CROI 2019 Abstract eBook

Abstract eBook

Oral Abstracts


individuals or immune suppressed transplant patients. The prevalence for both KSHV and KS are highest in sub-Saharan Africa where HIV-1 infection is also epidemic. Current therapies for KS are not effective, with high reoccurrence and mortality rate. Similar to other herpesviruses, KSHV’s ability to establish latency in the host presents a major challenge to KS treatment or prevention. Among KSHV genes, the latency-associated nuclear antigen (LANA) is absolutely required for latency. Hence, strategies to eliminate LANA from KSHV latently infected cells might lead to prevention or treatment of KS. Methods: We designed a replication-incompetent adenovirus to deliver LANA- specific CRISPR-Cas9 system (Ad-CC9-LANA) at high efficiency into various KSHV latently infected cells and monitored over a period of 32 days. The effects of Ad- CC9-LANA had on KSHV episome in latently infected cells were then determined by droplet digital PCR. Real-time PCR was utilized to measure the mRNA expressions for LANA and Cas9. Immunohistochemistry (IHC) was performed to demonstrate the reduction of KSHV latently infected cells in Ad-CC9-LANA transduced cultures. Results: Reduction in KSHV episome was evidence as early as 4 days of transduction by Ad-CC9-LANA. At 32 days post-transduction, the Ad-CC9-LANA transduced cultures demonstrated a substantial reduction in KSHV episome copy number in latently infected cells. These reductions were accompanied by decrease in the LANA mRNA expression and confirmed by IHC. These observations were not due to cell death due to adenovirus transduction as demonstrated by the similar growth kinetic between transduced and non- transduced cells. The Cas9 mRNA expression was also shown to be robust and detected throughout the study period. Conclusion: Our study demonstrated the feasibility of using a KSHV LANA- targeted CRISPR-Cas9 system to disrupt KSHV latency in infected epithelial and endothelial cell lines. This approach to limit KSHV latency may also represent a viable strategy for against other tumorigenic viruses such as HCV, HPV and EBV. Therefore, it will have significant benefits to human health worldwide and particularly in developing countries where the viral cancer burden is high. 19 THE ROLE OF WILMS' TUMOR 1 IN KAPOSI SARCOMA HERPESVIRUS ONCOGENESIS Ayana Morales 1 , Ethel Cesarman 1 , Paul Rubinstein 2 , Warren Phipps 3 1 Weill Cornell Medicine, New York, NY, USA, 2 Rush University Medical Center, Chicago, IL, USA, 3 University of Washington, Seattle, WA, USA Background: Kaposi Sarcoma (KS), caused by HHV-8, is the most common HIV associated malignancy globally. It occurs predominantly in sub-Saharan Africa where it has a high mortality rate. Despite the burden of KS, it is unknown if KSHV causes a reactive proliferative process or a clonal malignancy due to oncogenic genetic alterations that occur in latent infection due to genetic instability. Discovery of recurrent genetic alterations would provide an improved understanding of KS pathogenesis and may allow for the development of prognostic biomarkers and improved treatment options. A promising cancer antigen is WT1 (Wilms’ Tumor 1), for which WT1 therapeutic vaccines have demonstrated benefit in patients with leukemias and solid tumors, and has served as a prognostic marker in patients with myelodysplastic syndromes and leukemias. Different isoforms of WT1 are proposed in leukemias and in solid tumors to have both tumor suppressive and oncogenic roles. We propose that genetic alterations of WT1, a preliminary finding among a subset of KS patients play a role in KS tumorigenesis. Methods: KS biopsy samples are obtained fromWeill Cornell Medical College, Stroger Hospital in Chicago and from the HIPPOS study (Kampala, Uganda). Lentiviral transduction of WT1 shRNA of KSHV infected 293T and endothelial cells were used to explore the role of identified genetic alterations. Results: We identified a deletion of WT1 in 2/11 patients with KS. Loss was confirmed by immunohistochemistry in these cases, while WT1 overexpression was seen in non-mutated cases. In an expanded cohort, we found additional cases that overexpress WT1 while others had no expression. In addition, the ‘tumorigenic’ form, cugWT1, was upregulated in endothelial and 293T cells upon infection with KSHV. Similar to the role of the oncogenic form of WT1 in other cancers in regulation of secondary target genes, knockdown of WT1 decreased BCL-2 expression, an anti-apoptotic gene. Conclusion: Kaposi sarcoma may manifest along a spectrum, as an inflammatory lesion or as a clonal malignancy, due to transformation in the setting of chronic KSHV infection leading to genomic instability. Given the finding of WT1 deletions in a subset of cases, as well as overexpression in others, WT1 isoforms may have pro-oncogenic and tumor suppressive roles in KS. Our

Ramya Ramaswami , Kathryn Lurain, Priscila H. Gonçalves, Mark Polizzotto, Anaida Widell, Matthew Lindsley, Richard F. Little, Thomas S. Uldrick, Robert Yarchoan NIH, Bethesda, MD, USA Background: Multicentric Castleman disease (MCD) is a rare systemic lymphoproliferative disease caused by Kaposi sarcoma-associated herpesvirus (KSHV), also known as human herpes virus 8 (HHV-8). Patients with HIV and KSHV-MCD may also have Kaposi sarcoma (KS) and are at increased risk of developing non-Hodgkin lymphoma, especially primary effusion lymphoma (PEL). The historical overall survival was 2.5 years, but this has improved following the use of rituximab for KSHV-MCD and antiretroviral therapy for patients with HIV. Here, we present the long-term outcomes of the largest prospective study of KSHV-MCD and HIV+ patients in North America. Methods: We evaluated longterm outcomes and concurrent diagnoses (KS and PEL) that influenced overall survival for patients with HIV and KSHV-MCD in a natural history study with 5 optional treatment regimens for MCD flares. This included high-dose zidovudine and ganciclovir, sirolimus, rituximab (R) with liposomal doxorubicin (R-LD) followed by interferon-α or high-dose zidovudine with valganciclovir (AZT/VGC), or rituximab plus infusional chemotherapy (R-EPOCH). Results: There were 58 participants (54 male, 4 female) with a median (range) age of 44 years (26-68), HIV VL <50 copies/mL (50 – 64100) and CD4 count 180 cells/μL (3-1319) at MCD diagnosis. All patients were on combined antiretroviral therapy at study entry, 38 patients had received prior therapy for KSHV-MCD (18 patients with R-based therapy), and 39 patients had a concurrent diagnosis of KS. Nine patients (15%) developed PEL after entry and 1 patient had been diagnosed with PEL prior to KSHV-MCD. Patients diagnosed with PEL were treated with R-EPOCH. The median duration of follow up was 4.1 years. Of the treatment options available in this study, the majority (52 patients (89%)) received R-LD, usually followed by high-dose AZT/VGC. The 5-year overall survival was 80% (95% confidence interval (CI), 66% to 88%). Eleven patients died: 4 from PEL, 4 from KSHV-MCD and associated complications, 2 from KS and sepsis, and 1 died from pancreatic cancer. A concurrent diagnosis of KS was not clearly a prognostic factor (hazard ratio (HR) 2.4; 95% CI, 0.5-11.1, P=0.3). However, a coexistent diagnosis of PEL was associated with worse survival (HR 3.4; 95% CI, 0.99-11.6, P=0.05, figure 1). Conclusion: KSHV-MCD is an under diagnosed but highly treatable condition if recognized. Physicians need to identify and promptly treat concurrent diagnoses of PEL and KS that may contribute to morbidity and mortality.

Oral Abstracts

18 REDUCTION OF KAPOSI SARCOMA–ASSOCIATED HERPESVIRUS LATENCY USING CRISPR-CAS9 For Yue Tso , John T. West, Charles Wood University of Nebraska–Lincoln, Lincoln, NE, USA Background: Kaposi sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi sarcoma (KS), an AIDS defining cancer in HIV-1 infected


CROI 2019

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