To evaluate the proposed link between KSHV (also known as human herpes virus-8, or HHV-8) and MM without the limitations of a purely PCR-based experimental design, we studied a series of 40 Italian patients with MM by combining a nested PCR method that achieves single copy sensitivity (2) with serologic assays for antibodies against KSHV. For each case, 1 µg of DNA extracted from freshly purified bone marrow mononuclear cells (10 times the amount used by Rettig et al.) was tested in triplicate by nested PCR. Serum samples obtained at diagnosis (that is before therapy began) were available for half (20) of the patients and were tested by protein immunoblot (3) and immunoperoxydase (4) assays for the presence of KSHV-specific antibodies against orf65 lytic and latency-associated nuclear antigens, respectively. In addition, all sera were screened for antibodies against cytomegalovirus (HCMV) and hepatitis B (HBV) (5). We performed these assays in order to exclude false negative results since, as Rettig et al. correctly observe (1), patients with MM often exhibit a profound inhibition of the humoral immune response (Table 1).

Table 1. Demographic and serologic features of 20 Italian patients with MM. Ab, antibody; Neg., negative; Pos., positive; ND, no data. Patient Sex Age Monoclonal component Decreased serum polyclonal Ig classes* KSHV+ LANA KSHV orf65 HBV Ab HCMV Ab 1012 F 67 G,k 2 Neg. Neg. Neg. Pos. 1013 F 74 G,k 2 Neg. Neg. Pos. Pos. 1014 M 57 A,k 2 Neg. Neg. Neg. Pos. 1015 F 71 A,k ND Neg. Neg. ND Pos. 1017 F 80 G, ND Neg. Neg. ND Pos. 1019 F 57 G, 1 Neg. Neg. Pos. Pos. 1020 M 58 G,k 0 Neg. Neg. Neg. Neg. 1021 F 54 A,k 0 Neg. Neg. Pos. Pos. 1101 M 64 G,k 1 Pos. Pos. Neg. Pos.  167 F 67 k 2 Neg. Neg. ND Pos. 1734 M 65 A,k 0 Neg. Neg. Neg. Pos. 1735 F 70 G, 1 Neg. Neg. Neg. Pos. 1739 F 63 A,k 0 Neg. Neg. Pos. Pos. 1740 F 67 A, 0 Neg. Neg. Neg. Pos. 1745 F 79 A, 2 Neg. Neg. ND Pos. 1746 M 45 G,k 2 Neg. Neg. Neg. Pos. 1748 M 68 A,k 2 Neg. Neg. Neg. Pos. 1749 F 45 G,k 0 Neg. Neg. Pos. Pos. 1750 M 67 G,k ND Neg. Neg. Pos. Neg. 1761 F 78 A,k ND Neg. Neg. Neg. Pos. Positive/total sample 1/20 1/20 6/16 19/20 (Percent positive) (5) (5) (38) (90) * Serum polyclonal Ig classes were evaluated by nephelometry (BNA, Behring, Dusseldorf, Germany); 0, no Ig reduction; 1, reduction of one serum polyclonal Ig class; 2, reduction of two serum polyclonal Ig classes.

Our results show that none of the 40 patients with MM had KSHV DNA sequences that were detectable in bone marrow aspirates and that only one out of 20 (5%) had antibodies against KSHV (Table 1). By contrast, antibodies to HCMV and HBV were detectable in 18 out of 20 (90%) and 6 out of 15 (38%) of the sera samples, and seroprevalence rates were similar to those found in earlier studies of the Italian general population (6). The latter findings make it unlikely that the low prevalence of KSHV antibodies in MM patients was a result of concomitant inhibition of the humoral response, unless one assumes that this inhibition selectively involved the production of antibodies against KSHV antigens. In fact, KSHV seroprevalence rates are similar in MM patients (5%) and in Italian blood donors of similar geographic origin (4%) (7). Therefore, our findings indicate that most MM patients are not infected by KSHV and argue against there being a link between KSHV infection and the development of MM.

Carlo Parravicini
Eleonora Lauri
Servizio di Anatomia Patalogica,
Luigi Sacco Hospital,
20157, Milano, Italy
Luca Baldini
Antonino Neri
Servizio di Ematologia,
Centro Giovanni Marcora,
Institute of Medical Sciences,
University of Milan,
Istituto di Ricerca e Cura a
Carattere Scientifico
(IRCCS) Ospedale Policlinico,
Milano, Italy
Francesca Poli
Girolamo Sirchia
North Italian Transplant,
Centro Trasfusionale e di Immunologia dei Trapianti,
IRCCS Ospedale Policlinico
Milano, Italy
Mauro Moroni
Massimo Galli
Mario Corbellino
Institute of Infectious Diseases,
University of Milan,
Luigi Sacco Hospital,
20147, Milano, Italy
E-mail: mcorbell{at}imiucca.csi.unimi.it

REFERENCES AND NOTES

1. M. B. Rettig, et al., Science 276, 1851 (1997) [Abstract/Free Full Text] .
2. Bone marrow mononuclear cells were obtained from bone marrow aspirates by Ficoll density centrifugation and their DNA extracted with the use of standard protocols [J. Sambrook et al., in Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, ed. 2, 1989)]. Thirty-cycle PCR using human -globin specific primers [ M. Bauer et al., J. Am. Med. Assoc. 256, 472 (1991) ] and 1 µg of DNA as template was performed in order to rule out major Taq polymerase inhibitors and to verify the integrity of the extracted DNA. Nested PCR (35+35 amplification cycles) was performed as described in detail elsewhere [ M. Corbellino et al., AIDS Res. Hum. Retrovir. 12, 651 (1996) [Web of Science] [Medline] ]. One microgram of DNA was used as template, and each sample was tested in triplicate individual PCR reactions. This assay achieves single-copy sensitivity, as assessed by titration experiments and signal distribution analysis [Z. Wang and J. Spadoro in 94th Gen. Meet. Am. Soc. Microbiol., abstr. D256 (1994), p. 141]. According to the Poisson distribution, when the sensitivity of an analytical system achieves a single copy and the solution is repeatedly tested in this system, the concentration of the PCR target [which is referred to as Signal Generating Unit (SGU), or the smallest unit that gives a PCR-positive signal] is given by the formula SGU/PCR = ln (no. of negative results/no. of replicates). Thus, if the hypothesis of Rettig et al. is correct (that all MM bone marrow aspirates contain KSHV-infected bone marrow dendritic stromal cells), given that we tested 40 MM specimens in triplicate (that is, a total of 120 replicates) and they were all negative, then the concentration of KSHV DNA sequences in bone marrow aspirates should be very low (for example, with 1/120 positive replicates the concentration of KSHV sequences is 1 KSHV-SGU per 17,924,896 cell equivalents).
3. orf65 KSHV recombinant protein was provided by Y. Chang and P. Moore (Columbia University, New York). Protein immunoblot assay was performed according to the method published by G. R. Simpson et al. [Lancet 348, 1133 (1996)].
4. Immunoperoxydase assay against KSHV latency associated nuclear antigens (LANA) was performed on acetone-fixed cytospins of KSHV-infected BCBL-1 cells with the use of sera diluted 1:100, 1:500, 1:2500, and 1:10,000. Reaction was revealed by a peroxydase labeled antiserum specific for human immuno-globulin (IgG) (Dako, Glostrupp, DK) diluted 1:100, followed by tyramide signal amplification (Du Pont/NEN, Boston, MA). KSHV-infected BCBL-1 cells were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, from M. McGrath and D. Ganem [ R. Rennie et al., Nature Med. 2, 342 (1996) [CrossRef] [Web of Science] [Medline] ]. Cells were cultured with RPMI-1640 (Gibco-BRL, Grand Island, NY). Patient sera and peroxydase-labeled rabbit antibody to human IgG were diluted in 0.05M TBS (tris-buffered saline, pH 7.2) containing 1% BSA (bovine serum albumin, Fraction V, Sigma Chemical, St. Louis, MO) and 0.001% Nonidet P-40 (Sigma Chemical, St. Louis, MO). BCBL-1 immunoperoxidase (IPA) is identical to previously reported immunofluorescence (IFA) assays [ S. J. Gao et al., Nature Med. 2, 925 (1996) [CrossRef] [Web of Science] [Medline] ; D. H. Kedeset al., ibid., p. 918] and represents a technical modification of this test. For both protein immunoblot and IPA tests, sera from patients with Mediterranean Kaposi's sarcoma and blood donors previously demonstrated to be KSHV seropositive and seronegative, respectively, were used as controls. IPA assay was effective in detecting latent KSHV infection in patients who developed Kaposi's sarcoma after receiving solid organ allograft [ C. Parravicini et al., Blood 90, 2826 (1997) [Abstract/Free Full Text] ].
5. Serological tests were performed for detecting IgG antibodies against cytomegalovirus, IMX System (Abbott Diagnostics, Science Park, IL) and antibodies against hepatitus virus B surface antigen, MEIA, AXSYM system (Abbott Diagnostics).
6. M. Moroni, D'Arminio-Monforte, in Manuale di Malattie Infettive., M. Moroni, R. Esposito, F. De Lalla, Eds. (Masson S.p.A., Milano, 1994), p. 669; P. Pasquini et al., Am. J. Epidemiol. 118, 699 (1983) [Abstract/Free Full Text] .
7. S. J. Gao et al., Nature Med. 2, 925 (1996) .
8. We thank L. Cro and E. Locatelli for technical assistance. Supported by Associazione Nazionale per la Lotta contro l'AIDS (ANLAIDS) Seizione Lombarda, Federazione Italiana per la Ricerca sul Cancro (FIRC), and Associazione Italiana per la Ricerca sul Cancro (AIRC).

KSHV is associated with Kaposi's sarcoma (KS), Castleman's disease, and a rare form of body cavity lymphoma (1-4). Rettig et al. (5) found KSHV (also known as HHV-8) in bone marrow stromal cells, but not malignant plasma cells, in all 15 patients with MM that they examined. Their results suggest that KSHV may be required for the transformation of plasma cells. However, their study was limited to examination of bone marrow stromal and mononuclear cells by PCR for KSHV and showed viral DNA only in the bone marrow stromal cells. Bone marrow cells before stromal cell isolation were found not to contain KSHV. Serology for KSHV was not given for these patients with MM (5).

We blindly analyzed serum samples from patients with MM or with epidemic (classic) KS, and from healthy adult donors for antibodies against  [KSHV-8 and Epstein-Barr virus (EBV)] and  (HHV-6 and HHV-7) herpesviruses. We used (i) an enzyme-linked immunosorbent assay (ELISA) to detect whole virus lysate (2 × 10⁹ viral particles per liter) that contained the majority of the viral structural proteins (1.5 mg of viral protein per milliliter) and (ii) an indirect immunofluorescence assay (IFA) to detect KSVH lytic antigens with the KS-1 cell line [which comes from human immunodeficiency virus (HIV)-and EBV-negative body cavity lymphoma] as a substrate (6). The IFA to detect HHV-6, HHV-7, and EBV (also called HHV-4) was performed as described previously (7). We also examined peripheral blood mononuclear cells (six cases) and bone marrow mononuclear cells (five cases), bone marrow stromal cells (five cases) from patients with MM, and two myeloma cell lines for KSHV DNA with the use of PCR. Bone marrow stromal cells were isolated and studied as described previously (5) and were also analyzed for the presence of KSHV by IFA, with the use of the polyclonal antibody against KSHV (6). We did not observe any decline in KSHV antibody when we tested the polyclonal reference serum (which came from a patient with classic KS) for absorption of EBV antibodies with the EBV-producing cell line P3HR1 (Advanced Biotechnologies, Inc., Columbia, Maryland).

Antibodies against EBV, HHV-6, and HHV-7 IgG were detected in the majority of the sera from MM patients (Table 1). However, antibodies against KSHV were detected in only 7% of MM sera samples, as compared with 8% of samples from healthy adult donors, and over 85% of samples from patients with acquired immunodeficiency virus (AIDS) and KS. Thus, these patients with MM were able to mount an immune response to highly prevalent lymphotropic human herpesviruses (EBV, HHV-6, HHV-7). The low seropositivity to KSHV in MM is similar to that of the general population in North America and is not secondary to the lack of a humoral response. Furthermore, mononuclear cells (from peripheral blood and from bone marrow) from the 11 patients with MM that we tested were found not to contain KSHV DNA; all of these patients were also seronegative for KSHV serum antibodies (Tables 1 and 2). Last, the short term cultures of myeloma bone marrow stromal cells from five of these cases were found not to contain KSHV with the use of DNA PCR and IFA (Table 2, last row, and Fig. 1).

Table 1. Antibody assays of immune response.* Virus MM KS Blood donor Number positive/ number tested (percent positive) Number positive/ number tested (percent positive) Number positive/ number tested (percent positive) KSHV  2/28  (7) 22/25 (88)  2/24  (8) EBV 26/28 (93) 20/25 (80) 20/24 (83) HHV-6 22/28 (79) 21/25 (84) 21/24 (88) HHV-7 24/28 (86) 22/25 (88) 23/24 (96) * More than 60% of the samples were evaluated by IFA as well as by ELISA. IFA samples were tested at a 1:20 dilution; ELISA samples were tested at a 1:100 dilution. Appropriate negative and positive control sera for each herpes virus were tested simultaneously.

Table 2. Detection of KSHV DNA by PCR or by IFA. PB, peripheral blood; MNC, mononuclear cell; BM, bone marrow; ND, not done. Cell type Method MM Hematologic malignancies Number positive/ number tested Number positive/ number tested PB-MNC PCR 0/6 ND BM-MNC PCR 0/5 0/11 BM stroma* PCR 0/5 0/11 Cell lines PCR 0/2 0/5 BM stroma IFA 0/5 ND * Short-term cultures of bone marrow stromal cells were established and successive subpassages (P1-P3) were tested for presence of KSHV antigen by IFA with the use of polyclonal KSHV-positive and KSHV-negative human serum. Hematologic malignancy cell lines included HUT 78, Raji cells, U937, Daudi cells (all from the American Type Culture Collection), and 23-2 [a lymphoma cell line (8)]. Bone marrow mononuclear cells from hematologic malignancies consisted of chronic myelogenous leukemia and Hodgkin's disease.

These data contradict the study by Rettig et al. in which KSHV DNA was detected with the use of PCR in all 15 samples of bone marrow stromal cells taken from MM patients (5).

Rizwan Masood
Tong Zheng
Anil Tulpule
Naveen Arora
School of Medicine,
University of Southern California,
Los Angeles, CA 90033, USA
Louise Chatlynne
Michael Handy
James Whitman, Jr.
Advanced Biotechnologies Inc.,
Columbia MD 21046, USA
Mark Kaplan
North Shore University Hospital
(New York University),
Manhasset, NY 11030, USA
Michael Dosik
North Shore Oncology Group,
Smithtown NY 11787, USA
Dharam V. Ablashi
Advanced Biotechnologies Inc.,
and School of Medicine,
Georgetown University,
Washington, DC 20057, USA
Parkash S. Gill
School of Medicine,
University of Southern California

REFERENCES AND NOTES

1. Y. Chang et al., Science 266, 1865 (1994) [Abstract/Free Full Text] .
2. J. Souleir et al., Blood 86, 1276 (1995) [Abstract/Free Full Text] .
3. Y. Chang and P. S. Moore, Infectious Agents and Disease (Lippincott-Raven, Philadelphia, PA, 1996).
4. L. A. Brooks, A. Wilson, T. Crook, J. Pathol. 172, 262 (1997) .
5. M. B. Rettig et al., Science 276, 1851 (1997) .
6. D. V. Ablashi et al., J. AIDS Hum. Retroviol. 14, A29 (1997).
7. D. V. Ablashi et al., Int. J. Cancer 42, 787 (1988).
8. R. Masood et al., Blood 85, 3423 (1995) [Abstract/Free Full Text] .
9. Supported in part by NCI grant (UO1-CA66533).

Rettig et al. propose (1) that KSHV (2) is involved in the development of MM from its more indolent precursor MGUS. They argue that KSHV may influence the switch from benign MGUS to malignant myeloma through the secretion and induction of autocrine growth factors, because the virus itself is not present in the clonal plasma cells. This is an attractive hypothesis because KSHV encodes a homolog of one of the cytokines, IL-6 (3), that is involved in MM pathogenesis. A further link might be that patients with multicentric Castleman's disease, another lymphoproliferation associated with KSHV (4), often have immunoglobulin dyscrasias, and occasionally develop MM.

The curious epidemiologic distribution of KS implies that an agent causing this disease would certainly not be the same agent inducing myeloma, which is a common hematological cancer with a much more even distribution throughout different populations. Furthermore, current molecular and serologic assays indicate that KSHV is, in northern Europe and the United States at least, predominantly sexually acquired and is not like most other herpesviruses, which are common pathogens (5) (6). The incidence rates of MM are homogenous in the various regions of northern and southern Italy (7), in contrast to the rate of classic KS (8), which correlates with the seroprevalence rates of KSHV in the different regions (9).

An immunofluorescence assay (IFA) that detects antibodies against the latent nuclear antigen (LNA-1) of KSHV is the most sensitive and specific serologic assay that we know of for revealing past or present infection with this virus (10). The decrease in polyclonal immunoglobulins associated with MM may be global or specific, and antibodies against KSHV may therefore not be detectable in infected patients, even if they have latent or lytic-specific antibodies against other herpesviruses. Antibodies against viruses in patients with MGUS are not suppressed, as a near normal immunoglobulin profile distinguishes MGUS from MM. If KSHV plays any major and common pathogenic role in MM and a subset of MGUS cases, we would expect to find increased serologic markers of infection, particularly in those with MGUS.

We therefore investigated the prevalence of antibodies against KSHV LNA-1 by IFA in patients with MM or MGUS from the Po valley of northern Italy, an area with one of the highest incidence rates of classic (HIV-negative) KS in the world. We compared the detection rate of antibodies against KSHV in these patients with that in patients with lymphoma (Hodgkin's disease and nonHodgkin's lymphoma) and blood donors, all from the same Po valley area (Table 1). Sera were tested blindly at a dilution of 1:100 as previously described (11).

Table 1. Results from sera of patients and blood donors living in the Po valley of Italy, tested with an IFA specific for antibodies against KSHV. Sera type Number tested Number positive (percent positive) Myeloma 37 4  (10.8) MGUS 36 2  (5.5) Hodgkin's disease 42 7  (16.7) Non-Hodgkin's lymphoma 60 8  (13.3) Blood donors 139 18  (12.9)

The prevalence of antibodies against KSHV in the sera of patients with MM, with MGUS, with lymphoma, and in sera from blood donors was not significantly different (P > 0.01; Table 1). The detection rate in Po valley blood donors is significantly higher (12.9% as opposed to <3.0%) than that found with the use of the same assay in blood donors in the United States or the United Kingdom (6) (11), which reflects the much higher incidence of classic KS there.

Four of the MGUS samples with no detectable antibodies were from patients who subsequently developed overt MM, whereas the only two MGUS patients with detectable KSHV antibodies had no evidence of MM after 36 and 48 months, respectively.

Thus, we did not find evidence for an increased detection rate of antibodies against KSHV in patients with MM, and even less so for those with MGUS. Our data do not support a role for KSHV in the evolution of MGUS to MM.

Denise Whitby
Chris Boshoff
Institute of Cancer Research,
Chester Beatty Laboratories,
London, SW3 6JB, United Kingdom
E-mail: cboshoff{at}icr.ac.uk
Mario Luppi
Giuseppe Torelli
Department Medical Sciences,
University of Modena,
Via del Pozzo 71,
41100 Policlinico, Modena, Italy

REFERENCES AND NOTES

1. M. B. Rettig et al., Science 276, 1851 (1997) .
2. Y. Chang et al., ibid. 266, 1865 (1994) .
3. P. S. Moore, C. Boshoff, R. A. Weiss, Y. Chang, ibid. 274, 1739 (1996).
4. J. Soulier et al., Blood 86, 1276 (1995) .
5. S. J. Gao et al., Nature Med. 2, 925 (1996) .
6. D. H. Kedes et al., ibid., p. 918.
7. G. Masala et al., Int. J. Cancer 68, 156 (1996) [CrossRef] [Web of Science] [Medline] .
8. M. Geddes et al., J. Natl. Cancer Inst. 87, 1015 (1995) [Free Full Text] .
9. D. Whitby et al., ibid., in press.
10. C. S. Rabkin et al., 4th Conf. Retrovir. Opportunistic Inft. abstr. 699 (1997).
11. G. R. Simpson et al., Lancet 348, 1133 (1996) [CrossRef] [Web of Science] [Medline] .
12. Sera provided by G. Longo, Second Medical Clinic, Modena, and D. Vallisa and L. Cavanna, First Medical Division, Section of Hematology, Piacenza, Italy. Supported by the Medical Research Council and the Cancer Research Campaign.

As a clinical dermatologist involved in the study of classic KS, I (F.C.) have followed about 200 cases of this disease over 20 years in the University Hospital of Sassari, which is the referral center for this disease in northern Sardinia. My epidemiological study has shown a standardized incidence of 1.58 per 100,000 inhabitants per year (2.43 for males and 0.77 for females) (1); this incidence is significantly higher than that observed in nine other Italian regions (1.05 for males and 0.27 for females) (2). I have found cases of KS associated with Hodgkin's disease, chronic lymphocytic leukemia, and Castleman's disease, but not with MM. Furthermore, the incidence of MM in the Sassari district is 4.51 per 100,000 inhabitants per year (4.57 for males and 4.45 for females), which is no different from that observed in seven other Italian regions where classic KS is not frequently detected (incidence range from 3.3 to 7.6 for males and from 3.7 to 7.3 for females) (3).

With regard to KSHV epidemiology in northern Sardinia, we found that 12 out of 13 KS patients had KSHV in their peripheral blood as detected by PCR (4). We have recently found that 95% of KS patients had sera with antibodies against KSHV and that the healthy northern Sardinian population has a higher prevalence of antibodies against KSHV in the serum (18%) than do people in the rest of Italy (4%) (5). If KSHV had a role in MM, as suggested by Rettig et al. (6), then we would expect to find an increased rate of MM in the Sassari district and, therefore, a more frequent association of classic KS with MM. Our observations (although made on a small number of cases because of the rarity of classic KS) do not seem to support such an association.

Francesca Cottoni
Institute of Dermatology,
University of Sassari,
07100 Sassari, Italy
Stefania Uccini
Department of Experimental
Medicine and Pathology,
University La Sapienza,
00161 Rome, Italy

REFERENCES AND NOTES

1. F. Cottoni, R. De Marco, M. A. Montesu, Br. J. Cancer 72, 1132 (1996) .
2. M. Geddes et al., ibid. 69, 333 (1994) [Web of Science] [Medline].
3. Cancer in Italy: Incidence Data for Cancer Registries 1983-1987, R. Zanetti and P. Crosignani, Eds. (Lega Italiana Lotta Tumori, Torino, Italy, 1992).
4. S. Uccini et al., Invest. Dermatol. 108, 119 (1997).
5. A. Angeloni et al., II Internat. Conf. Hum. Herpesvirus 6,7,8, Abstr. p. 3 (1997).
6. M. B. Rettig et al., Science 276, 1851 (1997) .
7. I would like to thank an anonymous reviewer for helpful remarks.

Contrary to the report by Rettig et al. (1), other groups have not found antibodies against KSHV in serum from patients with MM (2).

We took acetone-fixed and paraffin-embedded bone marrow biopsies from patients with MM and performed PCR analysis in an attempt to detect KSHV. We investigated 20 cases of MM (at different stages) with a protocol previously described (3) with the use of the primer set within open reading frame (ORF) 26 (KS330₂₃₃). We carried out two rounds of amplification (2 × 30 cycles). This protocol yielded a positive amplification of 233 base pairs (bp) in 18 out of 20 patients. We also analyzed 15 bone marrow biopsy samples from patients with follicular lymphomas and 5 samples from patients with reactive processes; these samples did not show the presence of KSHV. In all cases, the integrity of the DNA was confirmed after amplification of a 258-bp-long fragment corresponding to the c-raf-1 gene as previously described (4). The specificity of the KS330₂₂₃ PCR was checked by sequencing the amplification products of five randomly selected cases. We consider our results to be specific because there were different point-mutations in each case as compared with the original sequence (3). These results are similar to those obtained by Rettig et al. (1). Our method would appear to eliminate technical problems such as contamination as a source of the results. Overall, we confirm that, at least in our French series of cases, KSHV is strongly associated with MM.

P. Brousset
F. Meggetto
M. Attal
G. Delsol
Centre Hospitalier Universitaire Purpan,
31059 Toulouse Cédex, France

REFERENCES

1. M. B. Rettig et al., Science 276, 1851 (1997) .
2. A. G. Marcelin et al., Lancet 350,1144 (1997); J. MacKenzie et al., ibid., p. 1144.
3. Y. Chang et al., Science 266, 1865 (1994) .
4. S. Galoin et al., Brit. J. Haematol. 94, 676 (1996).

Response: Several groups raise questions regarding our report that KSHV (also called HHV-8) infects the bone marrow dendritic cells of myeloma patients and may be involved in myeloma pathogenesis (1). Whitby et al. performed a serologic assay, and both Parravicini et al. and Massoud et al. used both serologic and PCR assays to conclude that a lack of evidence exists for the association of KSHV and myeloma. Cottoni et al. demonstrate a lack of clinical correlation between the incidence of classic KS and MM in a cohort of patients from Sardinia.

A possible mechanism for the lack of serological evidence for KSHV in MM relates to a change of environment for B cell development in these patients. Myeloma patients demonstrate panhypogammaglobulinemia (that is, decreased concentrations of antibodies other than the monoclonal antibody produced by the malignant plasma cells), which may reduce the titer of KSHV antibodies. In addition, the infection of dendritic cells within the bone marrow, the site of early B cell development, may lead to B cell tolerance to KSHV antigens. Clonal deletion, functional inactivation, and antigen receptor editing have been shown to be mechanisms for preventing the maturation of B cells that have been presented antigen in the bone marrow (2). Thus, the combination of global and viral antigen-specific immunological defects may contribute to the decreased seroprevalence to KSHV in myeloma patients.

With the use of PCR amplification, Parravicini et al. and Massoud et al. were unable to detect viral sequences in mononuclear cells obtained from aspirated bone marrow, peripheral blood mononuclear cells, or bone marrow stromal cells. The results from bone marrow mononuclear cells are consistent with our original report. Similar to the work of Parravicini et al., we have subsequently performed nested PCR on 1 µg of DNA from mononuclear cells from bone marrow aspirates and repeated the reaction up to 10 times for each patient; we have not detected the virus. Parravicini et al. alluded to some of the possible explanations for the inability to detect virus. They stated that the presence of Taq polymerase inhibitors were excluded because they amplified the  globin gene. However, the presence of heparin, which is the anticoagulant most often used for bone marrow aspirations, may inhibit the amplification of low copy number sequences by Taq polymerase (3). Moreover, heparin is not removed during standard DNA extraction techniques. We have used heparinase to remove contaminating heparin from our bone marrow mononuclear cell samples and have successfully amplified KSHV sequences in bone marrow mononuclear cell samples. Recently, the precise bone marrow culture conditions have been shown in an abstract to be essential for the detection of KSHV DNA (4). PCR signal for viral DNA may be lost with more prolonged culture, as has been seen in the case of KS.

To exclude the possibility that our observations of KSHV infection in dendritic cells were a result of an in vitro artifact of tissue culture, we have evaluated fresh uncultured bone marrow core biopsies for the presence of KSHV. Whereas none of the normal subjects (n = 4) or patients with other hematological malignancies (n = 23) demonstrated the presence of KSHV by in situ hybridization for ORF 72, 18 out of 21 myeloma patients showed viral staining (5). PCR for KS330₂₃₃ on these same tissue samples detected KSHV in 12 out of 16 myeloma patients (5). Our results have been replicated by Brousset et al., as described in their comment. In our cohort of myeloma patients, immunohistochemical staining on serial sections showed that the distribution of the presence of KSHV corresponded to the distribution of cells containing long cytoplasmic processes. The latter stained for fascin, a marker for dendritic cells).

Similar to results seen by Parravicini et al., we were unable to detect viral sequences in bone marrow mononuclear cells. Although we were also unable to detect KSHV in whole peripheral blood mononuclear cells, when we enriched these cells for macrophage and dendritic cell markers (CD68 and CD83, respectively) with the use of an immunomagnetic bead column, we identified viral sequences in 25 out of 32 myeloma patients, 0 out of 5 patients with other malignancies, and 1 out of 13 hematologically normal subjects (6).

Cottoni and Uccini discuss a cohort of classic KS patients in Sardinia in whom they did not detect an increased prevalence of myeloma. The extrapolation of these data to myeloma patients worldwide may not be valid. In addition to KSHV infection, other environmental and genetic factors may well contribute to the development of myeloma. By analogy, EBV is closely associated with nasopharyngeal myeloma in southern China and with Burkitt's lymphoma in Africa, yet the incidence of these malignancies among immigrants to the United States is equal to that of the general population. Three decades ago, Mazzaferri and Penn reviewed the literature and concluded that there was a correlation between KS and myeloma (7). Epidemiological studies in HIV-positive patients may provide important clinical information correlating KSHV infection, KS, and myeloma. As HIV patients live longer, they may manifest an increased incidence of myeloma.

M. B. Rettig
Division of Hematology and Oncology,
Veterans Affairs West
Los Angeles Medical Center,
11301 Wilshire Boulevard, 111-H,
Los Angeles, CA, 90073, USA, and
Jonsson Comprehensive Cancer Center and
Division of Hematology and Oncology,
University of California Los Angeles
School of Medicine,
10833 LeConte Avenue,
Los Angeles, CA, 90095 USA
J. W. Said R. Sun
Jonsson Comprehensive Cancer Center
R. A. Vescio J. R. Berenson
Division of Hematology and Oncology,
Veterans Affairs West
Los Angeles Medical Center and
Jonsson Comprehensive Cancer Center

REFERENCES

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