection but only low but detectable transient viremia between 4-14 days. Seroconversion occurred at 11-14 days following inoculation, with titers peaking around day 95. Virus was isolated from T- and B- lymphocytes and NK cells in blood (but not macrophages), prostate epithelial cells, vaginal tissue, and there was evidence for viral replication in spleen, lung, lymph nodes and liver (5).

Since the discovery of XMRV there have been several controversial reports about the association of XMRV with PC and CFS. Studies of an Irish PC cohort of 139 patients with the R462Q mutation showed no evidence of XMRV (6). In a German study, only 1/105 tissue samples were positive for XMRV DNA from non-familial PC while 1/70 samples were positive from men who did not have PC (7). In a different study of German PC patients, 589 PC tissues with the RNaseL R462Q mutation were tested using nested RT-PCR and all were found to be negative (8).

In regard to CFS, several recent studies have reported negative findings of XMRV in blood. A Dutch study failed to detect XMRV in PBMC of patients with CFS (9) and similar findings were reported in two studies from the UK although a few samples showed XMRV neutralizing activity in one study (10,11). Studies conducted by CDC scientists in a US population of CFS cases and blood donors showed no evidence of XMRV infection in PBMC and plasma. These data do not lend support to an association of XMRV with CFS or its presence in blood donors. (13)

Among other related findings are those recently reported by investigators in Germany that XMRV could be detected in respiratory secretions (14). In this study, 267 respiratory samples taken from German patients were screened for XMRV infection by PCR assay. The prevalence of XMRV DNA was 2.3% (3/75) in travelers from Asia who had respiratory tract infections; 3.2% (1/31) in patients with chronic obstructive pulmonary disease; 9.9% (16/161) in immunosuppressed patients with severe respiratory tract infections; 3.2% (2/62) in the healthy control group. It is not clear whether finding XMRV in the respiratory tract indicates that the virus can be transmitted by the respiratory route (14). In general, retroviruses, like HIV-1, are rarely transmitted through respiratory secretions.

A compilation of all the XMRV studies done so far is shown in appendix I. The reasons for discrepancies between findings are not clear. Differences in cohorts, populations and test methods have been considered as potential causes. In addition, differences in geographic prevalence, genetic variation of the virus or other factors could also potentially contribute to these conflicting findings. Additional studies are needed to confirm disease association of XMRV and to fully understand the role of XMRV in disease causation.

Discussion:

(a) Detection methods:

Currently there are no commercially approved/licensed tests for detection of XMRV infection. The assays used for research studies include PCR, EIA, Western blot, and immunohistochemical methods. The relative sensitivity and specificity of various assays have not been determined and standards for performance of assays have not yet been established.

(b) Evaluation of Transfusion Risk in the US:

Transmission through transfusion has not been shown, but is theoretically possible since virus has been detected in blood cells and there is evidence of cell-free virus. A seroprevalence study in Japan found 1.7% of random donors to be positive (12). Preliminary studies have shown that 3/2851 US blood donors (0.1%) were anti-XMRV antibody positive using a research immunoassay based on gag and env proteins (5).

A Blood XMRV Scientific Research Working Group, led by the National Heart Lung and Blood Institute (NHLBI), has been established to identify and design research studies to evaluate whether XMRV poses a threat to blood safety. An evaluation of blood safety risks includes 1) an evaluation of the prevalence of XMRV in blood donors; 2) if prevalent in blood donors, a determination of whether the virus is transfusion-transmitted; and 3) if transfusion-transmitted, whether clinical manifestations occur. An evaluation of the prevalence of XMRV in blood donors necessitates a way to measure or identify the agent in blood specimens. The fact that different laboratories obtain different results for XMRV detection in patients’ specimens and different prevalence estimates in normal controls strongly suggests the need for the development of standard XMRV reference and clinical case and healthy donor prevalence panels for XMRV detection. Thus, development of XMRV nucleic acid test (NAT) analytical and clinical panels was identified as the first priority by the Working Group. As a first step towards this goal, an analytical reference panel was developed to validate NAT assays for use in donor survey studies . The panel was developed by the Blood Systems Research Institute which is the Central Laboratory for the NHLBI Retrovirus Epidemiology Donor Study-II (REDS-II). Virus stocks were prepared from culture supernatants of the chronically infected prostate cancer cell line, 22Rv1. Following titration, reference infected cell preparations were established by spiking known numbers of infected cells into whole blood. Similarly, reference infected plasma preparations were established by spiking pedigreed-negative plasma with known viral titers of cell-free culture supernatants at serial dilutions. These coded analytical reference panels of specimens containing varying concentrations of XMRV virus as well as clinical panels which include biospecimens from blood donors, patients with Chronic Fatigue Syndrome whose blood was previously found to be positive for XMRV by the Whittemore Peterson Institute, and positive and negative control specimens are in the process of being tested by up to six participating laboratories to compare the sensitivity of their respective assays for XMRV. The study participants include laboratories from the NCI, FDA (two laboratories), CDC, Blood Systems Research Institute and the Whittemore Peterson Institute.

Summary:

The BPAC session will include several informational presentations on XMRV including a background and review of studies that led to the identification of the virus, ongoing validation studies of XMRV panels, updates of research efforts on test/panel development and other studies.

Finally, FDA will periodically update the committee on progress in our understanding of XMRV and transfusion safety. As additional data are obtained on prevalence in blood donors and any associated transfusion risks, BPAC may be asked to advise FDA on appropriate measures to maintain the safety and availability of the blood supply.

References:

  1. Urisman A, Molinaro RJ, Fischer N, Plummer SJ, Casey G, Klein EA, Malathi K, Magi-Galluzzi C, Tubbs RR, Ganem D, Silverman RH, DeRisi JL. Identification of a novel gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant. PLoS Pathog. 2006;2:e25.
  2. Dong B, Kim S, Hong S, Das Gupta J, Malathi K, Klein EA, Ganem D, Derisi JL, Chow SA, Silverman RH. An infectious retrovirus susceptible to an IFN antiviral pathway from human prostate tumors. Proc Natl Acad Sci U S A. 2007;104:1655-60.
  3. Schlaberg, R., Choe, D.J., Brown, K.R., Thaker, H.M., Singh, I.R. XMRV is present in malignant prostatic epithelium and is associated with prostate cancer, especially high grade tumors. PNAS, 2009, 106(38):16351-6
  4. Lombardi VC, Ruscetti FW, Das Gupta J, Pfost MA, Hagen KS, Peterson DL, Ruscetti SK, Bagni RK, Petrow-Sadowski C, Gold B, Dean M, Silverman RH, Mikovits JA. Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Science. 2009;326:585-589.
  5. Qui X, P Swanson, Luk K-C, Das Gupta J, Onlamoon N, Silverman R, Villanger F, Devare S, Schochetman G, Hackett, J Jr. . XMRV: examination of viral kinetics, tissue tropism, and serological markers of infection. The 17th Conference on Retroviruses and Opportunistic Infections (CROI 2010). At http://www.retroconference.org/2010/Abstracts/39393.htm. Accessed 3 March 2010.
  6. Fischer N, Hellwinkel O, Schulz C, Chun FK, Huland H, Aepfelbacher M, Schlomm T. Prevalence of human gammaretrovirus XMRV in sporadic prostate cancer. J Clin Virol. 2008;43:277-83.
  7. D’Arcy, F., Foley, R., Perry, A., Marignol, L., Lawler, M., Gaffney, E., Watson, RGW, Fitzpatrick, J.M., and Lynch, T.H., No evidence of XMRV in Irish prostate cancer patients with R462Q mutation, European Urology Supplements, Vol. 7, Issue 3, p271, 2008
  8. Hohn O, Krause H, Barbarotto P, Niederstadt L, Beimforde N, Denner J, Miller K, Kurth R, Bannert N. Lack of evidence for xenotropic murine leukemia virus-related virus(XMRV) in German prostate cancer patients. Retrovirology. 2009;6:92.
  9. van Kuppeveld F J M, de Jong AS, Lanke KH, Verhaegh GW, Melchers WJG, Swanink CMA, Bleijenberg G, Netea MG, Galama JMD, van der Meer JWM. Prevalence of xenotropic murine leukaemia virus-related virus in patients with chronic fatigue syndrome in the Netherlands: retrospective analysis of samples from an established cohort. BMJ 340, c1018 (2010)
  10. Groom HCT, Boucherit VC, Makinson K, Randal E, Baptista S, Hagan S, Gow GW, Mattes FM, Breuer J, Kerr JR, Stoye JP, Bishop KN. Absence of xenotropic murine leukaemia virus-related virus in UK patients with chronic fatigue syndrome. Retrovirology. 2010, 7:10 doi:10.1186/1742-4690-7-10.
  11. Erlwein O, Kaye S, McClure MO, Weber J, Wills G, Collier D, Wessely S, Cleare. Failure to detect the novel retrovirus XMRV in chronic fatigue syndrome. PLoS ONE. 2010, 5 (1):e8519.
  12. Furuta, RA, Miyazawa T., Sugiyama, T., Kimura, T, Hirayama, F et al. The prevalence of Xenotropic Murine Leukemia Virus-related virus in Healthy Blood Donors in Japan. Presented at the Cold Spring Harbor Retrovirus Symposium. 18-23 May 2009
  13. Switzer W, Jia H, Hohn O, Zheng H, Tang S, Shankar A, Bannert N, Simmons G, Hendry M, Falkenberg VR, Reeves WC, Heneine W. Absence of evidence of xenotropic murine leukemia virus-related virus infection in persons with chronic fatigue syndrome and healthy controls in the United States. Retrovirology. 2010; in press. http://www.retrovirology.com/content/7/1/57
  14. Fischer N, Schulz C, Stieler K, Hohn O, Lange C, Drosten C, Aepfelbacher M. Xenotropic murine leukemia virus-related gammaretrovirus in respiratory tract. Emerg Infect Dis. 2010; DOI: 10.3201/eid 1606.100066

Appendix I:

Table. Updates of Prevalence Studies on XMRV Infection in Human Diseases and General Populations

Study

Samples

Healthy subjects/controls XMRV positive 

Assay

Prostate cancer

Urisman A, et al. Identification of a Novel
Gammaretrovirus in Prostate Tumors of Patients
Homozygous for R462Q RNASEL Variant. PLOS. 2006;
2.

PROSTATE

8 of 20 (40%) RNASEL
R462Q- homozygous
cases
0 of 14 heterozygous
1 of 52 homozygous wild-type (familial PCa)

US study

 

Tissue

Nested RT-PCR

Fischer N, et al. Prevalence of human gammaretrovirus XMRV in sporadic prostate cancer. J Clin Virol. 2008; 43.

PROSTATE

1 of 105 (0.95%) positive
(non-familial PCa)

Germany study

1 of 70 (1.42%)
positive
(healthy prostate tissue)

Tissue, RNA
Nested RT-PCR

Hohn O, et al. Lack of evidence for xenotropic murine leukemia virus-related virus (XMRV) in German prostate cancer patients. Retrovirol. 2009; 6(92).

PROSTATE

0 of 589 DNA positive
0 of 146 Ab positive

Germany study

0 of 5 (Ab positive)

Tissue, DNA/RNA
Nested RT-PCR
ELISA

D'Arcy F, Foley R, Perry A, Marignol L, Lawler M, Gaffney E, Watson R, Fitzpatrick J, Lynch T: No evidence of XMRV in Irish prostate cancer patients with the R462Q mutation.European Urology supplements 2008, 7.

PROSTATE

0/139 by PCR

Irish study

 

PCR

Schlaberg R, et al. XMRV is present in malignant prostatic epithelium and is associated with prostate cancer, especially high-grade tumors. PNAS. 2009; 106 (38).

PROSTATE

14 of 233 (6.2%) PCR
54 of 233 (23%) IHC stain

US study

2 of 101 (2%) PCR
4 of 101 (4%) IHC stain

Tissue, DNA
Quantitative PCR
XMRV-specific stain

Switzer et al CDC
Paper #149, Abstract 17th CROI meeting, 2010
http://www.retroconference.org/2010/Abstracts/37160.htm

PROSTATE

2 of 162 (1.2%) tissues positive by PCR
0 of 162 plasma pos PCR
0 of 162 plasma pos WB

US study

All PCR negative tissues were neg (plasma) by PCR and WB

0 of 120 blood donors by WB

0 of 40 PBMC by PCR

Tissue, plasma

Nested PCR

Western Blot

Arnold RS, et al: XMRV infection in patients with prostate cancer: novel serologic assay and correlation with PCR and FISH. Urology, 75:755-761.

PROSTATE cancer

11 of 40 (27.5%) sera positive for neutralizing anti-XMRV antibodies.
8 of 20 (40%) RNASEL QQ
3 of 20 (15%) RNASEL RQ or RR

Consistent with PCR and FISH results

US study

 

Serological assay (Neutralizing antibodies)

Chronic fatigue syndrome (CFS)

Lombardi VC, et al. Detection of an Infectious Retrovirus, XMRV, in Blood Cells of Patients with Chronic Fatigue Syndrome. Science express. Oct 2009.

CFS

68 of 101 (67%) positive

US study.

8 of 218 (3.7%) positive

OR = 54.1(95%CI: 23.8-122)

PBMCs, DNA

Nested PCR

WPI

Erlwein O, et al. Failure to Detect the Novel Retrovirus XMRV in Chronic Fatigue Syndrome. PLoS ONE. Jan 2010; 5(1).

CFS

All negative.

UK study

186 samples.

DNA for XMRV and MLV. Nested PCR. Assay controls used.

Groom HC, et al. Absence of xenotropic murine leukaemia virus-related virus in UK patients with chronic fatigue syndrome. Retrovirology. 2010; 7(10).

CFS

All 170 CFS patients are negative by PCR.

1/28 (3.6%) CFS serum showed XMRV neutralizing activity.

UK study.

All 395 control samples are negative by PCR.

25/395 (6.3%) positive for neutralizing assay.

Strong cross reactive with VSV.

PBMCs. XMRV DNA. PCR (not nested) or real time PCR

Viral neutralization assay for XMRV and MLV.

Van Kuppeveld FJM, et al. Prevalence of xenotropic murine leukaemia virus-related virus in patients with chronic fatigue syndrome in the Netherlands: retrospective analysis of samples from an established cohort. BMJ. 2010; 340: c1018.

CFS

32 CFS pts.

All negative.

A matched case-control study

Dutch study.

43 controls. Retrospective.

PBMCs. DNA.

Nested PCR of gag gene.

Real time PCR of integrase gene

Switzer W, Jia H, Hohn O, Zheng H, Tang S, Shankar A, Bannert N, Simmons G, Hendry M, Falkenberg VR, Reeves WC, Heneine W. absence of evidence of xenotropic murine leukemia virus-related virus infection in persons with chronic fatigue syndrome and healthy controls in the United States. Retrovirology. 2010; in press

http://www.retrovirology.com/content/7/1/57

CFS, blood donors and general population

51 CFS cases,

All negative

US study

53 healthy controls

41 U.S. blood donors

All negative

Nested PCR, WB, ELISA, IFA

Tested in three labs

CDC study

Blood donors and general populations

Japanese Study; Furuta et al, Cold Spring Harbor symposium, 2009

Blood donors

In Japan

1-3% Ab pos in blood donors

Serum, Western blot

Qui et al.

Paper #151, Abstract 17th CROI 2010.

http://www.retroconference.org/2010/Abstracts/39393.htm

Blood donors

In the U.S.A.

3/2851 blood donor plasma specimens were positive for XMRV (0.1%)

Architect platform EIA, p15E, p30 and gp 70.

OTHER

   

Fischer N, Schulz C, Stieler K, Hohn O, Lange C, Drosten C, Aepfelbacher M. Xenotropic murine leukemia virus-related gammaretrovirus in respiratory tract. Emerg Infect Dis. 2010; DOI: 10.3201/eid 1606.100066

2.3% (3/75) in travelers from Asia who had respiratory tract infections;

3.2% (1/31) in patients with chronic obstructive pulmonary disease;

- 9.9% (16/161) in immunosuppressed patients with severe respiratory tract infections;

3.2% (2/62) in the healthy control group.

Nested PCR