To the Editor:
Rettig et al1 originally described the presence of Kaposi’s sarcoma-associated herpesvirus (KSHV) in cultured bone marrow (BM) stromal dendritic cells of patients with multiple myeloma (MM) using both in situ hybridization and simple, liquid-phase polymerase chain reaction (LP-PCR).
By contrast, we failed to obtain evidence of KSHV infection in 40 patients with MM using nested LP-PCR and serology.2However, in our study mononuclear cells obtained from BM aspirates were used as template DNA for LP-PCR.
The same authors strengthened their findings by the in vivo demonstration of KSHV DNA sequences in MM BM core biopsies.3 In particular, viral sequences were readily detectable (6 out of 7 cases; 86%) in DNA extracted from BM biopsies using simple LP-PCR and agarose gel electrophoresis of amplified PCR products.3
To definitively address this issue, we decided to look for KSHV DNA sequences in a new consecutive series of 12 Italian patients with MM. All were symptomatic and were enrolled after having obtained their informed consent at the Unit of Lymphoproliferative Disorders of the IRCCS Ospedale Maggiore, University of Milan, Italy. There were 6 men and 6 women, with a median age of 60.5 years. None had previously received therapy for MM. Clinicopathological details are available upon request.
BM core biopsies were placed after collection in 1.8 mL Nalgene Tubes (Nalge, Rochester, NY) and immediately stored at −80°C. In addition, coevally drawn BM aspirates and peripheral blood from each patient were subject to Ficoll density gradient centrifugation (Pharmacia, Uppsala, Sweden), and mononuclear cells were stored as dry pellets at −80°C until further processing. While collecting peripheral blood and BM aspirates, care was taken to use EDTA as anticoagulant. BM biopsies were processed for DNA extraction using the same protocol described by Said et al3 and the Easy DNA extraction KIT (Invitrogen, Carlsbad, CA). The DNA extraction procedure was begun only after complete lysis of bone specimens. The same kit was used for extraction of BM and peripheral blood mononuclear cell pellets.
We used simple LP-PCR and the conditions proposed by Said et al (ie, same KSHV330233 primers), except that amplification was allowed to proceed for 50 amplification cycles. In addition, a hot-start protocol was used employing Taq Gold DNA polymerase (Perkin-Elmer, Norwalk, CT). One microgram of spectrophotometrically measured DNA was loaded in each PCR reaction vessel. Each BM biopsy sample was tested in triplicate individual PCR reactions.
In addition, 1 μg of BM biopsy, BM and peripheral blood mononuclear cell DNA from each patient was tested using two KSHV-specific nested LP-PCR systems that amplify distant unrelated portions of the viral genome and that achieve single-copy sensitivity of the target after 35 + 35 amplification cycles.2,4-7 In this case, quintuplicate individual PCR reactions for every sample were performed with each of the two nested LP-PCR systems.
Absence of PCR inhibitors was ascertained by amplifying separately 1 μg of extracted DNA that was spiked with 10 copies of the KSHV genome. Accurate quantitation by limiting dilution and signal distribution analysis7 was performed on DNA extracted from the KSHV-positive BCBL-1 cell line to obtain a solution containing the appropriate concentration of KSHV genomes (BCBL-1 cells were obtained through the AIDS Research and Reference Program, Division of AIDS, NIAID, NIH from M. McGrath and D. Ganem8).
The presence and integrity of human DNA in the extracted samples was determined by performing simple LP-PCR using human β-globin–specific primers that amplify a DNA fragment greater in size than those obtained with the KSHV-specific PCR primers.
As negative control, each patient specimen was processed in parallel (ie, preceded and followed) with two tubes containing ultra-pure water. Negative controls were tested with the two nested PCR systems until exhaustion to exclude even minute (ie, single copy) levels of target DNA carry-over and/or amplimer contamination.
Simple and nested LP-PCR products were visualized by ultraviolet-light exposure after standard horizontal agarose gel electrophoresis.
All simple LP-PCR reactions performed on 1 μg of MM BM core biopsy DNA using the same conditions employed by Rettig et al were negative for KSHV DNA sequences (36 reactions or 12 MM core biopsy specimens each amplified individually three times).
A total of 120 μg of MM BM core biopsy DNA were probed by the two nested PCR systems for viral DNA sequences; none scored positive for KSHV.
Altogether, 360 nested PCR reactions performed on BM core biopsies, and BM and peripheral blood mononuclear cells obtained from 12 patients with MM gave invariably negative results for KSHV DNA sequences.
PCR inhibitors, if present, did not preclude the detection in all specimens of 10 copies of the KSHV genome in a background of 1 μg of extracted DNA.
None of the 72 negative controls (for a total of 144 nested PCR reactions) tested positive for KSHV DNA sequences.
The discrepancies observed between our results and those obtained by other authors are reminiscent of another controversy that has already seen KSHV at the center of attention (ie, the widely varying detection rates of KSHV DNA in semen).9 In that case, after a series of contradictory results and only one as yet official retraction,10 it is finally evident that PCR contamination was almost certainly the underlying explanation.11,12
In conclusion, our results argue against the presence of KSHV infection in MM patients. A similar approach to what has been done to assess the prevalence of KSHV in semen (ie, a blinded, multi-institutional study)12 is opportune to unequivocally settle this issue. Until then, the association between KSHV and MM has to be considered, in our opinion, unproven.