High-Risk Non-Small Cell Lung Cancer Treated With Active Scanning Proton Beam Radiation Therapy and Immunotherapy.

Purpose: Non-small cell lung cancer (NSCLC) is a deadly malignancy that is frequently diagnosed in patients with significant medical comorbidities. When delivering local and regional therapy, an exceedingly narrow therapeutic window is encountered, which often precludes patients from receiving aggressive curative therapy. Radiation therapy advances including particle therapy have been employed in an effort to expand this therapeutic window. Here we report outcomes with the use of proton therapy with curative intent and immunotherapy to treat patients diagnosed with high-risk NSCLC. Methods and Materials: Patients were determined to be high risk if they had severe underlying cardiopulmonary dysfunction, history of prior thoracic radiation therapy, and/or large volume or unfavorable location of disease (eg, bilateral hilar involvement, supraclavicular involvement). As such, patients were determined to be ineligible for conventional x-ray-based radiation therapy and were treated with pencil beam scanning proton beam therapy (PBS-PBT). Patients who demonstrated excess respiratory motion (ie, greater than 1 cm in any dimension noted on the 4-dimensional computed tomography simulation scan) were deemed to be ineligible for PBT. Toxicity was reported using the Common Terminology Criteria for Adverse Events (CTCAE), version 5.0. Overall survival and progression-free survival were calculated using the Kaplan-Meier method. Results: A total of 29 patients with high-risk NSCLC diagnoses were treated with PBS-PBT. The majority (55%) of patients were defined as high risk due to severe cardiopulmonary dysfunction. Most commonly, patients were treated definitively to a total dose of 6000 cGy (relative biological effectiveness) in 30 fractions with concurrent chemotherapy. Overall, there were a total of 6 acute grade 3 toxicities observed in our cohort. Acute high-grade toxicities included esophagitis (n = 4, 14%), dyspnea (n = 1, 3.5%), and cough (n = 1, 3.5%). No patients developed grade 4 or higher toxicity. The majority of patients went on to receive immunotherapy, and high-grade pneumonitis was rare. Two-year progression-free and overall survival was estimated to be 51% and 67%, respectively. COVID-19 was confirmed or suspected to be responsible for 2 patient deaths during the follow-up period. Conclusions: Radical PBS-PBT treatment delivered in a cohort of patients with high-risk lung cancer with immunotherapy is feasible with careful multidisciplinary evaluation and rigorous follow-up.

Remarkable Intracranial Response to Sotorasib in a Patient With KRAS G12C-Mutated Lung Adenocarcinoma and Untreated Brain Metastases: A Case Report.

Sotorasib is a KRAS G12C inhibitor that recently received approval for use in locally advanced or metastatic KRAS G12C-mutated NSCLC. CodeBreaK100, the phase 2 clinical trial leading to the approval of sotorasib, excluded patients with untreated brain metastases; there have been no reports describing efficacy of sotorasib on untreated brain metastases. We present a case of a patient with active untreated brain metastases with resulting disorientation and weakness who has radiographic response and complete resolution of neurologic symptoms with sotorasib. Our case illustrates the intracranial activity of sotorasib, but additional studies are needed to characterize the intracranial response rate and duration of response in these patients.

Management of invasive ductal carcinoma in hypermobile Ehlers-Danlos syndrome: a case report.

Literature is sparse regarding the management and long-term outcomes of breast cancer in patients with Ehlers-Danlos syndrome (EDS). Of the EDS subtypes, hypermobile Ehlers-Danlos Syndrome (hEDS) is associated with cardiovascular dysautonomia which manifests as spontaneous episodes of tachycardia and hypotension. Given this clinical autonomic system impact, hEDS is known to have significant intraoperative risk and postoperative complications. However, outcomes of hEDS patients have not been specifically studied in the field of breast cancer surgery. Here we present a case of a 62-year-old female with hEDS and node-positive invasive ductal breast carcinoma. Given the patient's medical history of hEDS, close attention was given to the patient's intraoperative vital signs and predisposition for poor wound healing. The patient underwent left Goldilocks mastectomy with left axillary lymph node dissection. Due to cardiac comorbidities, she was not a candidate for neoadjuvant or adjuvant chemotherapy. The patient tolerated adjuvant radiation and endocrine therapy without side effects, and has remained free of local, regional, and distant cancer recurrence following treatment. This case report highlights a literature gap in the surgical and radiation therapy management of breast cancer in patients with hEDS. Although breast surgery and radiation therapy in patients with invasive breast cancer and hEDS can be a safe management option, we discuss how perioperative complications must be cautiously navigated and how treatment must be tailored to individuals' specific hEDS variant to ensure optimal patient safety and positive long-term outcomes.

Early Experience of the First Single-Room Gantry Mounted Active Scanning Proton Therapy System at an Integrated Cancer Center.

Introduction: Review the early experience with a single-room gantry mounted active scanning proton therapy system. Material and Methods: All patients treated with proton beam radiotherapy (PBT) were enrolled in an institutional review board-approved patient registry. Proton beam radiotherapy was delivered with a 250 MeV gantry mounted synchrocyclotron in a single-room integrated facility within the pre-existing cancer center. Demographic data, cancer diagnoses, treatment technique, and geographic patterns were obtained for all patients. Treatment plans were evaluated for mixed modality therapy. Insurance approval data was collected for all patients treated with PBT. Results: A total of 132 patients were treated with PBT between March 2018 and June 2019. The most common oncologic subsites treated included the central nervous system (22%), gastrointestinal tract (20%), and genitourinary tract (20%). The most common histologies treated included prostate adenocarcinoma (19%), non-small cell lung cancer (10%), primary CNS gliomas (8%), and esophageal cancer (8%). Rationale for PBT treatment included limitation of dose to adjacent critical organs at risk (67%), reirradiation (19%), and patient comorbidities (11%). Patients received at least one x-ray fraction delivered as prescribed (36%) or less commonly due to unplanned machine downtime (34%). Concurrent systemic therapy was administered to 57 patients (43%). Twenty-six patients (20%) were initially denied insurance coverage and required peer-to-peers (65%), written appeals (12%), secondary insurance approval (12%), and comparison x-ray to proton plans (8%) for subsequent approval. Proton beam radiotherapy approval required a median of 17 days from insurance submission. Discussion: Incorporation of PBT into our existing cancer center allowed for multidisciplinary oncologic treatment of a diverse population of patients. Insurance coverage for PBT presents as a significant hurdle and improvements are needed to provide more timely access to necessary oncologic care.

Stereotactic Body Radiation Therapy: A Versatile, Well-Tolerated, and Effective Treatment Option for Extracranial Metastases From Primary Ovarian and Uterine Cancer.

PURPOSE: Single extracranial metastases from ovarian and uterine malignancies have historically been treated with surgery or conventional radiation. We report mature local control (LC), overall survival (OS), progression free survival (PFS), and toxicity for patients who completed 5-fraction stereotactic body radiation therapy (SBRT). METHODS: Patients with biopsy-proven, single extracranial metastases from primary ovarian and uterine malignancies treated with 5-fraction SBRT were included. Patients were stratified based on tumor volume (small < 50 cc or large ≥ 50 cc) and dose (low dose < 35 Gy or high ≥ 35 Gy). Kaplan-Meier method was used to estimate LC, OS, and PFS. RESULTS: Between July 2007 and July 2012, 20 patients underwent SBRT to a single extracranial metastasis. Primary site was divided evenly between ovarian and uterine (n = 10 each). Metastases involved the liver (30%), abdominal lymph nodes (25%), lung (20%), pelvic lymph nodes (10%), spine (10%), and extremity (5%). The median gross tumor volume (GTV) was 42.5 cc (range, 5-273 cc) and the median dose to the GTV was 35 Gy (range, 30-50 Gy). At a median follow-up of 56 months, the 5-year LC and OS estimates were 73 and 46%. When stratified by tumor volume, the 5-year LC and OS for small tumors were significantly better at 100% (p < 0.01) and 65% (p < 0.02). When stratified by dose, the 5-year LC was 87.5% with high dose and 53.6% with low dose (p = 0.035). The 5-year PFS for the entire cohort was 20%. Four patients with small metastases who had complete response remained disease free at study completion and were considered cured (median PFS > 10 years). Treatment was generally well tolerated, and only one patient experienced a late grade III musculoskeletal SBRT related toxicity. CONCLUSIONS: SBRT is a versatile, well-tolerated, and effective treatment option for single extracranial metastases from ovarian and uterine primary tumors. 35 Gy in five fractions appears to be a practical minimum effective dose. Four patients with small metastases were disease free at the study completion and considered cured. However, patients with larger metastases (≥50 cc) may require higher SBRT dosing or alternative treatments.

Stereotactic radiosurgery for the treatment of brain metastasis from gastrointestinal primary cancers.

PURPOSE: We aimed to determine the efficacy of gamma knife stereotactic radiosurgery (SRS) to control brain metastases (BM) from GI primaries and report on the patient outcomes. MATERIALS/METHODS: We retrospectively evaluated patients who had undergone SRS at our institution for the treatment BM from GI primaries from 2000 to 2016. Actuarial rates for overall survival (OS) and local control (LC) were calculated. Survival rates were computed via the Kaplan-Meier method from the day of SRS. Multivariate analysis (MVA) using proportional hazards regression was done to determine prognostic factors for OS and LC. RESULTS: 53 patients with a total of 148 BM were treated with SRS. The median age at SRS was 60 years and the median treatment dose was 18 Gy. SRS was given as an adjuvant treatment to 30.4% of lesions. Twelve patients underwent second course of SRS for new/recurrent lesions at a median of 8 months from the first SRS (range 2-25.5). Twelve patients (22.6%) received salvage whole brain radiation. The median follow up time from the diagnosis of BM was 6 months. Local control rate at 6 months was 74.33% and 57.21% at 12 months. The OS at 1 year was 34% and 8% at 3 years. On MVA, higher GK dose was associated with better LC, and and >10 BM trended towards higher risk of local recurrence (LR). None of the tested factors proved to be significant for OS on MVA. No radiographic radionecrosis was observed on follow up MRI. CONCLUSIONS: SRS is a safe treatment modality for the management of CNS metastases from GI primary. Consideration for dose-escalated approaches may improve LC rates.

Patterns of Failure and Survival Outcomes after Total Lymphoid Irradiation and High-Dose Chemotherapy with Autologous Stem Cell Transplantation for Relapsed or Refractory Classical Hodgkin Lymphoma.

PURPOSE: The patterns of failure and long-term outcomes of patients with relapsed or refractory classical Hodgkin lymphoma treated with total lymphoid irradiation (TLI) and high-dose chemotherapy followed by autologous stem cell transplantation (aSCT) are reported. METHODS AND MATERIALS: Patients with biopsy-proven primary refractory or relapsed classical Hodgkin lymphoma who received salvage chemotherapy and accelerated hyperfractionated TLI before high-dose chemotherapy and aSCT were included. Patterns of failure were delineated after fusing pretransplant planning computed tomography to the scan reporting the first failure. Survival rates were computed using the Kaplan-Meier method. Multivariate analysis using proportional hazards regression was done to determine prognostic factors for overall survival (OS) and progression-free survival (PFS). RESULTS: Between 1993 and 2016, 89 patients underwent salvage treatments. Twenty patients failed at a median of 6.1 months after aSCT. Posttreatment scans were available for 16 patients who failed in a combined 43 different sites, 11 of which were extranodal. Patients failed at multiple sites, mostly within radiation fields. The 5-, 10-, and 15-year OS rates were 72.8%, 68.0%, and 58.3%; PFS rates were 73.3%, 68.5%, and 58.7%; event-free survival rates were 72.3%, 67.5%, and 57.8% respectively. The 5- and 10- year actuarial local control rates were both 77.6%. Complete response (CR) to salvage chemotherapy was associated with statistically significant improvements in OS and PFS. Eight patients developed secondary malignancies; 5 were hematologic and 3 were solid tumors. CONCLUSIONS: Most failures were within the irradiated volume, which reflects the treatment-resistant disease biology. As part of a conditioning regimen, TLI yields good survival outcomes, particularly in patients achieving CR before transplant. However, need for RT in this setting should be assessed and new strategies should be developed to combat the treatment-resistant biology, especially in patients with less than CR after salvage chemotherapy.

Impact of breast MRI in women eligible for breast conservation surgery and intra-operative radiation therapy.

PURPOSE: The current standard of care for women diagnosed with early stage breast cancer is breast-conserving surgery (BCS) followed by external beam radiation therapy, commonly delivered over 3-6 weeks. As an alternative, select patients can undergo intra-operative radiation therapy (IORT) at the time of BCT. This technique delivers a single fraction of radiation at the time of surgery, enabling patients to undergo both surgery and radiation in a single session. Our current study analyzed the value of incorporating breast MRI into the routine work-up of patients deemed eligible for IORT, to quantify the impact on patient eligibility and requirement for additional work-up. MATERIALS AND METHODS: We retrospectively identified patients treated by a single surgeon who were eligible for IORT based on institutional eligibility criteria which included: women age ≥55, grades 1-2, size <3 cm, estrogen receptor (ER) positive, Her-2 neu non-amplified and low/intermediate Ki-67, unifocal invasive ductal/mixed histology carcinomas. All patients must have undergone a physical exam and bilateral diagnostic mammography with ultrasound. From this population, we identified all patients who had undergone bilateral breast MRI as part of pre-operative evaluation. RESULTS: A total of 215 women were identified who met all eligibility criteria. MRI detected additional abnormalities in the breast in 89 patients (41%). Sixty-eight women underwent additional biopsies, with a total of 117 separate lesions biopsied. Of these, pathology was benign in 61 (52.1%), atypical ductal hyperplasia (ADH) in 21 (18%), ductal carcinoma in-situ (DCIS) in 17 (14.5%) and invasive disease in 18 (15.4%). Six patients had MRI-detected abnormalities in the contralateral breast only, with biopsies identifying invasive disease (3), DCIS (1) and benign (2) findings. MRI showed abnormalities in both breasts in 6 patients and 18 additional lesions were biopsied which reveled invasive carcinoma (6), DCIS (7), ADH (3) and benign findings (2). Fifteen patients had either multifocal/multicentric disease or index lesion >3 cm on MRI and were deemed ineligible for IORT. Based on either MRI size or biopsy results, management was ultimately changed for 27 patients (12.5%). Extramammary findings were observed in 17 patients and 11 of these patients underwent further imaging studies all of which returned negative results. CONCLUSION: Preoperative bilateral breast MRI is a valuable tool in the proper selection of patients best suited for IORT. Even in highly selected, favorable risk patients, MRI detected additional lesions that changed surgical and radiation therapy recommendations in 12.5% of patients. However, the cost/benefit ratio needs to be taken into consideration given the high frequency of benign biopsies and additional radiological work-up.

Kaposi's sarcoma-associated herpesvirus latency in endothelial and B cells activates gamma interferon-inducible protein 16-mediated inflammasomes.

Kaposi's sarcoma-associated herpesvirus (KSHV) infections of endothelial and B cells are etiologically linked with Kaposi's sarcoma (KS) and primary effusion B-cell lymphoma (PEL), respectively. KS endothelial and PEL B cells carry multiple copies of the nuclear episomal latent KSHV genome and secrete a variety of inflammatory cytokines, including interleukin-1ß (IL-1ß) and IL-18. The maturation of IL-1ß and IL-18 depends upon active caspase-1, which is regulated by a multiprotein inflammasome complex induced by sensing of danger signals. During primary KSHV infection of endothelial cells, acting as a nuclear pattern recognition receptor, gamma interferon-inducible protein 16 (IFI16) colocalized with the KSHV genome in the nuclei and interacted with ASC and procaspase-1 to form a functional inflammasome (Kerur N et al., Cell Host Microbe 9:363-375, 2011). Here, we demonstrate that endothelial telomerase-immortalized human umbilical cells (TIVE) supporting KSHV stable latency (TIVE-LTC cells) and PEL (cavity-based B-cell lymphoma 1 [BCBL-1]) cells show evidence of inflammasome activation, such as the activation of caspase-1 and cleavage of pro-IL-1ß and pro-IL-18. Interaction of ASC with IFI16 but not with AIM2 or NOD-like receptor P3 (NLRP3) was detected. The KSHV latency-associated viral FLIP (vFLIP) gene induced the expression of IL-1ß, IL-18, and caspase-1 mRNAs in an NF-κB-dependent manner. IFI16 and cleaved IL-1ß were detected in the exosomes released from BCBL-1 cells. Exosomal release could be a KSHV-mediated strategy to subvert IL-1ß functions. In fluorescent in situ hybridization analyses, IFI16 colocalized with multiple copies of the KSHV genome in BCBL-1 cells. IFI16 colocalization with ASC was also detected in lung PEL sections from patients. Taken together, these findings demonstrated the constant sensing of the latent KSHV genome by IFI16-mediated innate defense and unraveled a potential mechanism of inflammation induction associated with KS and PEL lesions.

Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen interacts with multifunctional angiogenin to utilize its antiapoptotic functions.

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with the angioproliferative Kaposi's sarcoma (KS). KSHV infection and the expression of latency-associated nuclear antigen (LANA-1) upregulates the angiogenic multifunctional 123-amino-acid, 14-kDa protein angiogenin (ANG), which is detected in KS lesions and in KSHV-associated primary effusion lymphoma (PEL) cells. ANG knockdown or the inhibition of ANG's nuclear translocation resulted in decreased LANA-1 gene expression and reduced KSHV-infected endothelial and PEL cell survival (Sadagopan et al., J. Virol. 83:3342-3364, 2009). Further studies here demonstrate that LANA-1 and ANG colocalize and coimmunoprecipitate in de novo infected endothelial cells and in latently infected PEL (BCBL-1 and BC-3) cells. LANA-1 and ANG interaction occurred in the absence of the KSHV genome and other viral proteins. In gel filtration chromatography analyses of BC-3 cell lysates, ANG coeluted with LANA-1, p53, and Mdm2 in high-molecular-weight fractions, and LANA-1, p53, and Mdm2 also coimmunoprecipitated with ANG. LANA-1, ANG, and p53 colocalized in KSHV-infected cells, and colocalization between ANG and p53 was also observed in LANA-1-negative cells. The deletion constructs of ANG suggested that the C-terminal region of amino acids 104 to 123 is involved in LANA-1 and p53 interactions. Silencing ANG or inhibiting its nuclear translocation resulted in decreased nuclear LANA-1 and ANG levels, decreased interactions between ANG-LANA-1, ANG-p53, and LANA-1-p53, the induction of p53, p21, and Bax proteins, the increased cytoplasmic localization of p53, the downregulation of Bcl-2, the increased cleavage of caspase-3, and the apoptosis of cells. No such effects were observed in KSHV-negative BJAB cells. The phosphorylation of p53 at serine 15, which is essential for p53 stabilization and for p53's apoptotic and cell cycle regulation functions, was increased in BCBL-1 cells transduced with short hairpin RNA targeting ANG. Together, these studies suggest that the antiapoptosis observed in KSHV-infected cells and the suppression of p53 functions are mediated in part by ANG, and KSHV has probably evolved to utilize angiogenin's multiple functions for the maintenance of its latency and cell survival. Thus, targeting ANG to induce the apoptosis of cells latently infected with KSHV is an attractive therapeutic strategy against KSHV infection and associated malignancies.

Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen and angiogenin interact with common host proteins, including annexin A2, which is essential for survival of latently infected cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) infection and latency-associated nuclear antigen (LANA-1) upregulate the multifunctional protein angiogenin (ANG). Our studies demonstrate that silencing ANG or inhibiting its nuclear translocation downregulates KSHV LANA-1 expression and ANG is necessary for KSHV latency, anti-apoptosis and angiogenesis (Sadagopan et al., J. Virol. 83:3342-3364, 2009; Sadagopan et al., J Virol. 85:2666-2685, 2011). Here we show that LANA-1 interacts with ANG and colocalizes in latently infected endothelial telomerase-immortalized human umbilical vein endothelial (TIVE-LTC) cells. Mass spectrometric analyses of TIVE-LTC proteins immunoprecipitated by anti-LANA-1 and ANG antibodies identified 28 common cellular proteins such as ribosomal proteins, structural proteins, tRNA synthetases, metabolic pathway enzymes, chaperons, transcription factors, antioxidants, and ubiquitin proteosome proteins. LANA-1 and ANG interaction with one of the proteins, annexin A2, was validated. Annexin A2 has been shown to play roles in cell proliferation, apoptosis, plasmin generation, exocytosis, endocytosis, and cytoskeleton reorganization. It is also known to associate with glycolytic enzyme 3-phosphoglyceratekinase in the primer recognition protein (PRP) complex that interacts with DNA polymerase α in the lagging strand of DNA during replication. A higher level of annexin A2 is expressed in KSHV+ but not in Epstein-Barr virus (EBV)+ B-lymphoma cell lines. Annexin A2 colocalized with several LANA-1 punctate spots in KSHV+ body cavity B-cell lymphoma (BCBL-1) cells. In triple-staining analyses, we observed annexin A2-ANG-LANA-1, annexin A2-ANG, and ANG-LANA-1 colocalizations. Annexin A2 appeared as punctate nuclear dots in LANA-1-positive TIVE-LTC cells. In LANA-1-negative TIVE-LTC cells, annexin A2 was detected predominately in the cytoplasm, with some nuclear spots, and colocalization with ANG was observed mostly in the cytoplasm. Annexin A2 coimmunoprecipitated with LANA-1 and ANG in TIVE-LTC and BCBL-1 cells and with ANG in 293T cells independent of LANA-1. This suggested that annexin A2 forms a complex with LANA-1 and ANG as well as a separate complex with ANG. Silencing annexin A2 in BCBL-1 cells resulted in significant cell death, downregulation of cell cycle-associated Cdk6 and of cyclin D, E, and A proteins, and downregulation of LANA-1 and ANG expression. No effect was seen in KSHV⁻ lymphoma (BJAB and Ramos) and 293T cells. These studies suggest that LANA-1 association with annexin A2/ANG could be more important than ANG association with annexin A2, and KSHV probably uses annexin A2 to maintain the viability and cell cycle regulation of latently infected cells. Since the identified LANA-1- and ANG-interacting common cellular proteins are hitherto unknown to KSHV and ANG biology, this offers a starting point for further analysis of their roles in KSHV biology, which may lead to identification of potential therapeutic targets to control KSHV latency and associated malignancies.

c-Cbl-mediated selective virus-receptor translocations into lipid rafts regulate productive Kaposi's sarcoma-associated herpesvirus infection in endothelial cells.

During target cell entry and infection, many enveloped and nonenveloped viruses utilize cell surface receptors that translocate into lipid rafts (LRs). However, the mechanism behind this translocation is not known. Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with the human microvascular dermal endothelial (HMVEC-d) cell surface heparan sulfate (HS), integrins α3ß1, αVß3, and αVß5, and the amino acid transporter x-CT protein and enters via c-Cbl-bleb-mediated macropinocytosis (Veettil et al., J. Virol. 82:12126-12144, 2008; Veettil et al., PLoS Pathog. 6:e1001238, 2010). Here we have demonstrated that very early during infection (1 min postinfection), c-Cbl induced the selective translocation of KSHV into the LR along with the α3ß1, αVß3, and x-CT receptors but not αVß5. Activated c-Cbl localized with LRs at the junctional base of macropinocytic blebs. LR-translocated α3ß1 and αVß3 were monoubiquitinated, leading to productive macropinocytic entry, whereas non-LR-associated αVß5 was polyubiquitinated, leading to clathrin entry that was targeted to lysosomes. c-Cbl knockdown blocked the macropinocytosis and receptor translocation and diverted KSHV to a clathrin-lysosomal noninfectious pathway. Similar results were also seen by LR disruption with MßCD. These studies provide the first evidence that c-Cbl regulates selective KSHV-α3ß1, -αVß3, and -x-CT receptor translocations into the LRs and differential ubiquitination of receptors which are critical determinants of the macropinocytic entry route and productive infection of KSHV. Our studies suggest that interventions targeting c-Cbl and LRs are potential avenues to block KSHV infection of endothelial cells.

Kaposi's sarcoma-associated herpesvirus-induced angiogenin plays roles in latency via the phospholipase C gamma pathway: blocking angiogenin inhibits latent gene expression and induces the lytic cycle.

During de novo infection of human dermal microvascular endothelial cells (HMVEC-d), Kaposi's sarcoma-associated herpesvirus (KSHV) induced the multifunctional angiogenin (ANG) protein, which entered the nuclei and nucleoli of infected cells and stimulated 45S rRNA gene transcription, proliferation, and tube formation, which were inhibited by blocking ANG nuclear translocation with the antibiotic neomycin (S. Sadagopan et al., J. Virol. 83:3342-3364, 2009). ANG was induced by KSHV latency protein LANA-1 (open reading frame 73 [ORF73]). Here we examined the presence and functions of ANG in KSHV-positive (KSHV(+)) primary effusion lymphoma (PEL/BCBL) cells. Significant ANG gene expression and secretion were observed in KSHV(+) (BCBL-1 and BC-3) and KSHV(+) and Epstein-Barr virus-positive (KSHV(+) EBV(+)) (JSC-1) PEL cells and in BJAB-KSHV cells but not in EBV(-) KSHV(-) lymphoma cells (Akata, Loukes, Ramos, and BJAB), EBV(+) lymphoma cells (Akata-EBV and Raji), and cells from an EBV(+) lymphoblastoid cell line, thus suggesting a specific association of ANG in KSHV biology. Inhibition of nuclear translocation of ANG resulted in reduced BCBL-1 and TIVE-LTC (latently infected endothelial) cell survival and proliferation, while EBV(-) and EBV(+) Akata cells were unaffected. Blocking nuclear transport of ANG inhibited latent ORF73 gene expression and increased lytic switch ORF50 gene expression, both during de novo infection and in latently infected cells. A greater quantity of infectious KSHV was detected in the supernatants of neomycin-treated BCBL-1 cells than 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated cells. Neomycin treatment and ANG silencing inhibited phospholipase Cγ (PLC-γ) and AKT phosphorylation, and in contrast, ANG induced ORF73 expression and PLC-γ and AKT phosphorylation. Further studies provided evidence that blockage of PLC-γ activation by neomycin appears to be mediating the inhibition of latent gene expression, since treatment with the conventional PLC-γ inhibitor U73122 also showed similar results. Silencing of ANG also resulted in reduced cell survival, reduced ORF73 gene expression, and lytic gene activation in BCBL-1 and TIVE-LTC cells and during de novo infection. Taken together, these studies suggest that KSHV has evolved to exploit ANG for its advantage via a so-far-unexplored PLC-γ pathway for maintaining its latency.