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Advisory Committees

December 14-15, 2010: Blood Products Advisory Committee Meeting Issue Summary

99th Meeting, December 14-15, 2010
Hilton Washington D.C./North
620 Perry Parkway, Gaithersburg, MD

Topic: Murine Leukemia Virus (MLV) related human retroviruses and blood safety

Issue: FDA is seeking the advice of the Blood Products Advisory Committee (BPAC) on the deferral of donors based on a diagnosis of Chronic Fatigue Syndrome (CFS) or Prostate Cancer (PC) and testing for the newly identified MLV-related human retroviruses (that include XMRV) associated with CFS and PC that may pose a concern for blood safety.

  1. Background
    Murine Leukemia Viruses (MLVs) are gamma retroviruses belonging to the Retroviridae family. Gamma retroviruses are widespread in vertebrates and in animals they cause a wide range of diseases including cancers, immunodeficiency, and neurological disorders. Xenotropic murine leukemia virus-related virus (XMRV), the first gamma retrovirus known to infect humans, was originally identified in 2006 in prostatic tissue from patients with familial prostate cancer [1]. XMRV is a simple xenotropic MLV-related human retrovirus whose genome encodes gag, pro, pol, and env genes [2]. XMRV is unrelated to HIV and unlike Human Immunodeficiency Virus (HIV) or the Human T-lymphotropic Virus (HTLV), accessory or regulatory genes have not been identified to date[1]. XMRV is highly similar to mouse retroviruses, in particular Moloney Murine Leukemia Virus (MoMLV)[1, 3]. Virions have a complex structure and consist of an envelope, a nucleocapsid, and a nucleoid. Virions are spherical to pleomorphic measuring 90-100 nm in diameter[4] or 137 nm[3]. The viral genome is a dimer of linear, positive sense, single-stranded RNA with approximately 8100 nucleotides [1, 4]. Although early sequence data indicated that the genetic sequence of XMRV is highly conserved, recent studies have reported genetic variation in XMRV sequences[5]. Sequences of a new polytropic MLV-related virus which are clearly different from the XMRV strains previously reported were found in CFS samples[6], suggesting that there may be several MLV-related viruses in addition to XMRV. Therefore, the term MLV-related human retroviruses is used to include both xenotropic and polytropic viruses
  2. MLV-related viruses: Epidemiology and Disease Association
    1. Prostate Cancer
      Since the discovery of MLV-related human retroviruses, there have been several reports about their association with Prostate Cancer (PC) and Chronic Fatigue Syndrome (CFS) and other reports of a lack of association with these diseases. Using the viral sequence based array, XMRV sequences were identified in samples from 7/11 prostate cancer patients that were homozygous (QQ) for the R462 mutation for RNAse L which is an important molecule in the innate antiviral response [7]. 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 suggested an association of XMRV with prostate cancer [3].
      However, there are also studies which either did not find or found in low frequency MLV-related human retrovirus sequences in PC patients. Studies of an Irish PC cohort of 139 patients with the R462Q mutation showed no evidence of XMRV [8]. 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 [9]. 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 [10]. In a recent Dutch study, XMRV was found in 3 out of 74 (4%) prostate cancer patients[11].
      At the 1st International Workshop on XMRV held in September, 2010, several new findings were reported on the association of MLV-related human retroviruses with PC and they are presented below. A study of 3 separate PC tissues from 3 separate PC patients showed that all specimens contained integrated proviral sequences of MLV/XMRV [12]. A second study evaluated PC in 355 tissues, 40 PBMC from PC patients and 70 healthy controls in Northern Europe. Of the 355 PC tissues tested, 2 were positive for XMRV, one belonging to wild type and the other, heterozygous for R462Q[13]. PBMC from the two positive patients were found to be negative for XMRV sequences by PCR. In a separate study, prostatic tissue from 144 PC patients from the southern US were tested by nested env PCR on proviral DNA and for 55 of these patients, both tumor and normal prostate tissue specimens were available for testing. The findings were that 32/144 (22%) were positive for XMRV which was also detected in normal tissue. Positive findings were independent of R462Q in this patient population[14]. Finally, in a study of 161 PC patients, 452 prostate tissues with benign pathologies and 596 prostate tumor tissue specimens were tested and no XMRV was detected in any of the samples [15].
    2. Chronic Fatigue Syndrome (CFS)
      MLV-related human retroviruses have also been reported by a few laboratories in the blood of patients with Chronic Fatigue Syndrome (CFS), a disease of unknown etiology, known to affect several million people worldwide [4]. Other laboratories have failed to detect XMRV in blood samples from CFS patients. CFS is a clinically defined condition characterized by severe, debilitating fatigue and a combination of symptoms that prominently features self-reported impairments in concentration and short-term memory, sleep disturbances and muscle and joint pain and symptoms similar to those of infectious diseases [16]. A number of viruses, including human herpes virus-6 (HHV-6), Epstein-Barr virus (EBV), various enteroviruses, and the human T-lymphotropic virus type 2 (HTLV-2), have been cited as possible triggers of CFS; but, so far, no causative infectious agent has been conclusively identified [17]. Clinical diagnosis of CFS is largely a diagnosis of exclusion of other diseases.
      In a study reported in 2009 [4], DNA from peripheral blood mononuclear cells (PBMCs) of CFS patients and healthy controls from the US were tested for the presence of XMRV sequences. XMRV sequences were found in 68 out of 101 CFS patients (67%), as compared to 8 of 218 (3.7%) healthy US controls. 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 in LNCaP cells from 10/12 CFS patient plasma samples. 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).
      In August 2010, a second study provided further evidence for the presence of MLV-related human retroviral sequences in PBMC of CFS patients[6]. The study involved testing 41 PBMC samples from 37 CFS patients using gag primers; 32/37 (86.5%) patients were found to be positive for polytropic MLV-related viral sequences. The corresponding plasma samples from CFS patients showed evidence for MLV-related virus gag gene sequences in 42% when tested by RT-PCR. PBMC from 7/8 patients for whom a follow up sample obtained 15 years later showed presence of MLV related human retrovirus sequences by PCR[6]. Furthermore, MLV related sequences were also detected in PBMC of 3/44 (6.8%) of healthy blood donors. In contrast to the previously reported findings of close genetic relatedness of all XMRVs in patients with CFS and in PC from different geographic regions the amplified gag gene sequences revealed a genetically diverse group of MLV-related viruses. Phylogenetic analysis of 21 CFS patient sequences revealed 3 different MLV-related retroviral gag gene sequences and a fourth variant was identified in 1/3 blood donor samples all of them forming a cluster distinct from XMRV. The viral gag gene sequences were phylogenetically more closely related to polytropic or modified polytropic endogenous retroviruses. There was no evidence of mouse DNA contamination based on a mitochondrial DNA assay. These findings suggest the presence of a heterogeneous group of MLV-related human retroviruses in the blood of CFS patients and to a lesser extent in healthy blood donors. The sequences detected were polytropic MLV rather than XMRV.
      At the 1st International Workshop on XMRV held in September, 2010 several conflicting findings were reported on detection of MLV related viruses in the blood of CFS patients. In one study, PBMCs from 184 CFS and 25 controls were tested using Taqman PCR and nested PCR assays for XMRV gag, and conventional PCR for mouse DNA. Positive results were obtained using 1 of 2 nested gag PCR assays and sequences were identical to those observed in PC and CFS and closely related to endogenous MLVs. However, all samples that were positive for XMRV were also positive for mouse DNA indicating the need to test for mouse DNA sequences to control for contamination[18]. In a second study, PBMCs and plasma from 30 adults including 10 severe, 10 recovered and 10 healthy controls were tested using PCR for the gag gene and culture of virus from plasma using the LNCaP cells permissive for XMRV replication. MLV related sequences were detected in 8 severe and 3 recovered cases and 1 healthy control. In addition virus could be cultured from 6 specimens of which 5 were from CFS cases and 1 from the control. The sequences identified in this study were all polytropic MLV/XMRV[19]. In a third study of UK patients, plasma from 50 CFS patients were tested in a US laboratory by virus culture and PBMC DNA by nested PCR using primers from the gag and env region. Other methods used included Western blot, immuno-histochemistry and serology. Sixty percent of cases were positive by culture using co-cultivation with LNCaP cells. Furthermore, env sequences were detected in PBMC of 7 CFS patients before co-culture. Sequence analysis showed that they were identical to XMRV sequences previously reported. Plasma was positive for antibodies in all cases where virus was isolated[20].
      Despite these positive findings of MLV related human retroviruses in CFS patients, several other studies have reported negative findings in patients from the Netherlands[20], UK[22, 23] and China[24]. Furthermore, studies conducted in a US population of CFS cases and healthy blood donors showed no evidence of MLV related viruses in PBMC and plasma[25, 26]. Studies by other groups presented at the 1st International XMRV Workshop showed negative findings of MLV related viruses in CFS patients and healthy controls. A German study reported absence of XMRV antibodies in 36 CFS patients and 17 healthy controls using gag and env based ELISA. In addition, 50 sera from patients with multiple sclerosis (MS) were tested for serological evidence of XMRV infection and found to be negative. Furthermore, nested PCR on DNA from stimulated PBMC of 39 CFS and 50 MS patients, and 30 healthy controls gave negative results. Consistent with the PCR results, no reverse transcriptase activity was detectable in supernatants of cultured PBMC 7 days post-infection and no infectious virus could be detected by co-culture with susceptible LNCaP cells [27]. Finally, in a study of PBMC and plasma samples from 50 patients with myalgic encephalitis, 400 PC patients and 200 blood donors in Sweden by PCR, co-culture and serology methods, none were found to be positive for MLV related human retroviral sequences[28].
      The reasons for the discrepancies between findings of various epidemiologic studies of MLV related human retroviruses are not clear at the present time. Differences in cohorts, populations, sample preparation, and test methods have been considered as potential causes [29]. In addition, differences in geographic prevalence, genetic variation of the virus or other factors could also potentially contribute to these findings [2]. Additional studies are needed to confirm the presence of MLV-related human retroviruses in CFS and to fully understand the possible role of these retroviruses in disease causation. Finally, although XMRV and MLV sequences have been identified in CFS and prostate cancer patients in some studies, a causal relationship with disease has not yet been established.
    3. Other Epidemiologic Findings
      Among other epidemiologic findings are those recently reported by investigators in Germany that MLV related human retroviruses could be detected in respiratory secretions [30]. In this study, 267 respiratory samples from patients with various respiratory conditions and related illnesses were screened for XMRV infection using PCR methods. The prevalence of XMRV infection was found to be 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. In general, retroviruses are rarely transmitted through respiratory secretions.
  3. Discussion
    1. Evaluation of Transfusion Risk
      Transmission through blood transfusion of XMRV has not been shown, but is theoretically possible since XMRV virus has been detected in blood cells and there is evidence of cell-free virus. Recent studies using activated patient PBMC co-cultured with susceptible PC cells (LNCaP) showed that virus could be transmitted by both cells and tissue culture supernatant, suggesting that the virus was infectious [4]. Virus could also be cultured and isolated from CFS patient plasma, suggesting that both cell-associated and cell-free transmission of MLV-related human retroviruses is possible [4]. A preliminary animal study of XMRV performed in rhesus macaques showed disseminated infection with low but detectable transient viremia between 4-14 days. Seroconversion occurred at 11-14 days following inoculation, with titers peaking around day 95. Virus could be 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[31]. A seroprevalence study in Japan found 1.7% of random donors to be positive [32]. 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[31]. Additional prospective and retrospective studies to evaluate prevalence of MLV related human retroviruses in blood donors are ongoing and results will be discussed at the BPAC meeting.
    2. Donor Deferral for risk of MLV-related infection in patients with a diagnosis of CFS and PC
      Although as yet there is no demonstration of MLV-related sequence transmission through transfusion, as a precautionary measure, blood establishments in different countries are deferring individuals with diagnosed CFS. Since November 2009 the United Kingdom Blood Services has deferred individuals suffering from CFS from blood donation until the condition is resolved and they are feeling completely well. On April 23, 2010, Australia's Red Cross Blood Service recommended that donors who have been diagnosed with CFS will be indefinitely deferred and the decision reviewed in two years time to allow for further studies of XMRV. Since April, 2010, Canadian Blood Services has indefinitely deferred donors with a medical history of CFS. The New Zealand Blood Service (NZBS) is adopting a similar approach to that developed by the Canadian Blood Services in deferring donors with a history of CFS, also known as myalgic encephalitis (ME). In New Zealand, the present exclusion as blood donors of people still suffering CFS or who had been diagnosed in the past two years would be extended to also exclude donors who report ever having been diagnosed with CFS. The deferral policies in all of these countries involve donors volunteering CFS information; there is no specific donor question on the screening questionnaire.
      In the US, in June 2010, AABB (formerly known as the American Association of Blood Banks) published an Association Bulletin (10-03) which recommends as an interim measure that “blood collecting organizations make educational information available regarding the reasons why an individual diagnosed with CFS should not donate blood or blood components.” [33]. The AABB also developed donor education materials suitable for discouraging such donations. The AABB does not recommend adding a specific question to the donor history questionnaire. The Chronic Fatigue and Immune Dysfunction Syndrome (CFIDS) Association of America has a long-standing recommendation that individuals with CFS voluntarily not donate blood or organs based on both donor and recipient safety concerns which include low blood volume and infections common in CFS [34]. In May 2010, the HHS Chronic Fatigue Syndrome Advisory Committee (CFSAC) recommended to the Assistant Secretary for Health that blood collection facilities indefinitely defer donors with a history of CFS, or with active CFS, through a donor screening question specifically for CFS.
      Currently, FDA does not have a policy for deferring donors based on a history of cancer and there is no scientific evidence that cancer can be transmitted through blood transfusions [35]. In regard to PC, results of a large survey conducted in Sweden and Denmark (SCANDAT) of 888,843 blood transfusion recipients without a prior cancer diagnosis at the index transfusion showed no increase in prostate cancer [36]. Furthermore, statistically significant decreased risk was observed in anatomic site-specific analysis for prostate cancer 2-4 years after transfusion. For these reasons, donor deferral on the basis of a history of PC has not been recommended in the US or other countries except in Canada, where since 2003, a policy of permanent deferral for prostate cancer patients has been implemented and prior donations are retrieved and destroyed.
    3. Detection methods
      Currently there are no commercially approved/licensed tests for donor screening or for the detection of infection with MLV related human retroviruses. Research assays to detect XMRV include PCR, EIA, Western blot, immuno-histochemistry and virus culture. Purified recombinant antigens have been used to develop serology assays and preliminary data indicate that the gp70 antigen provided higher sensitivity than p15E or p30. Additionally, a virus infectivity assay based on green fluorescent protein detection in an indicator cell line has been developed to enable early and sensitive virus detection in samples, and determine infectivity and inactivation of virus. The relative sensitivity and specificity of various assays have not been determined and standards for their performance have not yet been established.
      A Scientific Working Group, led by the National Heart, Lung, and Blood Institute (NHLBI), was established to design and coordinate research studies to evaluate whether XMRV poses a threat to blood safety. As a first step towards this goal, an analytical reference panel was developed to validate the analytical sensitivity of assays used by different laboratories. Three additional phases are in progress or being planned to evaluate sample processing questions, clinical sensitivity and specificity, and obtain a preliminary estimate of the prevalence of XMRV in blood donors. The study is being sponsored by the NHLBI through the Retrovirus Epidemiology Donor Study-II (REDS-II) program with coordination by Blood Systems Research Institute, the REDS-II central laboratory. A coded analytical reference panel of plasma specimens containing varying concentrations of a reference culture supernatant from the chronically infected prostate cancer cell line 22Rv1 was tested in six participating laboratories to compare the analytical sensitivity of their respective assays for XMRV. Similarly, reference infected cell preparations were established by spiking known numbers of infected cells into whole blood and panels created for detection of XMRV DNA in these specimens. The study participants in this first phase included laboratories from the NCI, FDA (two laboratories), CDC and the Whittemore Peterson Institute. All assays detected at least 136 copies/ml of XMRV DNA in whole blood. Four out of five plasma RNA assays performed similarly, with limits of detection of at least 80 RNA copies/ml. Phase II which is being conducted to evaluate sample processing effects is currently underway.
      An appendix of studies that have published or reported thus far on the association of MLV/XMRV in prostate cancer, CFS and in the healthy population is attached for reference.


The science of MLV related viruses in humans is still evolving. While many laboratories have detected MLV-related sequences in prostate cancer samples, others have not. The frequency of positive samples varies widely between laboratories. There is controversy about the detection of MLV-related sequences in CFS with the majority of laboratories failing to detect MLV sequences. There are ongoing studies to determine whether technical issues, such as extraction methods or primers and/or subject selection including clinical criteria and geographic issues might influence the detection of MLV sequences. Potential transmission through blood has not been demonstrated. There is no known causative relationship between MLV infection in humans and disease.

The BPAC session will include several presentations on MLV-related human retroviruses. The committee will hear presentations on the background of the issues to be discussed, an update of current research on epidemiology, disease association, animal transmission studies, ongoing validation studies of assays and panels for MLV related human retroviruses, and other relevant studies on the association of MLV-related viruses with CFS, prostate cancer and blood donors.

Based on the current scientific knowledge of MLV-related viruses in humans and their reported disease association, FDA is seeking recommendations from the BPAC on 1) indefinite deferral for blood and plasma donors with a diagnosis of CFS or PC, and 2) donor screening for MLV-related virus in absence of confirmed disease causation. FDA is also seeking the advice of the BPAC on the potential suitability of NAT and/or serology testing of blood donations as safeguards against transmission of MLV related viruses by blood transfusion.

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.
  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.


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