Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3164-3166
CONTROVERSIES IN HEMATOLOGY
Rebuttal to Berenson and Vescio
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ARTICLE |
REPRODUCIBILITY is a requirement in establishing
whether a scientific finding is valid. Not only must results be
reproducible, but they must be routinely reproducible in multiple
laboratories. The field of KSHV research in its short history has a
number of examples of startling reports of disease associations,
including sarcoidosis, posttransplant skin tumors, angiosarcoma, and
T-cell lymphomas that have not been reproducible.1 The
dispute over whether KSHV plays a role in multiple myeloma involves the
reproducibility of the initial findings based on Rettig et
al.2
Aside from Berenson's group, three other groups have reported results
that could be consistent with the hypothesis that KSHV is causally
associated with multiple myeloma.3-5 Brousset et al3 have been cited as a group that was able to confirm the association in their French series. Although Berenson has asserted that
KSHV/HHV8 cannot be detected by PCR on fresh bone marrow biopsies,
nevertheless, Brousset et al3 were able to detect KSHV/HHV8
KS330233 from 18 of 20 acetone-fixed and paraffin-embedded bone marrow biopsies. Agbaliki et al5 were also able to
detect KSHV from 5 of 10 paraffin-embedded bone marrows from patients with MM. In contrast, many other groups have not been able to detect
KSHV/HHV8 even in fresh bone marrow cores or aspirates.6-12
The debate then shifts to the type of cell that is permissive for
KSHV/HHV8 infection in the bone marrow. Apparently, the virus is only
readily detectable after long-term culture in a subpopulation of cells
of "unknown phenotype."13 Several laboratories have
tried culturing stromal cells, some using the protocol of Rettig et al,
to amplify the virus-carrying cell type and have failed to detect
KSHV/HHV8.6,8,11,14-16 Contrary to Berenson et al's
rebuttal, the sensitivity of the PCR assays used in some of these
studies was quantified and was found to be extremely sensitive, in the
case of KS330233, detecting a few ORF 26 copies of
105 cells.6,7,14 Berenson et al mention that
the presence of viral DNA was "confirmed" using primers to
various KSHV ORFs, referring to their unpublished or in press reports.
Furthermore, the original basis for suggesting a connection between
KSHV/HHV8 and multiple myeloma was the possibility that the
virus-encoded vIL-6 protein acts as a paracrine factor for myeloma
cells.2 However, even assuming that KSHV/HHV8 is present,
no KSHV vIL-6 protein is detectable by immunohistochemistry in any
multiple myeloma lesions,6 eliminating the theoretical
underpinning for this hypothesis.
Berenson et al suggest that KSHV in situ hybridization studies confirm
their PCR-based observations.2,17 These studies purport to
show a majority of cultured cells infected with KSHV. Furthermore, the
virus must be present in multiple myeloma tissues at greater than
homeopathic amounts to drive tumor cell replication through a paracrine
mechanism. When only one virus copy is present per cell (the level of
infection for mixed tumor and nontumor KS lesions), virus is readily
detectable by Southern hybridization and does not require PCR
amplification for detection.18 If the strong in situ
hybridization signal is truly reflective of the degree of infection by
KSHV, then any laboratory should be able to detect virus by PCR or even
Southern hybridization. A 
phage library could be easily made from
the degree of infection reported by Berenson in in situ hybridization
studies. Have larger portions of the viral genome been pulled out from
such libraries?
With respect to serologic testing, only Gao et al4 have
found any significant positivity for KSHV/HHV8-specific antibodies in
multiple myeloma patients. Careful examination of this report shows
that the immunofluorescent assay (IFA) detecting LANA (ORF73) antigen
is positive in only 3 of 27 (11%) of multiple myeloma patients,
whereas this group found 14 of 27 (52%) positive by LANA
immunoblotting. Examination of the "LANA" immunoblot bands in Fig
1 of this report is not convincing for true seropositivity. Additionally, they also found the sera of control cancer patients to
have a prevalence of KSHV/HHV8 antibodies higher than expected for the
general population. In contrast, many other groups familiar with these
seroassays have not been able to show an association between KSHV/HHV8
and multiple myeloma.6-9,12,16,19-21 Berenson et al make a
point that there are basepair changes in the sequence of ORF 65 in
multiple myeloma patients that would result in a negative or
insensitive ORF 65.2 Western seroassay. It should be stressed that the
indirect IFA frequently used in KSHV serologic testing detects ORF73
not ORF65, so basepair changes in ORF65 should not affect the results
using IFA.
In summary, neither viral DNA nor antibodies can be reproducibly found
in multiple myeloma patients. There now exists a large enough body of
data from multiple laboratories that do not support the findings of
Rettig et al,6-12,14-16,19-24 and the known epidemiology of
KSHV does not make sense with the known epidemiology of multiple myeloma.25 KSHV research is young, and several very
competent laboratories have been misled by technical artifacts that are part of the natural growing pains for any new field. Although we would
be delighted if Berenson et al's results implicating KSHV in the
pathogenesis of multiple myeloma were correct, however, this does not
appear to be the case.
| |
REFERENCES |
1.
Moore PS:
Human herpesvirus 8 variants.
Lancet
351:679, 1998[Medline]
[Order article via Infotrieve]
2.
Rettig MB, Ma HJ, Vescio RA, Pold M, Schiller G, Belson D, Savage A, Nishikubo C, Wu C, Fraser J, Said JW, Berenson JR:
Kaposi's sarcoma-associated herpesvirus infection of bone marrow dendritic cells from multiple myeloma patients.
Science
276:1851, 1997[Abstract/Free Full Text]
3.
Brousset P, Meggetto F, Attal M, Delsol G:
Kaposi's sarcoma-associated herpesvirus infection and multiple myeloma.
Science
278:1972, 1997
4.
Gao SJ, Alsina M, Deng JH, Harrison CR, Montalvo EA, Leach CT, Roodman GD, Jenson HB:
Antibodies to Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) in patients with multiple myeloma.
J Infect Dis
178:846, 1998[Medline]
[Order article via Infotrieve]
5.
Agbalika F, Mariette X, Marolleau J-P, Fermand J-P, Brouet J-C:
Detection of human herpesvirus-8 DNA in bone marrow biopsies from patients with multiple myeloma and Waldenström's macroglobulineamia.
Blood
91:4393, 1998[Free Full Text]
6.
Olsen SJ, Tarte K, Sherman W, Hale EE, Weisse MT, Orazi A, Klein B, Chang Y:
Evidence against KSHV infection in the pathogenesis of multiple myeloma.
Virus Res
57:197, 1998[Medline]
[Order article via Infotrieve]
7.
Parravicini C, Lauri E, Baldini L, Neri A, Poli F, Sirchia G, Moroni M, Galli M, Corbellino M:
Kaposi's sarcoma-associated herpesvirus and multiple myeloma.
Science
278:1969, 1997[Free Full Text]
8.
Masood R, Zheng T, Tulpule A, Arora N, Chatlynne L, Handy M, Whitman JJ, Kaplan M, Dosik M, Ablashi D, Gill P:
Kaposi's sarcoma-associated herpesvirus and multiple myeloma.
Science
278:1970, 1997
9.
Cathomas G, Stalder A, Kurrer MO, Regamey N, Erb P, Joller-Jemelka H:
Multiple myeloma and HHV8 infection.
Blood
91:4391, 1998[Free Full Text]
10.
Perna AM, Viviano E, Iannitto E, Marceno R, Romano N:
No association between human herpesvirus type 8 infection and multiple myeloma.
J Natl Cancer Inst
90:1013, 1998[Free Full Text]
11.
Yi Q, Ekman M, Anton D, Bergenbrant S, Osterborg A, Georgii HP, Holm G, Nilsson K, Biberfeld P:
Blood dendritic cells from myeloma patients are not infected with Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8).
Blood
92:402, 1998[Abstract/Free Full Text]
12.
Schonrich G, Raftery M, Schnitzler P, Rohr U, Goldschmidt H:
Absence of a correlation between Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) and multiple myeloma.
Blood
92:3474, 1998[Free Full Text]
13.
Berenson JR, Vescio RA:
HHV-8 is present in multiple myeloma patients.
Blood
93:3157, 1999[Free Full Text]
14.
Tarte K, Olsen SJ, Yang LZ, Legouffe E, Rossi JF, Chang Y, Klein B:
Clinical-grade functional dendritic cells from patients with multiple myeloma are not infected with Kaposi's sarcoma-associated herpesvirus.
Blood
91:1852, 1998[Abstract/Free Full Text]
15.
Cull GM, Timms JM, Haynes AP, Russell NH, Irving WL, Ball JK, Thomson BJ:
Dendritic cells cultured from mononuclear cells and CD34 cells in myeloma do not harbour human herpesvirus 8.
Br J Haematol
100:793, 1998[Medline]
[Order article via Infotrieve]
16.
Mitterer M, Mair W, Gatti D, Sheldon J, Vachula M, Coser P, Schultz TF:
Dendritic cells derived from bone marrow and CD34+ selected blood progenitor cells of myeloma patients, cultured in serum-free media, do not contain the Kaposi sarcoma herpesvirus genome.
Br J Haematol
102:1338, 1998[Medline]
[Order article via Infotrieve]
17.
Said JW, Rettig MR, Heppner K, Vescio RA, Schiller G, Belson D, Savage A, Nishikubo C, Wu C, Fraser J, Said JW, Berenson JR:
Localization of Kaposi's sarcoma-associated herpesvirus in bone marrow biopsy samples from patients with multiple myeloma.
Blood
90:4278, 1997[Abstract/Free Full Text]
18.
Chang Y, Cesarman E, Pessin MS, Lee F, Culpepper J, Knowles DM, Moore PS:
Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma.
Science
265:1865, 1994
19.
Whitby D, Boshoff C, Luppi M, Torelli G:
Kaposi's sarcoma-associated herpesvirus and multiple myeloma.
Science
278:1871, 1997
20.
Santarelli R, Angeloni A, Farina A, Gonnella R, Gentile G, Martino P, Petrucci MT, Mandelli F, Frati L, Faggioni A:
Lack of serologic association between human herpesvirus-8 infection and multiple myeloma and monoclonal gammopathies of undetermined significance.
J Natl Cancer Inst
90:781, 1998[Free Full Text]
21.
Marcelin A-G, Dupin N, Bouscary D, Bossi P, Cacoub P, Ravaud P, Calvez V:
HHV-8 and multiple myeloma in France.
Lancet
350:1144, 1997[Medline]
[Order article via Infotrieve]
22.
MacKenzie J, Sheldon J, Morgan G, Cook G, Schultz TF, Jarrett RF:
HHV-8 and multiple myeloma in the UK.
Lancet
350:1144, 1997
23.
Bouscary D, Dupin N, Fichelson S, Grandadam M, Fontenay-Roupie M, Marcelin AG, Blanche P, Picard F, Freyssinier JM, Ravaud P, Dreyfus F, Calvez V:
Lack of evidence of an association between HHV-8 and multiple myeloma.
Leukemia
12:1840, 1998[Medline]
[Order article via Infotrieve]
24.
Tarte K, Olsen SJ, Rossi JF, Legouffe E, Lu ZY, Jourdan M, Chang Y, Klein B:
Kaposi's sarcoma-associated herpesvirus is not detected with immunosuppression in multiple myeloma.
Blood
92:2186, 1998[Free Full Text]
25.
Hjalgrim H, Frisch M, Melbye M:
Incidence rates of classical Kaposi's sarcoma and multiple myeloma do not correlate.
Br J Cancer
78:419, 1998[Medline]
[Order article via Infotrieve]
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