KSHV-associated Multicentric Castleman Disease Research Articles

Deconstructing the liquid architecture of human herpesvirus 8 diseases.

Human herpesvirus 8 (HHV-8), also called Kaposi sarcoma herspesvirus (KSHV), causes several human tumours, which may be associated with systemic inflammation and body cavity effusions, including a form of multicentric Castleman disease (MCD) and a novel inflammatory syndrome, KSHV inflammatory cytokine syndrome (KICS). In this issue, Zhou et al. demonstrate that HHV-8-infected lambda-restricted plasmablasts can be detected in these effusions and can be used to distinguish MCD from other HHV-8 tumours as well as to categorise KICS into distinct clinicopathological groups. These findings open a path to an integrated clinicopathological approach to HHV-8-associated inflammatory diseases and may have clinical implications. Commentary on: Zhou et al. A novel approach for characterisation of KSHV-associated multicentric Castleman disease from effusions. Br J Haematol 2023;200:462-475.

A novel approach for characterization of KSHV-associated multicentric Castleman disease from effusions.

A biopsy of lymphoid tissue is currently required to diagnose Kaposi sarcoma-associated herpesvirus (KSHV)-associated multicentric Castleman disease (KSHV-MCD). Patients showing clinical manifestations of KSHV-MCD but no pathological changes of KSHV-MCD are diagnosed as KSHV inflammatory cytokine syndrome. However, a lymph node biopsy is not always feasible to make the distinction. A pathognomonic feature of lymph nodes in KSHV-MCD is the expansion of KSHV-infected, lambda-restricted but polyclonal plasmablasts. To investigate whether these cells also reside in extra-nodal sites, effusion from 11 patients with KSHV-MCD and 19 with KSHV inflammatory cytokine syndrome was analysed by multiparametric flow cytometry. A distinct, lambda-restricted plasmablastic population (LRP) with highly consistent immunophenotype was detected in effusions in 8/11 patients with KSHV-MCD. The same population was also observed in 7/19 patients with KSHV inflammatory cytokine syndrome. The detection of LRP stratified KSHV inflammatory cytokine syndrome into two clinically distinct subgroups; those with detectable LRP closely resembled KSHV-MCD, showing similar KSHV viral load, comparable severity of thrombocytopenia and hypoalbuminaemia, and similar incidences of hepatosplenomegaly. Collectively, the detection of LRP by flow cytometry can serve as a valuable tool in diagnosing KSHV-MCD. KSHV inflammatory cytokine syndrome with LRP in effusions may represent a liquid-form of KSHV-MCD.

Characteristics of patients admitted to the ICU with Kaposi sarcoma herpesvirus-associated diseases.

There are four conditions caused by Kaposi sarcoma herpesvirus (KSHV): Kaposi sarcoma, KSHV-associated multicentric Castleman disease (MCD), primary effusion lymphoma (PEL), and KSHV inflammatory cytokine syndrome (KICS). These KSHV-associated disorders (KADs) often occur in people with HIV and can lead to multiorgan dysfunction requiring admission to the ICU. However, little is known about patient outcomes in this setting. A retrospective study of patients with KADs admitted to the ICU between 2010 and 2021 was conducted, examining KAD admission diagnoses, HIV characteristics, selected cytokine profiles, and ICU interventions. Primary outcomes were 60-day and median overall survival from ICU admission to death from any cause. Forty-seven patients (all but one with HIV coinfection) were included. At ICU admission, 44 patients (94%) were on antiretroviral therapy with a median CD4 + count of 88 cells/μl and HIV viral load of 23 copies/ml. The most common presentation was respiratory failure alone (19%) or with hypotension (17%). Twenty-two (47%) patients had presumed KICS (with or without Kaposi sarcoma) at admission and an additional KAD was diagnosed in 36% of these patients. IL-6 levels did not vary across KAD subtype. Twenty (43%) patients received KAD-directed therapy in the ICU. Sixty-day survival was 70% and median overall survival was 9 months. The majority of patients with HIV and KADs admitted to the ICU had well controlled HIV. Additional KAD were diagnosed during ICU admission in a proportion of patients who presented with presumed KICS. Critical illness did not preclude a subset of patients from receiving KAD-directed therapy in the ICU.

KSHV/HHV8-Associated Lymphoproliferative Disorders: Lessons Learnt from People Living with HIV

In 1992, Kaposi sarcoma herpesvirus (KSHV/HHV8) was discovered and identified as the causative agent for Kaposi sarcoma. Subsequently, the presence of this virus has been detected in a number of lymphoproliferative disorders in people living with HIV (PLWH), including: KSHV-associated multicentric Castleman disease, primary effusion lymphoma, KSHV-positive diffuse large B-cell lymphoma, and germinotropic lymphoproliferative disorder. Each of these rare entities has subsequently been diagnosed in HIV-negative individuals. The recognition of some of these KSHV/HHV8-associated lymphoproliferative disorders has led to their inclusion in the WHO classification of lymphomas in 2008 and the revision of 2016; however, further revision is under way to update the classification. The relatively recent recognition of these lymphoproliferative disorders and their low incidence, particularly in the HIV-negative population, means that there is little published evidence and consensus on their clinical features and management. The publication of a new WHO classification of lymphomas should yield diagnostic clarity, providing an impetus for retrospective case series and prospective clinical trials in these KSHV/HHV8-associated lymphoproliferative disorders.

Characteristics and outcomes of KSHV-associated multicentric Castleman disease with or without other KSHV diseases

Kaposi sarcoma (KS)-associated herpesvirus (KSHV)–associated multicentric Castleman disease (MCD) is a relapsing and remitting systemic lymphoproliferative disorder characterized by severe inflammatory symptoms most common among people living with HIV (PLWH). Patients with KSHV-MCD may present with concurrent KSHV-associated diseases, such as KS and/or primary effusion lymphoma (PEL). We evaluated clinical and immunologic characteristics, the effects of concurrent KSHV malignancies, and treatments from the largest prospective natural history study of participants with KSHV-MCD within the United States. Treatment options administered at investigator discretion included high-dose zidovudine with valganciclovir (AZT/VGC), rituximab, or rituximab with liposomal doxorubicin (R-Dox) during KSHV-MCD flares. Survival analyses and prognostic factors were explored for all participants. Sixty-two participants with HIV were enrolled, including 20 with KSHV-MCD alone, 34 with KSHV-MCD and KS, 1 with KSHV-MCD and PEL, and 7 with all KSHV-associated diseases. Forty-four percent of KSHV-MCD diagnoses were made at our institution. Forty-four participants received rituximab-based therapies, 20 of whom had maintenance AZT/VGC or interferon. Participants receiving R-Dox and then maintenance AZT/VGC had the highest 5-year progression-free survival (89%). Cytokine profiles during KSHV-MCD flares did not differ by the presence of concurrent KSHV-associated diseases. The 10-year survival was 71% (95% confidence interval [CI], 56% to 82%) for all participants. A concurrent diagnosis of PEL negatively impacted survival (PEL hazard ratio, 5.4; 95% CI, 1.8 to 16.8). KSHV-MCD is an underdiagnosed condition among PLWH, including those with KS. KSHV-MCD has an excellent prognosis with appropriate treatment. Physicians should be alert for patients with multiple KSHV diseases, which impact optimal treatment and survival outcomes. This study was registered at www.clinicaltrials.gov as #NCT00099073.

Elevated IL-13 in effusions of patients with HIV and primary effusion lymphoma as compared with other Kaposi sarcoma herpesvirus-associated disorders.

To assess the cytokine and viral profiles of effusions and peripheral blood among patients diagnosed with HIV and Kaposi sarcoma herpesvirus [KSHV, also known as human herpesvirus 8 (HHV-8)]-associated conditions. Retrospective comparative study evaluating clinicopathologic findings in patients with HIV and KSHV-associated conditions presenting with an effusion between 2010 and 2018. Paired peripheral blood and effusion samples collected at the time of pathological diagnosis of KSHV-associated conditions [Kaposi sarcoma, KSHV-associated multicentric Castleman disease (KSHV-MCD), primary effusion lymphoma (PEL), or KSHV-associated inflammatory cytokine syndrome (KICS)] were evaluated for disease-specific and compartment-specific (effusion vs. blood) characteristics. We assessed 12 cytokines, KSHV viral DNA (KSHV-VL), and Epstein--Barr virus (EBV) viral DNA (EBV-VL). Nine patients had PEL, five patients had KSHV-MCD, and eight patients met criteria for KICS; all but one patient had concurrent Kaposi sarcoma in addition to these conditions. PEL effusions had substantially higher levels of IL-13 (median 16.9 pg/ml; interquartile range 9.7--26.9 pg/ml) compared with KSHV-MCD (median <0.114 pg/ml; P = 0.0037) or KICS (median <0.114 pg/ml; P = 0.0003) effusions. IL-13 was also higher in PEL effusions as compared with serum (median <0.12 ng/ml; P = 0.007). KSHV-VL levels were significantly higher in PEL effusions as compared with KICS effusions (median 31 × 10 vs. 569 copies/million-cell equivalent; P = 0.0005) or KSHV-MCD effusions (median 231,884 copies/million-cell equivalent; P = 0.02). PEL effusions had a distinct profile as compared to other KSHV-associated diseases with regard to elevated IL-13 and KSHV-VL. These findings may provide insights into PEL pathogenesis and aid in diagnosis.

Phase I study of pembrolizumab in people with HIV and cancer.

2500 Background: People with HIV have been excluded from immuno-oncology (IO) studies. Anti- PD-1/PD-L1 therapies are approved for a growing number of cancers. We evaluated pembrolizumab (pembro) in people with HIV and cancer. Methods: CITN-12 is a multicenter phase 1 trial. Key eligibility: advanced cancer; ECOG ≤1; CD4 ≥100 cells/μL; ≥4 weeks antiretroviral therapy (ART), HIV viral load (VL) &lt; 200 copies/mL. Exclusion: uncontrolled HBV/HCV, autoimmune disease. Participants (pts) accrued into CD4 based cohorts (C): C1 100-199; C2 200-350; C3 &gt; 350 CD4 cells/μL. Pembro 200 mg IV administered Q3W for up to 35 doses. Adverse events (AE) evaluated by CTCAE. Immune related AE ≥ grade (Gr) 2 were events of clinical interest (irECI). Clinical benefit (tumor shrinkage or stable disease [SD] ≥24 weeks) was estimated. Data were locked for safety analysis and publication once C2 and C3 completed accrual and all pts completed ≥2 cycles. Accrual continued for 6 C1 pts and a new phase 1b Kaposi sarcoma (KS) cohort (C4). Results: 30 pts, characteristics: C1 (6), C2 and 3 (12 each), median (med) age 57 years (range 39-77), 28 men, 2 women, 60% White, 30% Black, 10% Hispanic. Med CD4 285 cells/μL (132 - 966). Cancers: KS (6), non-Hodgkin lymphoma (NHL) (5), non-AIDS defining (19) – most common: anal (6) and squamous cell skin (3). Prior radiation (19), med prior systemic therapies 2 (0-8). Safety observed over 183 cycles, med 5 (1-32). Treatment emergent AE ≥ possibly attributed to pembro mostly Gr 1-2, with 20% of pts having Gr 3. irECI: hypothyroidism (6), elevated AST/ALT (1), pneumonitis (3), rash (2), musculoskeletal (1).1 KS pt developed KSHV-associated multicentric Castleman disease (KSHV-MCD) and died of the AE. HIV was controlled and increasing CD4 counts were observed. Best response: complete (lung, 1), partial (NHL, 2), SD ≥24 weeks (KS, 2), SD &lt; 24 weeks (13), progressive disease (10), not evaluable (2). Conclusions: Pembro has acceptable safety in cancer pts with HIV on ART and &gt; 100 CD4 cells/µL, similar to patients without HIV. Anti-PD-1 may unmask KSHV-MCD and such KSHV-viremic patients should be excluded. Clinical benefit was noted in several tumor types. Anti-PD1 is appropriate for FDA-approved indications in this population. Patients with HIV meeting appropriate eligibility criteria should be included in IO studies. Clinical trial information: NCT02595866.

The Contribution of Kaposi's Sarcoma-Associated Herpesvirus to Mortality in Hospitalized Human Immunodeficiency Virus-Infected Patients Being Investigated for Tuberculosis in South Africa.

Despite increasing numbers of human immunodeficiency virus (HIV)-infected South Africans receiving antiretroviral therapy (ART), tuberculosis (TB) remains the leading cause of mortality. Approximately 25% of patients treated for TB have microbiologically unconfirmed diagnoses. We assessed whether elevated Kaposi's sarcoma-associated herpesvirus (KSHV) viral load (VL) contributes to mortality in hospitalized HIV-infected patients investigated for TB. Six hundred eighty-two HIV-infected patients admitted to Khayelitsha Hospital, South Africa, were recruited, investigated for TB, and followed for 12 weeks. KSHV serostatus, peripheral blood KSHV-VL, and KSHV-associated clinical correlates were evaluated. Median CD4 count was 62 (range, 0-526) cells/μL; KSHV seropositivity was 30.7% (95% confidence interval [CI], 27%-34%); 5.8% had detectable KSHV-VL (median, 199.1 [range, 13.4-2.2 × 106] copies/106 cells); 22% died. Elevated KSHV-VL was associated with mortality (adjusted odds ratio, 6.5 [95% CI, 1.3-32.4]) in patients without TB or other microbiologically confirmed coinfections (n = 159). Six patients had "possible KSHV-inflammatory cytokine syndrome" (KICS): 5 died, representing significantly worse survival (P < .0001), and 1 patient was diagnosed with KSHV-associated multicentric Castleman disease at autopsy. Given the association of mortality with elevated KSHV-VL in critically ill HIV-infected patients with suspected but not microbiologically confirmed TB, KSHV-VL and KICS criteria may guide diagnostic and therapeutic evaluation.

Viral, immunologic, and clinical features of primary effusion lymphoma

AbstractPrimary effusion lymphoma (PEL) is an aggressive HIV-associated lymphoma with a relatively poor prognosis in the era of effective HIV therapy. Kaposi sarcoma herpesvirus (KSHV) is the etiologic agent, and ∼80% of tumors are coinfected with Epstein-Barr virus (EBV). A better understanding of how KSHV-related immune dysregulation contributes to the natural history of PEL will improve outcomes. Twenty patients with PEL diagnosed between 2000 and 2013, including 19 treated with modified infusional etoposide, vincristine, and doxorubicin with cyclophosphamide and prednisone (EPOCH), were identified. We compared their clinical, virologic, and immunologic features vs 20 patients with HIV-associated diffuse large B-cell lymphoma and 19 patients with symptomatic interleukin (IL)-6 related KSHV-associated multicentric Castleman disease. Survival analyses of treated patients with PEL were then performed to identify prognostic factors and cancer-specific mortality. Compared with HIV-associated diffuse large B-cell lymphoma, PEL was associated with significant hypoalbuminemia (P < .0027), thrombocytopenia (P = .0045), and elevated IL-10 levels (P < .0001). There were no significant differences in these parameters between PEL and KSHV-associated multicentric Castleman disease. Median overall survival in treated patients with PEL was 22 months, with a plateau in survival noted after 2 years. Three-year cancer-specific survival was 47%. EBV-positive tumor status was associated with improved survival (hazard ratio, 0.27; P = .038), and elevated IL-6 level was associated with inferior survival (hazard ratio, 6.1; P = .024). Our analysis shows that IL-6 and IL-10 levels contribute to the natural history of PEL. Inflammatory cytokines and tumor EBV status are the strongest prognostic factors. Pathogenesis-directed first-line regimens are needed to improve overall survival in PEL.

Induction of Kaposi's Sarcoma-Associated Herpesvirus-Encoded Viral Interleukin-6 by X-Box Binding Protein 1.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent for Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and a subset of multicentric Castleman disease (MCD). The KSHV life cycle has two principal gene repertoires, latent and lytic. KSHV viral interleukin-6 (vIL-6), an analog of human IL-6, is usually lytic; production of vIL-6 by involved plasmablasts is a central feature of KSHV-MCD. vIL-6 also plays a role in PEL and KS. We show that a number of plasmablasts from lymph nodes of patients with KSHV-MCD express vIL-6 but not ORF45, a KSHV lytic gene. We further show that vIL-6 is directly induced by the spliced (active) X-box binding protein-1 (XBP-1s), a transcription factor activated by endoplasmic reticulum (ER) stress and differentiation of B cells in lymph nodes. The promoter region of vIL-6 contains several potential XBP-response elements (XREs), and two of these elements in particular mediate the effect of XBP-1s. Mutation of these elements abrogates the response to XBP-1s but not to the KSHV replication and transcription activator (RTA). Also, XBP-1s binds to the vIL-6 promoter in the region of these XREs. Exposure of PEL cells to a chemical inducer of XBP-1s can induce vIL-6. Patient-derived PEL tumor cells that produce vIL-6 frequently coexpress XBP-1, and immunofluorescence staining of involved KSHV-MCD lymph nodes reveals that most plasmablasts expressing vIL-6 also coexpress XBP-1. These results provide evidence that XBP-1s is a direct activator of KSHV vIL-6 and that this is an important step in the pathogenesis of KSHV-MCD and PEL. Kaposi sarcoma herpesvirus (KSHV)-associated multicentric Castleman disease (KSHV-MCD) is characterized by severe inflammatory symptoms caused by an excess of cytokines, particularly KSHV-encoded viral interleukin-6 (vIL-6) produced by lymph node plasmablasts. vIL-6 is usually a lytic gene. We show that a number of KSHV-MCD lymph node plasmablasts express vIL-6 but do not have full lytic KSHV replication. Differentiating lymph node B cells express spliced (active) X-box binding protein-1 (XBP-1s). We show that XBP-1s binds to the promoter of vIL-6 and can directly induce production of vIL-6 through X-box protein response elements on the vIL-6 promoter region. We further show that chemical inducers of XBP-1s can upregulate production of vIL-6. Finally, we show that most vIL-6-producing plasmablasts from lymph nodes of KSHV-MCD patients coexpress XBP-1s. These results demonstrate that XBP-1s can directly induce vIL-6 and provide evidence that this is a key step in the pathogenesis of KSHV-MCD and other KSHV-induced diseases.

Rituximab plus liposomal doxorubicin in HIV-infected patients with KSHV-associated multicentric Castleman disease

AbstractKaposi sarcoma (KS) herpesvirus–associated multicentric Castleman disease (KSHV-MCD) is a lymphoproliferative disorder, most commonly seen in HIV-infected patients, that has a high mortality if untreated. Concurrent KS is common. Although rituximab has reported activity in KSHV-MCD, its use is often associated with KS progression. Within a natural history study of KSHV-MCD, we prospectively evaluated rituximab 375 mg/m2 combined with liposomal doxorubicin 20 mg/m2 (R-Dox) every 3 weeks in 17 patients. Patients received a median of 4 cycles (range 3-9). All received antiretroviral therapy, 11 received consolidation interferon-α, and 6 received consolidation high-dose zidovudine with valganciclovir. Using NCI KSHV-MCD response criteria, major clinical and biochemical responses were attained in 94% and 88% of patients, respectively. With a median 58 months’ potential follow-up, 3-year event-free survival was 69% and 3-year overall survival was 81%. During R-Dox therapy, cutaneous KS developed in 1 patient, whereas 5 of 6 patients with it had clinical improvement. R-Dox was associated with significant improvement in anemia and hypoalbuminemia. KSHV viral load, KSHV viral interleukin-6, C-reactive protein, human interleukin-6, and serum immunoglobulin free light chains decreased with therapy. R-Dox is effective in symptomatic KSHV-MCD and may be useful in patients with concurrent KS. This trial was registered at www.clinicaltrials.gov as #NCT00092222.

KSHV‐associated multicentric Castleman disease: A tangle of different entities requiring multitarget treatment strategies

Multicentric Castleman Disease (MCD) is a lymphoproliferative disorder presenting with heterogeneous pathological and clinical features. It comprises disease entities with a complex aetiology and overlapping pathogenesis. MCD can be found in association with HIV infection, plasma-cell dyscrasias, Kaposi sarcoma (KS), B-cell lymphomas including primary effusion lymphoma (PEL) and its solid variant, and Hodgkin lymphoma. In KSHV-associated MCD cases, a common association is KS and a specific variant of lymphoma referred to as "plasmablastic lymphoma," also called "large B-cell lymphoma arising in KSHV-associated MCD" lacking EBV infection. MCD is often referred to as human interleukin-6 (hIL-6) syndrome, since an overproduction of IL-6 occurs in MCD-associated diseases as well as in MCD itself. hIL-6 and a viral IL-6 (vIL-6) homolog encoded by KSHV can independently or together lead to flares of KSHV-associated MCD. Recently, a new clinical entity was proposed to describe a severe systemic infection/reactivation of KSHV: KSHV inflammatory syndrome (KICS). KICS may contribute in inducing the inflammatory symptoms seen in some patients with severe KS or PEL. The precise relationship of KICS to KSHV-associated MCD is unclear and it is possible that KICS may be prodromal symptoms to frank KSHV-associated MCD. Options for treatment of KSHV-associated MCD and related diseases include monoclonal antibodies, chemotherapy, immune modulators, virus-activated cytotoxic therapy and antiviral therapies. A comprehensive understanding of the intricacies of the HIV-KSHV coinfection will probably lead to additional advances in therapy and managements for these disorders.

The Use of Monoclonal Antibodies to Treat Castleman‘s Disease

Multicentric Castleman's disease (MCD) is a rare lymphoproliferative disorder presenting with heterogeneous clinical features and with a complex etiology. MCD incidence is increased in people living with HIV/AIDS when it is causally associated with Kaposi's sarcoma-associated herpes virus (KSHV). HIV-seronegative individuals present with either idiopathic or KSHV-associated MCD. Central to MCD pathology is altered expression and signaling of IL-6, which promotes B-cell proliferation and causes systemic manifestations. KSHV encodes a viral homolog of human IL-6, accounting for its role in MCD, while recent evidence shows an association between IL-6 receptor polymorphisms and idiopathic MCD. The increased understanding of mechanisms underlying the pathogenesis of MCD has guided the use of new monoclonal antibody therapies for treating this complex disorder.

Clinical, Immunologic, and Virologic Findings in Kaposi Sarcoma Herpesvirus (KSHV)-Associated Lymphomas Suggest KSHV-Associated Inflammatory Syndromes Contribute to Symptoms and Disease Pathogenesis.

AbstractAbstract 2708 Background:KSHV, also known as human herpesvirus-8, is the etiologic agent of a lymphoproliferative disorder, KSHV-associated multicentric Castleman disease (KSHV-MCD), two lymphomas: primary effusion lymphoma (PEL) and large cell lymphoma arising in KSHV-MCD (together, KSHV-NHL) and Kaposi sarcoma (KS). KSHV-associated diseases mainly occur in patients with HIV. KSHV is notable for its modulation of host immune response, including induction of IL6 and IL10; and an IL6-related KSHV-associated inflammatory cytokine syndrome (KICS) has been described in HIV/KSHV infected patients without KSHV-MCD. The clinical features and natural history of KSHV-NHL are not well understood. We hypothesize KSHV-NHL is associated with pathophysiologic features similar to those of KSHV-MCD, and is unique among HIV-associated lymphomas. Methods:Clinical records of patients with KSHV-NHL diagnosed 2000-12, treated in the HIV and AIDS Malignancy Branch Clinic were reviewed, and evaluated for clinical, and laboratory abnormalities using our criteria for KICS (NCT01419561): at least 2 select clinical and laboratory manifestations, not otherwise explained; elevated c-reactive protein [CRP]; and KSHV viral load greater than 100 copies/106 PBMC; Additionally, serum inflammatory cytokines, (IFN-gamma, TNF-alpha, IL-1beta, IL-6, IL-8, IL-10, IL12p70; Mesoscale Discovery, Gathersburg, MD) as well as PBMC-associated KSHV viral load, platelets, hemoglobin, and albumin were evaluated in KSHV-NHL patients, and compared to patients with A) Symptomatic KSHV-MCD and no KSHV-NHL, and B) Other HIV-associated lymphomas. Comparisons used exact two-tailed Wilcoxon rank sum tests, p<0.005 was considered statistically significant, p<0.05 strong trends. Results:Study cohort: 14 patients with HIV and KSHV-NHL; men (13), woman (1); median (med) age 42 yrs (26, 60); African American (4), African (2), Hispanic (4), white (4). Diagnoses included PEL (12, 5 with extracavitary manifestations) and large cell lymphoma in KSHV-MCD (2). Of the 14, 5 had pathologically confirmed KSHV-MCD, and 10 had cutaneous KS. Laboratory data: med CD4 126 cells/uL (15,1072), HIV viral load <100 copies/ml (7). All 10 KSHV-NHL with complete CRP and KSHV data either met criteria for KICS or had a history of KSHV-MCD; med CRP 48.8 g/dL (4.6, 114). Controls: A) KSHV-MCD (n=19): med age 43 (29, 54), med CD4 266 (67,1319). B) Other HIV-associated lymphoma (n=28): med age 37 (21, 60); med CD4 29 cells/uL (0, 730). Laboratory, immunologic, and virologic parameters were comparable between KSHV-NHL and KSHV-MCD, with no significant differences or trends towards differences in CRP, hemoglobin, platelets, CD4 count, albumin, sodium, KSHV viral load, or any measured cytokine. Compared to patients with other HIV-associated lymphomas, those with KSHV-NHL had statistically significant elevated serum IL10 (med 513 vs 12.2 pg/ml, p<0.0001), IL6 (29 vs 4.1 pg/dl, p=0.0011), KSHV viral load (med 3913 vs. 0 copies/106 PBMC, p<0.0001), and hypoalbuminemia (med 1.8 vs 3.5 mg/dL, p=0.001) (Figure). Strong trends towards statistically significant thrombocytopenia (med 87 vs 246 K/uL, p = 0.0064), anemia (med 8.9 vs 11.1 gm/dl, p=0.019), hyponatremia (med 134 vs 137 mEq/L, p=0.03), and elevated IL1beta (1.6 vs. 0.5 pg/dL, p=0.024), and IFNgamma (med 6 vs 2.9 pg/dl, p=0.038) were also observed. [Display omitted] Conclusions:In the era of antiretroviral therapy, KSHV-NHL in HIV occurs at a broader range of CD4 counts than previously appreciated, often in the setting of suppressed HIV. Significant overlap exists between KSHV-associated lymphoproliferative disease and KSHV-NHL. Essentially all KSHV-NHL patients presented with inflammatory symptoms, elevated CRP, hypoalbuminemia, and cytopenias, and either had KSHV-MCD or met our working criteria for KICS. Elevated KSHV-infected PBMCs and associated KSHV modulation of host immune response leading to marked elevations in IL10 and IL6 likely contribute to symptoms and KSHV-NHL pathogenesis. KSHV-MCD and KICS Natural History studies are ongoing, and evaluation of curative-intent regimens for KSHV-NHL incorporating strategies to target these unique immunologic and virologic abnormalities is warranted. Disclosures:No relevant conflicts of interest to declare.

Recent advances in Kaposi sarcoma herpesvirus-associated multicentric Castleman disease.

The discovery of Kaposi sarcoma herpesvirus (KSHV) led to recognition of KSHV-associated multicentric Castleman disease (MCD) as a distinct lymphoproliferative disorder. The pathogenesis of KSHV-MCD is attributed to proliferation of KSHV-infected B cells, production of KSHV-encoded viral interleukin 6 by these cells, and dysregulation of human interleukin 6 and interleukin 10. This article reviews advances in the field of disease pathogenesis and targeted therapies. Our understanding of the pathogenesis of KSHV-MCD has increased in recent years and improved therapies have been developed. Recent studies demonstrate that the anti-CD20 monoclonal antibody, rituximab, as well as virus-activated cytotoxic therapy using high-dose zidovudine and valganciclovir, can control symptoms and decrease adenopathy. With treatment, 1-year survival now exceeds 85%. Interestingly, even in the absence of pathologic findings of MCD, KSHV-infected patients may have inflammatory symptoms, excess cytokine production, and elevated KSHV viral load similar to KSHV-associated MCD. The term KSHV-associated inflammatory cytokine syndrome has been proposed to describe such patients. Recent advances in targeted therapy have improved outcomes in KSHV-MCD, and decreased need for cytotoxic chemotherapy. Improved understanding of the pathogenesis of KSHV-MCD and KSHV-associated inflammatory cytokine syndrome is needed, and will likely lead to additional advances in therapy for these disorders.

Distinct Human and Viral Interleukin-6 Profiles and Other Viral and Immunologic Abnormalities In KSHV-Associated Multicentric Castleman Disease: Relationship with Disease Activity and Individual Disease Manifestations

Abstract 1573 Background:Multicentric Castleman disease (MCD) is a polyclonal B-cell lymphoproliferative disorder characterized by flares of severe inflammatory symptoms, including fever and cachexia, with cytopenias and biochemical abnormalities. In the idiopathic form, its pathogenesis is linked to overproduction of interleukin (IL)-6. A distinct form of MCD is caused by Kaposi sarcoma-associated herpesvirus (KSHV, also called human herpesvirus [HHV]-8). KSHV encodes a viral homolog of IL-6, vIL-6, and this has been hypothesized to be central to KSHV-MCD pathogenesis. However, viral and human cytokines have not been examined together in KSHV-MCD, and their contribution to disease activity and symptoms is not known. Methods:Patients with pathologically-proven KSHV-MCD were enrolled on a prospective natural history study incorporating pilot evaluation of novel therapies (NCT00099073). Factors potentially important in pathogenesis were assayed at flare and complete clinical and laboratory remission: KSHV viral load (VL) in peripheral blood mononuclear cells, vIL-6, IL-1β, IL-5, human IL-6 (hIL-6), IL-8, IL-10, IL-12p70, IFN-γ and TNF-α. Paired analyses were performed for each patient comparing initial flare and remission. Associations between cytokines and individual disease manifestations were explored across all flares. Results:21 patients had at least one flare for analysis (19 [90%] male; med age 44 [range 29–52]). 34 flares were observed (range 1–3 per patient) and followed to remission (20 patients) or death (1). All were HIV infected: CD4 med 252cells/μL (range 24–1319), HIV VL med <48 copies/mL (<48–64,100), 94% receiving antiretrovirals. Clinical symptoms included fever (present in 62%, med 38°C [range 36.1–40.5]); fatigue (91%, med CTC grade 2 [0–3]), gastrointestinal (GI) symptoms (68%, med grade 1 [0–3]) and respiratory symptoms (61%, med grade 1 [0–2]). Laboratory findings included anemia (97%, 9.9g/L [6.8–14.4]), thrombocytopenia (68%, 100×103cells/μL [6–567]), hypoalbuminemia (97%, 2.7g/L [1.2–3.9]) hyponatremia (68%, 133mEg/L [127–143]) and C-reactive protein (CRP) elevation (100%, 87.3g/L [6.3–339.5]).Factors elevated during initial flares were: KSHV VL (med 14,700/106 PBMCs [range 0–3,913,000] P <0.0001 compared with remission); vIL-6 (detected in 48%, <1560pg/mL [<1560-20,500] P= 0.0039); hIL-6 (15.9pg/mL [1.4-171.5] P= 0.0006); IL-10 (449pg/mL [2.8-85,900] P= 0.0007); TNF-α (29.0 pg/ml [7.9–90.8] P= 0.0083) and IL-1β (1.2 pg/ml [0.1–5.7] P= 0.0027). IL-5 was decreased compared with remission (0.6 [0.1–15.4] P= 0.016). Differences were most marked for vIL-6 (undetectable in all remissions), hIL-6 (med increase from remission 520%) and IL-10 (med increase from remission 5000%). KSHV VL was correlated with hIL-6 (R = 0.68, P= 0.001) and IL-10 (R=0.82, P= 0.001) across all flares and remissions; other correlations were weaker. There was also a strong association between KSHV VL and vIL-6 (P= 0.001 by Jonckheere-Terpstra). For hIL-6 and vIL-6 only, we observed distinct profiles across flares: vIL-6 elevation only (2 flares, 6%), hIL-6 only (17 flares, 50%), and hIL-6 with vIL-6 (13 flares, 38%). In 2 flares (6%), neither vIL-6 nor hIL-6 were detected at onset, but in both hIL-6 alone soon became elevated. In contrast, KSHV VL, IL-10, TNF-α and IL-1β were consistently elevated during flares. Compared with hIL-6-only flares, hIL-6 with vIL-6 flares exhibited significantly higher CRP (P =0.0009); worse hyponatremia (P =0.02); higher KSHV VL (P =0.016) and IL-10 (P= 0.012); and lower IL-5 (P= 0.009). In linear/logistic regression models of disease manifestations with cytokines, best predictors were: GI symptoms, respiratory symptoms, temperature, platelet count: hIL-6 alone; hemoglobin: hIL-6 and vIL-6; sodium: hIL-6, vIL-6 and IL-10; albumin: IL-1β and vIL-6. Conclusions:This prospective analysis shows for the first time that vIL-6 and hIL-6 can independently or together lead to flares of KSHV-MCD. It shows novel associations of KSHV-MCD flares with IL-1β and TNF-α elevation and IL-5 depression while confirming associations with KSHV VL and IL-10. It further suggests that vIL-6 and hIL-6 may jointly contribute to the severity of some symptoms during flares. As vIL-6 can signal independently of the ligand-binding IL-6 receptor-a, these findings have implications for the development of novel KSHV-MCD therapies targeting IL-6 and its downstream signaling. Disclosures:No relevant conflicts of interest to declare.

Kaposi's sarcoma-associated herpesviral IL-6 and human IL-6 open reading frames contain miRNA binding sites and are subject to cellular miRNA regulation.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a viral interleukin 6 (vIL-6) that mimics many activities of human IL-6 (hIL-6). Both vIL-6 and hIL-6 play important roles in stimulating the proliferation of tumours caused by KSHV. Here, we provide evidence that a miRNA pathway is involved in regulation of vIL-6 and hIL-6 expression through binding sites in their open reading frames (ORFs). We show a direct repression of vIL-6 by hsa-miR-1293 and hIL-6 by hsa-miR-608. The repression of vIL-6 by miR-1293 was reversed by disruption of the vIL-6 miR-1293 seed match through the introduction of point mutations. In addition, expression of vIL-6 or hIL-6 in KSHV-infected cells could be enhanced by transfection of the respective miRNA inhibitors. In situ hybridization of human lymph node sections revealed that miR-1293 is primarily expressed in the germinal centre but is deficient in the mantle zone of lymph nodes, where the expression of vIL-6 is often found in patients with KSHV-associated multicentric Castleman's disease, providing evidence of an anatomical correlation. Taking these factors together, our study indicates that IL-6 expression can be regulated by miRNA interactions in its ORF and provides evidence for the role of these interactions in the pathogenesis of KSHV-associated diseases.

KSHV activation, human and viral IL-6 production, and other cytokine dysregulation: Association with the symptomatology of KSHV-associated multicentric Castleman's disease

KSHV activation, human and viral IL-6 production, and other cytokine dysregulation: Association with the symptomatology of KSHV-associated multicentric Castleman’s disease Mark N Polizzotto, Thomas S Uldrick, Victoria Wang, Karen Aleman, Kathleen M Wyvill, Vickie Marshall, Stefania Pittaluga, Dierdre O’Mahony, Denise Whitby, Giovanna Tosato, Seth M Steinberg, Richard F Little, Robert Yarchoan From 12 International Conference on Malignancies in AIDS and Other Acquired Immunodeficiencies (ICMAOI) Bethesda, MD, USA. 26-27 April, 2010

Generation of vFLIP Transgenic Mice: A Model to Study KSHV-Associated Lymphomagenesis

Primary effusion lymphoma (PEL) is a distinct subtype of aggressive non-Hodgkin's lymphoma (NHL), specifically associated with infection by Kaposi's sarcoma-associated herpesvirus (KSHV) and occurring more frequently in HIV-infected individuals. Several in vitro observations suggest that vFLIP, a viral protein expressed during latency, is an important viral oncogene. It is essential for the survival of KSHV-infected PEL cells, mainly by constitutively activating the NF-kB pathway. In order to assess the role of vFLIP in the pathogenesis of PEL, we knocked a cDNA encoding vFLIP, preceded by a loxP-flanked neoR-Stop cassette and followed by Frt-flanked IRES-eGFP sequences, into the ubiquitously expressed ROSA26 locus. A specifically restricted expression of the transgene in CD19+ B-cells has been achieved by crossing the ROSA26. vFLIP knock-in mice with mice expressing cre recombinase under the control of the CD19 promoter. These mice have also been crossed with transgenic mice expressing KSHV LANA, which is considered to also be a viral oncogene, to assess a potential synergistic effect of these two KSHV latent proteins in the lymphomagenic process of PEL. vFLIP expression in the CD19+ B-cells results in splenomegaly, with an increase in both T and B-cells, and with a relative increase of the T versus B-cell ratio. Although primary follicles were enlarged, the expression of vFLIP in the CD19+ B-cells results in lack of germinal center formation in the spleen, lymph nodes and intestine, and in partially impaired class-switching recombination. vFLIP transgenic mice had an increased number of plasmablast-like cells expressing lambda light chain, reminiscent of a phenomenon seen in KSHV-associated multicentric Castleman's disease (MCD). These results indicate that by constitutively activating the NF-κB pathway in pre-germinal center B-cells expressing CD19, the normal B-cell differentiation is impaired, and provide clues about possible aberrant differentiation in PEL and MCD.

Early Results of a Clinical Trial Using Targeted Oncolytic Virotherapy in Kaposi Sarcoma Herpes Virus (KSHV) Associated Multicentric Castleman's Disease (MCD).

Human herpes virus 8, also termed KSHV, is an oncogenic gamma herpesvirus associated with Kaposi's sarcoma (KS), primary effusion lymphoma and MCD. KSHV-associated MCD (KSHV-MCD), a rare B-cell lymphoproliferative disease almost universally found in association with HIV infection, is characterized by recurrent flares of a systemic syndrome of fatigue, fevers, cytopenias, elevated serum C-reactive protein (CRP), and lytic KSHV replication. Prognosis is poor (median survival 14 months). KSHV open reading frames (ORF) 21 and 36 respectively have the ability to phosphorylate zidovudine and ganciclovir, leading to cell death (Cancer Res 2007; 67(14):7003–10), and this may be exploitable for oncolytic virotherapy. Ten patients (pts) with symptomatic and biopsy confirmed KSHV-associated MCD have been treated with high dose oral zidovudine (HDAZT) 600mg every 6 hours and valganciclovir (VGCV) 900mg every 12 hours. Treatment length of first cycle is dependent on response, and ranges from 7–21 days. Subsequent cycle length is 21 days with 7 treatment days. Pt characteristics: median age 40 (range 33–56); ECOG PS 2 (1–3); median enrollment CD4 count 189 (range 19–1319 UL); median HIV viral load <50 copies/ml3 plasma (range <50 to 27,500). All pts were on highly active antiretroviral therapy; this was adjusted during time pts were on AZT. 8 pts had a history of KS. Median duration of MCD 3.5 months (range 0.5–45 months); seven pts had received at least one prior therapy for MCD (range 1–6). All had MCD-related constitutional symptoms and CRP above 0.8mg/dl (median 13.1, range 1.06–38.7 mg/dl) at treatment initiation. A total of 83 cycles have been administered to date, with a median of 6 (range 4–23) cycles per pt. 9/10 had documented improvement in constitutional symptoms, C-reactive protein levels or cytopenias. Therapy continues in 5 patients who have had sustained responses (median duration on therapy 7 months, range 3–18). Response was only short-lived in 4 (median duration on therapy 4.5 months, range 3.5–6.5) who then received alternative treatments. Grade (gr) 3 or 4 hematologic toxicity not attributable to disease was seen in only 2 patients. Two infectious events occurred, a staphylococcal skin abscess and streptococcal meningitis. There were no neutropenia associated infections. Treatment toxicity included two patients with fatigue (gr3), 1 with nausea (gr3), 1 with transaminitis (gr3) and one with insomnia (gr3). In summary, this preliminary data suggests that HDAZT and VGCV has activity useful in the management of KSHV-associated MCD. Accrual continues.