KSHV genomes were detected in PBMC and in plasma from most patients with AIDS-KS. KSHV genomes were also detected in PBMC and in plasma in some patients with AIDS-NHL who had no history of AIDS-KS. As expected, the differences between patients with KS and NHL were significant. However, among patients with KSHV detected in either PBMCs or in plasma, the copy number range and interquartile range did not distinguish between patients with KS and NHL (B). This finding—that, among AIDS patients with detectable KSHV DNA in plasma, KSHV copy number did not differentiate patients with KS from those with NHL—suggests that the KSHV DNA being measured in plasma is unlikely to be tumor-derived, even in patients with KS. Campbell et al16
reached similar conclusions, and they showed that among patients in Zimbabwe coinfected with HIV and KSHV, plasma KSHV levels did not differ between patients with and without KS.
Insofar as DNA tumor markers in cell-free blood are attracting increasing attention in breast, lung, gastrointestinal, head and neck, and other cancers,17–19
it is worth considering how the situation with AIDS-KS and AIDS-NHL patients differs from viral DNA markers in some other settings. EBV DNA markers are among the best studied. In patients with nasopharyngeal carcinoma, a series of studies has shown that EBV DNA in cell-free blood indicates the presence of tumor.15,20
In rare instances when tumor can be surgically excised, viral DNA is cleared within hours. With more standard treatments (ie, external-beam radiotherapy, chemotherapy), viral DNA clears with successful treatment.21
Quantification of viral DNA at diagnosis augments TNM tumor stage in predicting clinical outcome.22
The inability to clear viral DNA, or the reappearance of viral DNA after it is cleared, signals refractory or relapsing disease, respectively.21
The molecular character of the KSHV DNA in plasma in patients with AIDS malignancy differs from that of EBV DNA in nasopharyngeal cancer and some other EBV-associated malignancies. DNase protection indicative of the presence of virion DNA in AIDS-KS and AIDS-NHL, as shown here, has not been reported in nasopharyngeal cancer.15
The different assay results are not likely to simply reflect technical differences between laboratories, as our own studies show that there is no protection from DNase in plasma from EBV-associated Hodgkin's lymphoma (data not shown). DNase resistance data in patients in Zimbabwe who have HIV with and without KS also led others to conclude that virion DNA was present in plasma.16
Intracellular nuclease activity in the presence of nucleosomes, such as accompanies apoptotic cell death, yields DNA fragments less than 180 bp. Our finding of a high fractional concentration of KSHV fragment sizes in plasma larger than 180 bp also suggests an origin other than release of free DNA from cells undergoing apoptotic cell death. Again, the contrast with EBV DNA in plasma from patients with nasopharyngeal carcinoma, as reported by others,15
and with EBV-associated Hodgkin's lymphoma in our laboratory (unpublished data) is striking.
Thus, the clinical observation that the presence of plasma KSHV DNA does not correspond to the presence or absence of tumor and the molecular characterization that suggests that the KSHV DNA in plasma is virion DNA rather than tumor-derived free DNA are congruent. These results are consistent with our previous observation that the clinical outcome of interferon therapy for AIDS-KS was not correlated with changes in KSHV copy number.23
This finding does not preclude the possibility suggested by others that, among patients with AIDS KS, there may be a relationship between copy number and KS disease activity.24,25
Our findings did not suggest a relationship between CD4 cell count or HIV RNA and KSHV DNA. Some investigators have reported such a relationship,24,26
whereas others have not.27
Clearly, the determinants of KSHV copy number in PBMCs and in plasma requires additional investigation.
Several investigators have reported that KSHV copy number corresponds with KS tumor burden or activity.24
Our investigations yielded similar results (). Thus, our results confirm the observations already reported by others. However, when the analysis is restricted to patients in whom virus is detected, patients with AIDS-KS and patients with AIDS-NHL can no longer be distinguished (B).
Why did KSHV DNA copy number decrease after the initiation of chemotherapy in patients with lymphoma? The impact of various pharmacologic therapies on KSHV copy number has been investigated in other settings. Trials with antiherpesvirus agents (ie, ganciclovir, foscarnet, cidofovir) have shown no impact on KSHV copy number in blood, although a dramatic effect on oral shedding of virus has been reported.27–29
In contrast, antiretroviral therapy lowers KSHV copy number in PBMCs and in plasma, but the decrease is much less rapid than that documented here and generally begins after a year of therapy.30
Of the six patients in our study with detectable KSHV in PBMCs before the initiation of lymphoma therapy, all but one were already on highly active antiretroviral therapy.
It has been suggested that lymphoid tissue sustains KSHV viremia and that B cells are the lymphoid reservoir.31
We have previously reported the rapid decline (within 24 hours) of EBV viral copy number in PBMCs of patients with post-transplant lymphoma who were treated with rituximab alone.32
This corresponds to a disappearance of B cells from blood that accompanies rituximab treatment. Thus, we entertained the possibility that, in this study, the decrease in viral copy number reflected the disappearance of B cells from the blood. In a recent report that detailed treatment and re-treatment of Castleman disease in HIV-infected patients, the clinical response to rituximab treatment was accompanied by a marked decrease in KSHV copy number in blood.33
Disease recurrence was accompanied by an increase in KSHV copy number, and rituximab monotherapy again led to a clinical response and a decrease in KSHV copy number in blood.
In this study, depletion of B cells coincided with a decrease in KSHV in PBMCs and in plasma. In two patients treated with CHOP-R chemotherapy, flow cytometry confirmed depletion of CD19+
B cells to less than 0.1% of PBMCs just before cycle 4 (data not shown). Flow cytometry also showed a relative depletion of B cells in four patients treated with CHOP alone. CD19+
cells constituted less than 1% of mononuclear cells in three of these patients. In the remaining patient, CD19+
cells accounted for 4% of PBMCs. Of note, this latter patient is the patient with residual virus DNA detectable in PBMCs and in plasma (). Similarly, the reappearance of KSHV approximately a year after the conclusion of treatment with CHOP-R may correspond to a time when B-cell levels return to pretreatment levels. Thus, KSHV copy number in PBMCs and in plasma may reflect the size of the B lymphocyte pool. It should be noted, however, that in a study of infusional chemotherapy (without rituximab) for AIDS-NHL combined with antiretroviral therapy, in which 68 patients survived more than 3 months, two of the surviving patients developed KS.34
Thus, the decrease in KSHV identified in this study notwithstanding, it should not be presumed that lymphoma chemotherapy without rituximab confers protection against the development of KS. Indeed, there are a series of reports of KS exacerbation or de novo occurrence in HIV-infected patients with Castleman disease who were treated with rituximab.35–38
In addition, exacerbation of classic Castleman disease has been reported in a patient with autoimmune hemolytic anemia who was treated with rituximab.39
How the KSHV genome accesses the endothelial cells that lead to KS tumors is not understood. Viral DNA episomes might be transported in latently infected lymphocytes, and a direct cell-cell interaction might lead to infection of the precursor cells. Alternatively, virions produced elsewhere might be carried in cell-free blood to precursor cells. The suggestion has even been made that sustained tumor cell proliferation might require continuous reinfection.32
In any of these scenarios, lymphoma therapy might limit the delivery of virus (as infected PBMCs or virions) to premalignant or malignant cells and might prevent or delay the development of KS.
In summary, KSHV DNA is commonly detected in PBMCs and in plasma of patients with AIDS-NHL. Among patients in whom viral DNA is detected, viral copy number does not serve to discriminate between patients with AIDS -KS and AIDS -NHL. The characteristics of the viral DNA detected in plasma are consistent with the presence of virion DNA. Standard lymphoma chemotherapy with CHOP or CHOP-R led to rapid declines in viral DNA in PBMCs and in plasma. The changes in viral copy number may reflect changes in the size of the B-cell pool.