Karen Aleman

An Interleukin-6-Related Systemic Inflammatory Syndrome in Patients Co-Infected with Kaposi Sarcoma-Associated Herpesvirus and HIV but without Multicentric Castleman Disease

BACKGROUND Kaposi sarcoma-associated herpesvirus (KSHV) is the causal agent for Kaposi sarcoma (KS) and multicentric Castleman disease (MCD) in human immunodeficiency virus (HIV)-infected patients. Patients with KSHV-MCD develop fevers, wasting, hypoalbuminemia, cytopenias, and hyponatremia that are related to overproduction of KSHV-encoded viral interleukin (IL)-6 (vIL-6) and human IL-6 (hIL-6). METHODS We identified 6 HIV-infected patients with KS or serological evidence of KSHV infection who had severe inflammatory MCD-like symptoms but in whom we could not diagnose MCD, and we hypothesized that these symptoms resulted from vIL-6 overproduction. Serum vIL-6 levels were assessed in these 6 patients and compared with levels in 8 control patients with symptomatic KSHV-MCD and 32 control patients with KS. KSHV viral load, serum hIL-6 level, and human IL-10 level were also evaluated. RESULTS Patients with inflammatory MCD-like symptoms but without MCD had elevated vIL-6 levels, comparable with levels in patients with symptomatic KSHV-MCD, and had levels that were significantly greater than those in control patients with KS (P = .003). Elevated hIL-6, IL-10, and KSHV viral loads were also comparable to patients with symptomatic KSHV-MCD and significantly greater than those with KS. CONCLUSIONS A subset of patients with HIV and KSHV co-infection, but without MCD, can develop severe systemic inflammatory symptoms associated with elevated levels of KSHV vIL-6, IL-6, and KSHV viral loads. Excess lytic activation of KSHV, production of the lytic gene product vIL6, and associated immunologic dysregulation may underlie the pathophysiology of these symptoms. This IL-6-related inflammatory syndrome is important to consider in critically ill patients with HIV and KSHV co-infection.

Conservation of Virally Encoded MicroRNAs in Kaposi Sarcoma-Associated Herpesvirus in Primary Effusion Lymphoma Cell Lines and in Patients with Kaposi Sarcoma or Multicentric Castleman Disease

BACKGROUND MicroRNAs are small noncoding RNAs that posttranscriptionally regulate gene expression. Kaposi sarcoma (KS)-associated herpesvirus (KSHV) encodes 12 distinct microRNA genes, all of which are located within the latency-associated region that is highly expressed in all KSHV-associated malignancies. METHODS We amplified, cloned, and sequenced a 2.8-kbp-long region containing a cluster of 10 microRNAs plus a 646-bp fragment of K12/T0.7 containing the remaining 2 microRNAs from 5 primary effusion lymphoma-derived cell lines and from 17 patient samples. The patients included 2 with classic KS, 12 with AIDS-KS (8 from the United States, 1 from Europe, 3 from Africa, and 4 from Central/South America), and 2 with multicentric Castleman disease (MCD). Additionally, we analyzed the K1, open reading frame 75, and K15 genes to determine KSHV subtypes, and we performed a phylogenetic analysis. RESULTS Phylogenetic analysis of the 2.8-kbp microRNA region revealed 2 distinct clusters of sequences: a major (A/C) and a variant (B/Q) cluster. The variant cluster included sequences from 3 patients of African origin and both patients with MCD. Some microRNAs were highly conserved, whereas others had changes that could affect processing and, therefore, biological activity. CONCLUSIONS These data demonstrate that KSHV microRNA genes are under tight selection in vivo and suggest that they contribute to the biological activity and possibly the pathogenesis of KSHV-associated malignancies.

High-dose zidovudine plus valganciclovir for Kaposi sarcoma herpesvirus-associated multicentric Castleman disease: a pilot study of virus-activated cytotoxic therapy

Kaposi sarcoma herpesvirus (KSHV)-associated multicentric Castleman disease (MCD) is a lymphoproliferative disorder most commonly observed in HIV-infected patients. It is characterized by KSHV-infected plasmablasts that frequently express lytic genes. Patients manifest inflammatory symptoms attributed to overproduction of KSHV viral IL-6, human IL-6, and human IL-6. There is no standard therapy and no established response criteria. We investigated an approach targeting 2 KSHV lytic genes, ORF36 and ORF21, the protein of which, respectively, phosphorylate ganciclovir and zidovudine to toxic moieties. In a pilot study, 14 HIV-infected patients with symptomatic KSHV-MCD received high-dose zidovudine (600 mg orally every 6 hours) and the oral prodrug, valganciclovir (900 mg orally every 12 hours). Responses were evaluated using new response criteria. A total of 86% of patients attained major clinical responses and 50% attained major biochemical responses. Median progression-free survival was 6 months. With 43 months of median follow-up, overall survival was 86% at 12 months and beyond. At the time of best response, the patients showed significant improvements in C-reactive protein, albumin, platelets, human IL-6, IL-10, and KSHV viral load. The most common toxicities were hematologic. These observations provide evidence that therapy designed to target cells with lytic KSHV replication has activity in KSHV-MCD. This trial was registered at www.clinicaltrials.gov as #NCT00099073.

Human and viral interleukin-6 and other cytokines in Kaposi sarcoma herpesvirus-associated multicentric Castleman disease.

Kaposi sarcoma herpesvirus (KSHV)-associated multicentric Castleman disease (MCD) is a polyclonal B-cell lymphoproliferative disorder. Human (h) IL-6 and a KSHV-encoded homolog, viral IL-6, have been hypothesized to contribute to its pathogenesis, but their relative contributions to disease activity is not well understood. We prospectively characterized KSHV viral load (VL), viral (v) and hIL-6, and other cytokines during KSHV-MCD flare and remission in 21 patients with 34 flares and 20 remissions. KSHV-VL, vIL-6, hIL-6, IL-10, and to a lesser extent TNF-α, and IL-1β were each elevated during initial flares compared with remission. Flares fell into 3 distinct IL-6 profiles: those associated with elevations of vIL6-only (2 flares, 6%), hIL-6 elevations only (17 flares, 50%), and elevations in both hIL-6 and vIL-6 (13 flares, 38%). Compared with hIL-6-only flares, flares with elevated hIL-6 plus vIL-6 exhibited higher C-reactive protein (CRP) (P = .0009); worse hyponatremia (P = .02); higher KSHV VL (P = .016), and higher IL-10 (P = .012). This analysis shows vIL-6 and hIL-6 can independently or together lead to KSHV-MCD flares, and suggests that vIL-6 and hIL-6 may jointly contribute to disease severity. These findings have implications for the development of novel KSHV-MCD therapies targeting IL-6 and its downstream signaling. This trial was registered at clinicaltrials.gov as #NCT099073.

Phase II Study of Bevacizumab in Patients With HIV-Associated Kaposi's Sarcoma Receiving Antiretroviral Therapy

PURPOSE Alternatives to cytotoxic agents are desirable for patients with HIV-associated Kaposis sarcoma (KS). Vascular endothelial growth factor-A (VEGF-A) contributes to KS pathogenesis. We evaluated the humanized anti-VEGF-A monoclonal antibody, bevacizumab, in patients with HIV-KS. PATIENTS AND METHODS Patients with HIV-KS who either experienced progression while receiving highly active antiretroviral therapy (HAART) for at least 1 month or did not regress despite HAART for at least 4 months were administered bevacizumab 15 mg/kg intravenously on days 1 and 8 and then every 3 weeks. The primary objective was assessment of antitumor activity using modified AIDS Clinical Trial Group (ACTG) criteria for HIV-KS. HIV-uninfected patients were also eligible and observed separately. RESULTS Seventeen HIV-infected patients were enrolled. Fourteen patients had been receiving effective HAART for at least 6 months (median, 1 year). Thirteen patients had advanced disease (ACTG T(1)), 13 patients had received prior chemotherapy for KS, and seven patients had CD4 count less than 200 cells/μL. Median number of cycles was 10 (range, 1 to 37 cycles); median follow-up was 8.3 months (range, 3 to 36 months). Of 16 assessable patients, best tumor responses observed were complete response (CR) in three patients (19%), partial response (PR) in two patients (12%), stable disease in nine patients (56%), and progressive disease in two patients (12%). Overall response rate (CR + PR) was 31% (95% CI, 11% to 58.7%). Four of five responders had received prior chemotherapy for KS. Over 202 cycles, grade 3 to 4 adverse events at least possibly attributed to therapy included hypertension (n = 7), neutropenia (n = 5), cellulitis (n = 3), and headache (n = 2). CONCLUSION Bevacizumab is tolerated in patients with HIV-KS and has activity in a subset of patients.

Quantitative assessment of tumor vasculature and response to therapy in kaposi's sarcoma using functional noninvasive imaging.

Two noninvasive methods, thermography and laser Doppler imaging (LDI), were assessed for their ability to quantitatively assess parameters of vascularity in lesions of HIV-associated Kaposis sarcoma (KS). Thermography and LDI images of a representative KS lesion were recorded in 16 patients and compared to normal skin either adjacent to the lesion or on the contralateral side. Eleven of the 16 patients had greater than 0.5 °C increased temperature and 12 of the 16 patients had increased flux (measured by LDI) as compared to normal skin. There was a strong correlation between these two parameters (R = 0.81, p < 0.001). In ten patients, measurements were obtained prior to therapy and after receiving a regimen of liposomal doxorubicin and interleukin-12. After 18 weeks of therapy, temperature and blood flow of the lesions were significantly reduced from the baseline (p = 0.004 and 0.002 respectively). These techniques hold promise to assess physiologic parameters in KS lesions and their changes with therapy.

Sequence Analysis of Kaposi Sarcoma–Associated Herpesvirus (KSHV) MicroRNAs in Patients with Multicentric Castleman Disease and KSHV-Associated Inflammatory Cytokine Syndrome

BACKGROUND Kaposi sarcoma-associated herpesvirus (KSHV) encodes 12 pre-microRNAs that yield 25 mature microRNAs. We previously reported phylogenetic analysis of the microRNA-coding region of KSHV from Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD) patients. We observed a high level of conservation for most sequences but also a divergent cluster of 5 KSHV sequences, including 2 from MCD patients. METHODS KSHV microRNA sequences from 23 MCD patients and 7 patients with a newly described KSHV-associated inflammatory cytokine syndrome (KICS) were examined by amplification, cloning, and sequencing of a 646-bp fragment of K12/T0.7 encoding microRNA-K12-10 and microRNA-K12-12 and a 2.8-kbp fragment containing the remaining 10 pre-microRNAs. RESULTS Phylogenetic analysis showed a distinct variant cluster consisting exclusively of MCD and KICS patients in all trees. Pearson χ(2) analysis revealed that 40 single-nucleotide polymorphisms (SNPs) at various loci were significantly associated with MCD and KICS risk. Cluster analysis of these SNPs generated several combinations of 3 SNPs as putative indicators of MCD and KICS risk. CONCLUSIONS These findings show that MCD and KICS patients frequently have unusual KSHV microRNA sequences and suggest an association between the observed sequence variation and risk of MCD and KICS.

Clinical Features and Outcomes of Patients With Symptomatic Kaposi Sarcoma Herpesvirus (KSHV)-associated Inflammation: Prospective Characterization of KSHV Inflammatory Cytokine Syndrome (KICS)

BACKGROUND Kaposi sarcoma herpesvirus (KSHV) is the cause of Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and a form of Castleman disease (KSHV-MCD). Recently a KSHV-associated inflammatory cytokine syndrome (KICS) distinct from KSHV-MCD was reported. METHODS We prospectively characterized the clinical, laboratory, virologic and immunologic features of KICS by evaluating symptomatic adults with KSHV using a prespecified definition. These features and overall survival were compared with controls from 2 prospectively characterized human immunodeficiency virus (HIV)-infected cohorts, including 1 with KSHV coinfection. RESULTS All 10 KICS subjects were HIV infected males; 5 had HIV viral load (VL) suppressed <50 copies mL (median 72, range <50-74 375); all had KS and 2 also had PEL. All had multiple severe symptoms attributable to KICS: median number of symptoms 8 (6-11), median grade of worst symptom 3 (2-4). These included gastrointestinal disturbance (present in 9); edema (9); respiratory (6); and effusions (5). Laboratory abnormalities included anemia (all); hypoalbuminemia (all) and thrombocytopenia (6). None developed KSHV-MCD; 6 died with median survival from KICS diagnosis 13.6 months. KICS subjects compared with controls had more severe symptoms; lower hemoglobin and albumin; higher C-reactive protein; higher KSHV VL; elevated interleukin (IL)-6 and IL-10; and an increased risk of death (all P < .05). Anemia and hypoalbuminemia at presentation were independently associated with early death. CONCLUSIONS KICS subjects demonstrated diverse severe symptoms, a high rate of KSHV-associated tumors, high mortality, and a distinct IL-6/IL-10 signature. KICS may be an important unrecognized cause of morbidity and mortality, including symptoms previously ascribed to HIV. Exploration of KSHV-directed therapy is warranted.

Bone Marrow Findings in HIV-Positive Patients With Kaposi Sarcoma Herpesvirus-Associated Multicentric Castleman Disease

Kaposi sarcoma herpesvirus (KSHV), also known as human herpesvirus-8, is associated with 1 form of multicentric Castleman disease (MCD) and is the etiologic agent for most MCD in human immunodeficiency virus (HIV)-infected patients. Diagnosis is usually determined by lymph node biopsy. Bone marrow findings in KSHV-MCD are not well characterized. We conducted histomorphologic and immunohistochemical evaluation of bone marrow biopsy specimens in HIV-infected patients with KSHV-MCD, including evaluation for KSHV latency-associated nuclear antigen. Findings were correlated with clinical features and KSHV viral load. Reactive plasmacytosis was the predominant feature. Lymphoid aggregates were less common and not diagnostic of KSHV-MCD. Forty-eight percent of cases contained scattered KSHV-infected mononuclear cells. Although patients were generally cytopenic, bone marrow biopsy specimens were normocellular to hypercellular except in patients receiving hematotoxic therapy. Bone marrow biopsy specimens in KSHV-MCD patients recapitulate findings of interleukin-6 excess. In patients with HIV, unexplained cytopenias, and bone marrow plasmacytosis, evaluation for KSHV-MCD is warranted.

18F-fluorodeoxyglucose Positron Emission Tomography in Kaposi Sarcoma Herpesvirus–Associated Multicentric Castleman Disease: Correlation With Activity, Severity, Inflammatory and Virologic Parameters

BACKGROUND Kaposi sarcoma herpesvirus (KSHV)-associated multicentric Castleman disease (MCD) is a lymphoproliferative inflammatory disorder commonly associated with human immunodeficiency virus (HIV). Its presentation may be difficult to distinguish from HIV and its complications, including lymphoma. Novel imaging strategies could address these problems. METHODS We prospectively characterized (18)F-fluorodeoxyglucose positron emission tomography (PET) findings in 27 patients with KSHV-MCD. Patients were imaged with disease activity and at remission with scans evaluated blind to clinical status. Symptoms, C-reactive protein level, and HIV and KSHV loads were assessed in relation to imaging findings. RESULTS KSHV-MCD activity was associated with hypermetabolic symmetric lymphadenopathy (median maximal standardized uptake value [SUVmax], 6.0; range, 2.0-8.0) and splenomegaly (3.4; 1.2-11.0), with increased metabolism also noted in the marrow (2.1; range, 1.0-3.5) and salivary glands (3.0; range, 2.0-6.0). The (18)F-fluorodeoxyglucose PET abnormalities improved at remission, with significant SUVmax decreases in the lymph nodes (P = .004), spleen (P = .008), marrow (P = .004), and salivary glands (P = .004). Nodal SUVmax correlated with symptom severity (P = .005), C-reactive protein level (R = 0.62; P = .004), and KSHV load (R = 0.54; P = .02) but not HIV load (P = .52). CONCLUSIONS KSHV-MCD activity is associated with (18)F-FDG PET abnormalities of the lymph nodes, spleen, marrow, and salivary glands. These findings have clinical implications for the diagnosis and monitoring of KSHV-MCD and shed light on its pathobiologic mechanism.