To the Editor:

Kaposi’s sarcoma-associated herpesvirus (KSHV) is involved in the pathogenesis of all forms of Kaposi’s sarcoma (KS).1 In acquired immunodeficiency syndrome (AIDS)-associated KS, KSHV detection in peripheral blood mononuclear cells increases with immunosuppression.2 Posttransplant KS are generally due to KSHV reactivation,3 and complete KS remission is often achieved after reduction or cessation of immunosuppressive therapy.4 Serologic studies have shown that 80% to 90% of KS patients have detectable antibodies against KSHV.5 These data clearly demonstrate that KSHV is under immunological control in KS patients.

Recently, KSHV was detected in long-term cultures of bone marrow stromal cells (BMSC) with a phenotype of dendritic cells (DC)6 and in bone marrow (BM) core biopsies from patients with multiple myeloma (MM).7,8 The physiopathological relevance of KSHV in this interleukin-6 (IL-6)–related disease could be that it encodes for a viral IL-6 (vIL-6) able to stimulate the growth of human MM cell lines.9 However, these results contradict what is known about KSHV infection and MM. Epidemiological studies show that KSHV and non-AIDS KS are found at higher incidence in Italy5 and that this is clearly not the case for MM.10 In addition, five groups reported a lack of antibodies against KSHV antigens in MM patients despite a normal humoral response to other herpesvirus.11-15 Finally, we and others were recently unable to found KSHV in DC samples obtained from apheresis cells of MM patients,16,17 and Masood et al14 failed to detect KSHV DNA in long-term BMSC cultures from MM patients. This discrepancy led us to explore the possibility that an extremely low level of KSHV infection in MM patients, leading to variable detection, may be reactivated during severe immunosuppression.

Ten patients with MM were treated with a double high-dose chemotherapy (HDC; 140 mg/m2 melphalan plus 8 Gy total body irradiation) supported by autograft with purified CD34+ cells (reinjection of 4.02 ± 1.03 × 106CD34+/kg; range, 2.88 to 5.73 × 106/kg). CD34+ progenitors were purified by the clinical-grade method from Cellpro (Bothell, WA), leading to a 35.6-fold enrichment in hematopoietic progenitors from a mean value of 2.4% ± 1.08% CD34+ cells (range, 0.99% to 3.47%) before purification to 85.4% ± 7.1% CD34+ cells (range, 72.4% to 92.8%) after purification. The resulting graft was 1,407-fold depleted of T cells (reinjection of 0.11 ± 0.08 × 106CD3+ cells/kg; range, 0.05 to 0.25 × 106/kg). Four of 10 patients relapsed within 1 year. The peripheral blood CD4+ T-cell count was monitored at 3, 6, and 12 months after the second purified autograft. Eight of 10 patients had less than 200 CD4+ cells/μL for at least 3 months, with a mean duration of 7 months for the 6 evaluable over 1 year (Table 1). Many infectious events arose during this first year after second HDC (median of 3 episodes per patient). In particular, 7 of 10 patients suffered from herpesvirus reactivation (Table 1). Because KS has rarely been associated with hematopoietic stem cell transplantation, no kinetic study was available; thus, repeated polymerase chain reaction (PCR) amplification was performed to detect KSHV in BM samples harvested before and 90, 180, and 360 days after the second HDC. KSHV DNA was monitored in 1 μg of genomic DNA (ie, 150,000 cells), in a blinded fashion and in two different laboratories, using a PCR assay against the KS330233 KSHV sequence.1 This sensitive method allowed the detection of KSHV DNA in less than 1 pg of genomic DNA from the KSHV-infected BCBL-1 cell line that corresponds to approximately 5 KSHV genome copies.16 18 KSHV DNA was not detected in any of the 35 BM samples tested (Fig 1). The lack of KSHV detection was not due to the presence of Taq polymerase inhibitors, in particular heparin, because the sensitivity of KSHV PCR was the same when assayed with either DNA harvested from heparinized BM mononuclear cells or DNA from cells collected without heparin (Fig 1).

Table 1.

CD4+ T-Cell Counts and Infectious Events in Autografted MM Patients

Patient No. No. of CD4+ T Cells/μL Cumulative No. of Months With CD4+ T Cells <200/μLInfectious Events After Second HDC-150
Before First HDC Day 90 After Second HDC Day 180 After Second HDC Day 360 After Second HDC
 1  892  170 291  266  4  VZV, CMV  
 2  361  91  11 351  6  VZV, CMV  
 3  350  95  150  146  HZV, CMV  
 4  315  327  307  234  0  HSV 
 5  259  77  75  88  12  Other  
 6  369 168  148  160  12  CMV, Other  
 7  430  140 149  -151 11/11-152 Other  
 8  593 310  ND  -151 0  CMV  
 9  124 64  199  -153 6/6-152 
10  412  128 -151 -151 4/4-152 CMV 
Mean ± SD  410.5 ± 207.5 157 ± 92.5  166.2 ± 99.8  207.5 ± 94.9 
Patient No. No. of CD4+ T Cells/μL Cumulative No. of Months With CD4+ T Cells <200/μLInfectious Events After Second HDC-150
Before First HDC Day 90 After Second HDC Day 180 After Second HDC Day 360 After Second HDC
 1  892  170 291  266  4  VZV, CMV  
 2  361  91  11 351  6  VZV, CMV  
 3  350  95  150  146  HZV, CMV  
 4  315  327  307  234  0  HSV 
 5  259  77  75  88  12  Other  
 6  369 168  148  160  12  CMV, Other  
 7  430  140 149  -151 11/11-152 Other  
 8  593 310  ND  -151 0  CMV  
 9  124 64  199  -153 6/6-152 
10  412  128 -151 -151 4/4-152 CMV 
Mean ± SD  410.5 ± 207.5 157 ± 92.5  166.2 ± 99.8  207.5 ± 94.9 

CD4+ T-cell count was monitored by flow cytometry.

Abbreviation: ND, not done.

F0-150

Viral manifestation of varicella (VZV), zooster (HZV), herpes (HSV), other virus (Other), or cytomegalovirus antigen detection (CMV).

F0-151

Patient died before evaluation.

F0-152

CD4 count could be evaluated only during the indicated time.

F0-153

Patient has not reached day 360 after HDC.

Fig. 1.

Lack of KSHV reactivation during treatment-induced immunosuppression in MM patients. (A) DNA samples extracted from BM before and 90, 180, and 360 days after second HDC were amplified by PCR using the KSHV 330233 primers. Each PCR was performed on 1 μg of genomic DNA and the amplification products were transferred to a nylon membrane and hybridized with a 32P end-labeled internal probe. The positive control (lane C) was the PCR product from the KSHV-infected BCBL-1 cell line. (B) Lanes 1 through 5 contain 10-fold dilutions of BCBL-1 DNA from 1 ng (lane 1) to 0.1 pg (lane 5). BCBL-1 DNA was diluted in the DNA extracted from heparinized BM mononuclear cells from patient no. 1 90 days after the second HDC, so that all PCR were run on 1 μg of total DNA. These data are representative of 10 experiments performed with the heparinized DNA samples extracted from the 10 patients 90 days after second HDC.

Fig. 1.

Lack of KSHV reactivation during treatment-induced immunosuppression in MM patients. (A) DNA samples extracted from BM before and 90, 180, and 360 days after second HDC were amplified by PCR using the KSHV 330233 primers. Each PCR was performed on 1 μg of genomic DNA and the amplification products were transferred to a nylon membrane and hybridized with a 32P end-labeled internal probe. The positive control (lane C) was the PCR product from the KSHV-infected BCBL-1 cell line. (B) Lanes 1 through 5 contain 10-fold dilutions of BCBL-1 DNA from 1 ng (lane 1) to 0.1 pg (lane 5). BCBL-1 DNA was diluted in the DNA extracted from heparinized BM mononuclear cells from patient no. 1 90 days after the second HDC, so that all PCR were run on 1 μg of total DNA. These data are representative of 10 experiments performed with the heparinized DNA samples extracted from the 10 patients 90 days after second HDC.

Close modal

Three explanations may account for the discrepancy between the negative PCR with BM aspirates, the negative serological results, the lack of KSHV reactivation in immunosuppressed MM patients, and the positive PCR with stromal cultures and BM biopsies. (1) MM patients could be infected with a variant of KSHV that can escape the immune system or that encodes for antigens not recognized by the available immunological assays. This could explain the failure to detect anti-KSHV antibodies. (2) KSHV could be under a strict T-cell–mediated immune control in MM patients, leading to a very difficult detection by sensitive PCR. In this case, because infected cells remain undetectable in whole BM samples after double HDC and graft of purified CD34+ cells, one could hypothesize that this treatment has not destroyed anti-KSHV–specific CD4+ and CD8+ T cells, contrary to other anti-herpesvirus T cells. (3) KSHV could be not involved in MM patients, and its detection could be linked to false-positive PCR, as pointed out recently by Moore.19 

These contradictions need to be elucidated, but our present results emphasize that, if KSHV or a variant of KSHV is really involved in MM, it is not a major factor in relapse occuring in immunosuppressed patients after autologous graft and raises the question of its causal role in MM.

1
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
266
1994
1865
2
Whitby
D
Howard
MR
Tenant-Flowers
M
Brink
NS
Copas
A
Boshoff
C
Hatzioannou
T
Suggett
FE
Aldam
DM
Denton
AS
Detection of Kaposi sarcoma associated herpesvirus in peripheral blood of HIV-infected individuals and progression to Kaposi’s sarcoma.
Lancet
346
1995
799
3
Parravicini
C
Olsen
SJ
Capra
M
Poli
F
Sirchia
G
Gao
SJ
Berti
E
Nocera
A
Rossi
E
Bestetti
G
Pizzuto
M
Galli
M
Moroni
M
Moore
PS
Corbellino
M
Risk of Kaposi’s sarcoma-associated herpes virus transmission from donor allografts among Italian posttransplant Kaposi’s sarcoma patients.
Blood
90
1997
2826
4
Penn
I
Sarcomas in organ allograft recipients.
Transplantation
60
1995
1485
5
Gao
SJ
Kingsley
L
Li
M
Zheng
W
Parravicini
C
Ziegler
J
Newton
R
Rinaldo
CR
Saah
A
Phair
J
Detels
R
Chang
Y
Moore
PS
KSHV antibodies among Americans, Italians and Ugandans with and without Kaposi’s sarcoma.
Nat Med
2
1996
925
6
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
1997
1851
7
Said
JW
Rettig
MR
Heppner
K
Vescio
RA
Schiller
G
Ma
HJ
Belson
D
Savage
A
Shintaku
IP
Koeffler
HP
Asou
H
Pinkus
G
Pinkus
J
Schrage
M
Green
E
Berenson
JR
Localization of Kaposi’s sarcoma-associated herpesvirus in bone marrow biopsy samples from patients with multiple myeloma.
Blood
90
1997
4278
8
Brousset
P
Meggetto
F
Attal
M
Delsol
G
Kaposi’s sarcoma-associated herpesvirus infection and multiple myeloma [letter].
Science
278
1997
1972
9
Burger
R
Neipel
F
Fleckenstein
B
Savino
R
Ciliberto
G
Kalden
JR
Gramatzki
M
Human herpesvirus type 8 interleukin-6 homologue is functionally active on human myeloma cells.
Blood
91
1998
1858
10
Masala
G
Di Lollo
S
Picoco
C
Crosignani
P
Demicheli
V
Fontana
A
Funto
I
Miligi
L
Nanni
O
Papucci
A
Ramazzotti
V
Rodella
S
Stagnaro
E
Tumino
R
Vigano
C
Vindigni
C
Seniori Costantini
A
Vineis
P
Incidence rates of leukemias, lymphomas and myelomas in Italy: Geographic distribution and NHL histotypes.
Int J Cancer
68
1996
156
11
Marcelin
AG
Dupin
N
Bouscary
D
Bossi
P
Cacoub
P
Ravaud
P
Calvez
V
HHV-8 and multiple myeloma in France [letter].
Lancet
350
1997
1144
12
MacKenzie
J
Sheldon
J
Morgan
G
Cook
G
Schulz
TF
Jarrett
RF
HHV-8 and multiple myeloma in the UK [letter].
Lancet
350
1997
1144
13
Parravicini
C
Lauri
E
Baldini
L
Neri
A
Poli
F
Sirchia
G
Moroni
M
Galli
M
Corbellino
M
Kaposi’s sarcoma-associated herpesvirus infection and multiple myeloma [letter].
Science
278
1997
1969
14
Masood
R
Zheng
T
Tupule
A
Arora
N
Chatlynne
L
Handy
M
Whitman
J
Jr.
Kaposi’s sarcoma-associated herpesvirus infection and multiple myeloma [letter].
Science
278
1997
1970
15
Whitby
D
Boshoff
C
Luppi
M
Torelli
G
Kaposi’s sarcoma-associated herpesvirus infection and multiple myeloma [letter].
Science
278
1997
1971
16
Tarte
K
Olsen
SJ
Lu
ZY
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
1998
1852
17
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
1998
793
18
Parry
JP
Moore
PS
Corrected prevalence of Kaposi’s sarcoma (KS)-associated herpesvirus infection prior to onset of KS [letter].
AIDS
11
1997
127
19
Moore
PS
Human herpesvirus 8 variants [letter].
Lancet
351
1998
679
Sign in via your Institution