Faculty Bibliography

CD73, a cell-surface N-linked glycoprotein that produces extracellular adenosine, is a novel target for cancer immunotherapy. Although anti-CD73 antibodies have entered clinical development, CD73 has both protumor and antitumor functions, depending on the target cell and tumor type. The aim of this study was to characterize CD73 regulation in human hepatocellular carcinoma (HCC). We examined CD73 expression, localization, and activity using molecular, biochemical, and cellular analyses on primary HCC surgical specimens, coupled with mechanistic studies in HCC cells. We analyzed CD73 glycan signatures and global alterations in transcripts encoding other N-linked glycoproteins by using mass spectrometry glycomics and RNA sequencing (RNAseq), respectively. CD73 was expressed on tumor hepatocytes where it exhibited abnormal N-linked glycosylation, independent of HCC etiology, tumor stage, or fibrosis presence. Aberrant glycosylation of tumor-associated CD73 resulted in a 3-fold decrease in 5'-nucleotidase activity (P < 0.0001). Biochemically, tumor-associated CD73 was deficient in hybrid and complex glycans specifically on residues N311 and N333 located in the C-terminal catalytic domain. Blocking N311/N333 glycosylation by site-directed mutagenesis produced CD73 with significantly decreased 5'-nucleotidase activity in vitro, similar to the primary tumors. Glycosylation-deficient CD73 partially colocalized with the Golgi structural protein GM130, which was strongly induced in HCC tumors. RNAseq analysis further revealed that N-linked glycoprotein-encoding genes represented the largest category of differentially expressed genes between HCC tumor and adjacent tissue. Conclusion: We provide the first detailed characterization of CD73 glycosylation in normal and tumor tissue, revealing a novel mechanism that leads to the functional suppression of CD73 in human HCC tumor cells. The present findings have translational implications for therapeutic candidate antibodies targeting cell-surface CD73 in solid tumors and small-molecule adenosine receptor agonists that are in clinical development for HCC.

Background and aims: Hepatocellular carcinoma (HCC), the predominant form of primary liver cancer, is a leading cause of [Figure Presented] cancer-related deaths globally. Adenosine-3 receptor (A3R) activation blocks HCC progression in rodents, and small molecule A3R agonists are undergoing clinical development for HCC patients. Here, we examined the regulation of ecto-5'-nucleotidase (CD73), a novel target of cancer immunotherapy that produces extracellular adenosine, in human HCC.
Method(s): We analyzed CD73 expression and correlation with tumor characteristics and patient outcomes using The Cancer Genome Atlas (TCGA). In addition, we extracted CD73 from paired surgical specimens of HCC tumor and non-tumor adjacent liver tissues and normal control liver tissues collected under approved protocols, and performed glycopeptide mass spectrometry analysis. RNAseq analysis was performed to compare the expression of glycoprotein-encoding genes in addition to molecular, biochemical, and cell biological assays using multiple human HCC cell lines.
Result(s): High expression of the CD73-encoding gene (NT5E) correlated with poor survival in HCC patients (N = 365; p = 0.012) but did not correlate with a specific HCC immune subtype, based on TCGA data. We show using analysis of surgical specimens from HCC and adjacent liver tissues similar CD73 protein levels between normal and tumor hepatocytes. However, tumor-associated CD73 was primarily intracellular and exhibited altered N-linked glycosylation, independently of HCC etiology or fibrosis presence. The altered CD73 glycosylation correlated with a 2.7-fold decrease in tumor associated 5'-nucleotidase activity (p < 0.0001), which was otherwise necessary to induce cell death in HCC cultured cells. Mass spectrometry profiling of CD73 from tumors demonstrated a significant increase in high-mannose CD73 glycans on residues N311/N333. Blocking CD73 N311/N333 glycosylation caused intracellular CD73 accumulation and decreased 5'-nucleotidase activity in vitro, similar to the phenotypes observed in the primary human HCC tumors. Aberrant Golgi function was implicated as a potential mechanism, based on altered tumor expression and organization of the structural protein GM130 and genes encoding other N-linked glycoproteins, as determined by RNAseq analysis.
Conclusion(s): Our results reveal a novel glycosylation mechanism for regulating endogenous purinergic signaling in HCC tumors.
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Purpose: As Y90 radioembolization has been shown to activate an immune response, synergistic effects with check-point inhibitor immunotherapy have been proposed. However, the safety of concurrent Y90 and immunotherapy has not been reported. This study retrospectively evaluated the safety of Y90 with concurrent immunotherapy in patients with primary or metastatic liver cancer. Materials: The retrospective study was conducted with IRB approval. Patients who received Y90 treatment within 30 days of immunotherapy were considered to have concurrent therapy. Baseline laboratory values obtained within one month prior to Y90 and at 1 and 3 months after Y90 were evaluated. Hepatobiliary and immunotherapy-related adverse events were characterized according to NCI CTCAE v5.0. Patient survival was estimated using Kaplan-Meier analysis.
Result(s): Between June 2015 and March 2018, 18 patients received concurrent therapy. 14 patients had hepatocellular carcinoma (3 BCLC B, and 11 BCLC C), and 4 had metastatic disease to the liver (3 melanoma, 1 gastric cancer). The median interval between Y90 and immunotherapy was 7 days. Grade >=3 hepatobiliary toxicity occurred in 1 patient at 1 month (6%) and in 3 patients at 3 months (17%) after Y90. Grade >=3 toxicities occurred only in patients with advanced HCC (BCLC C). No grade >=3 immune-associated toxicities occurred at 1 or 3 months in any patients. Median overall survival from first Y90 was 27.4 months for patients with HCC and 13.7 months for patients with metastatic disease to the liver.
Conclusion(s): Concurrent Y90 radioembolization and checkpoint-inhibitor immunotherapy appears to be safe with a low incidence of toxicity. Toxicities were limited to patients with advanced HCC and may be confounded by disease progression.
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Orthotopic liver transplantation (OLT) and resection are effective treatments for hepatocellular carcinoma (HCC). However, optimizing OLT and limiting HCC recurrence remains a vexing problem. New HCC MELD and allocation algorithms provide greater observation of HCC patients, many while receiving local-regional treatments. Potential benefits of local-regional treatment for limiting HCC recurrence post-OLT remain incompletely understood. Therefore we aimed to define HCC specific prognostic factors affecting recurrence in a contemporary, multi-center cohort of HCC patients undergoing OLT and specifically whether local-regional therapies limited recurrence. We identified 441 patients undergoing OLT for HCC at three major transplant centers from 2008-2013. Cox regression was used to analyze covariate-adjusted recurrence and mortality rates post-OLT. "Bridging" or "down-staging" therapy was used in 238 patients (54%) with transarterial chemoembolization (TACE) being used in 170 (71%) of treated patients. The survival rate post-OLT was 88% and 78% at 1 and 3 years, respectively, with HCC recurrence (28% of deaths) significantly increasing mortality rate (HR=19.87, p<0.0001). Tumor size, not tumor number, either at presentation or on explant independently predicted HCC recurrence (HR 1.36 and 1.73, respectively, p<0.05) with a threshold effect noted at 4.0 cm size. Local-regional therapy (TACE) reduced HCC recurrence by 64% when adjusting for presenting tumor size (HR 0.36, p<0.05). Explant tumor size and microvascular invasion predicted mortality (HR 1.19 and 1.51, respectively, p<0.05) and pathologic response to therapy (TACE or RFA) significantly decreased explant tumor size (0.56-1.62 cm diameter reduction, p<0.05).

Histotripsy fractionates tissue through a mechanical, non-invasive ultrasonic ablation process that precisely controls acoustic cavitation while utilizing real-time ultrasound (US) imaging guidance. This study investigates the potential, feasibility and tumor volume reduction effects of histotripsy for liver cancer ablation in a subcutaneous in vivo murine Hepatocellular Carcinoma (HCC) model. Hep3B tumors were generated in the right flanks of 14 NSG and 7 NOD-SCID mice. The mice were grouped as follows: A (acute, NSG with n=9 treatment and n=1 control), B (chronic, NSG with n=2 treatment and n=2 control) and C (chronic NODSCID, with n=6 treatment and n=1 control). Treatment was performed when the tumor diameters reached >5 mm. 1-2 cycle histotripsy pulses at 100 Hz PRF (p- >30 MPa) were delivered using a custom built 1 MHz therapy transducer attached to a motorized positioner, which scanned the transducer focus to traverse the targeted tumor volume, guided by real-time US imaging. Tumor ablation effectiveness was assessed by obtaining T1, T2 and T2* weighted MR images. Post euthanasia, treated tumor, brain, and lung tissue samples were harvested for histology. Histology of acute group A showed fractionation of targeted region with a sharp boundary separating it from untreated tissue. Groups B and C demonstrated effective tumor volume reduction post treatment on MRI as the homogenate and edema were resorbed within 23 weeks. However, as the tumor was subcutaneous, it was not possible to set adequate treatment margin and since the mice were immune-compromised, residual viable tumor cells eventually developed into tumor regrowth at 3-9 weeks after histotripsy. Groups B and C showed no signs of metastasis in the lung and brain. Our study successfully demonstrated the potential of histotripsy for non-invasive HCC ablation in a subcutaneous murine model. Additional work is ongoing to study the response of histotripsy in immune-competent orthotopic liver tumor models.

Background and Aims: The majority of patients (pts) with advanced HCC (aHCC) progress on sorafenib (sor) therapy. Nivolumab (NIVO) is a fully human anti-PD-1 IgG4 mAb that demonstrated durable responses, manageable safety, and long-term survival in pts with aHCC in CheckMate-040 (El-Khoueiry AB, Sangro B, et al. Lancet 2017). Here we present updated hepatic safety and biomarker analyses in sor-experienced (sor-exp) pts with aHCC in CheckMate- 040. Method: Sor-exp pts with or without chronic viral hepatitis received NIVO 3 mg/kg Q2W in the dose-expansion phase (EXP) regardless of PD-L1 status. Primary endpoint was objective response rate (ORR) reported by blinded independent central review using RECIST v1.1 (EXP). Secondary endpoints included overall survival (OS), disease control rate (DCR), and safety. Exploratory analyses of on-treatment HCV and HBV viral kinetics and alpha-fetoprotein (AFP) levels were performed. Results: Median duration of follow-up was 14.9 mo in sor-exp pts (N = 145), 132 (91%) of whom had progressed on sor. Baseline Child- Pugh scores of 5 or 6 and extrahepatic metastases were observed in 99% and 71% of pts, respectively. The ORR with NIVO was 14%; the DCR was 56%; median OS was 15.6 mo. Any-grade and grade 3-4 hepatic treatment-related AEs (TRAEs) occurred in 12 (8%) and 5 (3%) pts, respectively; 100% of grade 3-4 hepatic TRAEs resolved. Frequencies of grade 3-4 treatment-related ALT/AST elevations were 2-3%. No drug-related deaths due to hepatic AEs occurred, and no new safety signals were observed. Among HBV-infected pts, 8% (3 of 38) had a >1 log decrease in HBsAg, and 12% (5 of 41) had a >1 log increase in HBV DNA. HBV DNA increases did not result in changes in hepatic parameters or serious AEs. Among HCV-infected pts, 30% (8 of 27) had a >1 log decrease in HCV RNA. Median baseline AFP in responders (165 mug/L) and nonresponders (85 mug/L) was similar (p = 0.5898). For pts with baseline AFP >=10 mug/L, ontreatment AFP declines >1 log occurred in 94% of responders and 10% of nonresponders. Updated data will be presented. Conclusion: NIVO demonstrated long-term survival and objective responses across etiologies in sor-exp pts with aHCC. The manageable safety profile of NIVO, including immune-mediated and hepatic AEs, was consistent with other tumor types in which NIVO is approved. NIVO had limited impact on viral kinetics in HBV and HCV-infected pts. Responses occurred irrespective of baseline AFP levels, and AFP declines were associated with response

Purpose: Histotripsy is a non-Thermal, non-invasive ultrasound (US) ablation method that fractionates tissue through the precise control of acoustic cavitation guided by real-Time US imaging. Histotripsy has the potential to improve treatment consistency and precision compared to thermal-based ablation methods. This study evaluates the feasibility and tumor volume reduction effects of histotripsy for liver cancer ablation in an in vivo murine HCC model. Materials: Subcutaneous xenograft tumors were generated by injecting human HCC Hep3B cells into 14 NSG mice (acute group A: Treated n = 9, control n = 1 and chronic group B: Treated n = 2, control n = 2) and 6 NOD-SCID mice (chronic group C: Treated n = 6). Once tumors reached >5 mm, mice were treated by histotripsy using a custom built 1 MHz histotripsy transducer attached to a motorized positioner guided by US imaging system. 1-2 cycle histotripsy pulses at 100 Hz PRF (p->30 MPa) were applied to the tumor volume. MRI was performed pre-and posttreatment to assess tumor ablation. Group A was sacrificed within 3 days post treatment. Groups B and C were monitored weekly using caliper measurements and MRI for 3 months or until tumors reached ~1.8 cm. Tumor, brain and lung tissues were harvested for histology. Results: Histotripsy-generated cavitation cloud and the treated region were visible on US imaging enabling real-Time feedback. In group A, histopathology showed that the targeted region was completely fractionated into acellular debris with a sharp boundary. In groups B and C, MRI revealed effective tumor volume reduction post treatment as the homogenate and edema were resorbed within 3 weeks. However, as the subcutaneous tumor does not allow sufficient treatment margin, residual viable tumor cells developed into tumor regrowth at 3-9 weeks after treatment. Treated mice in group B survived 2-3 times longer than the control mice and showed no signs of metastasis in the lung and brain. At the time of submission, group C mice are being monitored until study endpoint. Conclusions: This study demonstrates the potential of histotripsy for non-invasive liver tumor ablation. Future work will study the biological response in immune competent orthotopic liver tumor models

Importance: Patients with preexisting liver dysfunction could benefit the most from personalized therapy for liver tumors to balance maximal tumor control and minimal risk of liver failure. We designed an individualized adaptive trial testing the hypothesis that adapting treatment based on change in liver function could optimize the therapeutic index for each patient. Objective: To characterize the safety and efficacy of individualized adaptive stereotactic body radiotherapy (SRBT) for liver tumors in patients who have preexisting liver dysfunction. Design, Setting, and Participants: From 2010 to 2014, 90 patients with intrahepatic cancer treated with prior liver-directed therapy were enrolled in this large phase 2, single-arm, clinical trial at an academic medical center. All patients had at least 1 year of potential follow-up. Interventions: Using indocyanine green retention at 15 minutes (ICGR15) as a direct biomarker of liver function and a Bayesian adaptive model, planned SBRT was individually modified midway through the course of therapy to maintain liver function after the complete course. Main Outcomes and Measures: The primary outcome was local control; the secondary outcome was safety and overall survival. Results: Patients were 34 to 85 years of age, and 70% (63) were male. Ninety patients (69 [77%] with hepatocellular carcinoma, 4 [4%] with intrahepatic cholangiocarcinoma, and 17 [19%] with metastatic) received treatment to 116 tumors. Sixty-two patients (69%) had cirrhosis, 21 (23%) were Child-Pugh (CP) grade B. The median tumor size was 3 cm; 16 patients (18%) had portal vein involvement. Sixty-two (69%) received all 5 fractions (47 full dose, 15 dose-reduced owing to rising ICGR15). Treatment was well tolerated, with a lower than expected complication rate without adaptation: 6 (7%) experienced a 2-point decline in CP 6 months post-SBRT. The 1- and 2-year local control rates were 99% (95% CI, 97%-100%) and 95% (95% CI, 91%-99%), respectively. Conclusions and Relevance: We demonstrated that the treatment strategy of individualized adaptive therapy based on a direct biomarker of liver function can be used to achieve both high rates of local control and a high degree of safety without sacrificing either. Individualized adaptive radiotherapy may represent a new treatment paradigm in which dose is based on individual, rather than population-based, tolerance to treatment. Trial Registration: clinicaltrials.gov Identifier: NCT01522937.

ADAM17 (a disintegrin and metalloproteinase 17)/TACE (TNFalpha converting enzyme) has emerged as a potential therapeutic target in colorectal cancer (CRC) and other cancers, due in part to its role in regulating various tumor cell surface proteins and growth factors and cytokines in the tumor microenvironment. The emergence of MEDI3622, a highly potent and specific antibody-based ADAM17 inhibitor, has allowed testing of the concept that targeting ADAM17 may be an important new therapeutic approach for CRC patients. We demonstrate that MEDI3622 is highly efficacious on tumor growth in multiple human CRC PDX models, resulting in improved survival of animals bearing tumor xenografts. MEDI3622 was further found to impact Notch pathway activity and tumor-initiating cells. The promising preclinical activity seen here supports further clinical investigation of this treatment approach to improve therapeutic outcome for patients diagnosed with metastatic CRC, including patients with KRAS-mutant tumors for whom other therapeutic options are currently limited.

BACKGROUND: For patients with advanced hepatocellular carcinoma, sorafenib is the only approved drug worldwide, and outcomes remain poor. We aimed to assess the safety and efficacy of nivolumab, a programmed cell death protein-1 (PD-1) immune checkpoint inhibitor, in patients with advanced hepatocellular carcinoma with or without chronic viral hepatitis. METHODS: We did a phase 1/2, open-label, non-comparative, dose escalation and expansion trial (CheckMate 040) of nivolumab in adults (>/=18 years) with histologically confirmed advanced hepatocellular carcinoma with or without hepatitis C or B (HCV or HBV) infection. Previous sorafenib treatment was allowed. A dose-escalation phase was conducted at seven hospitals or academic centres in four countries or territories (USA, Spain, Hong Kong, and Singapore) and a dose-expansion phase was conducted at an additional 39 sites in 11 countries (Canada, UK, Germany, Italy, Japan, South Korea, Taiwan). At screening, eligible patients had Child-Pugh scores of 7 or less (Child-Pugh A or B7) for the dose-escalation phase and 6 or less (Child-Pugh A) for the dose-expansion phase, and an Eastern Cooperative Oncology Group performance status of 1 or less. Patients with HBV infection had to be receiving effective antiviral therapy (viral load <100 IU/mL); antiviral therapy was not required for patients with HCV infection. We excluded patients previously treated with an agent targeting T-cell costimulation or checkpoint pathways. Patients received intravenous nivolumab 0.1-10 mg/kg every 2 weeks in the dose-escalation phase (3+3 design). Nivolumab 3 mg/kg was given every 2 weeks in the dose-expansion phase to patients in four cohorts: sorafenib untreated or intolerant without viral hepatitis, sorafenib progressor without viral hepatitis, HCV infected, and HBV infected. Primary endpoints were safety and tolerability for the escalation phase and objective response rate (Response Evaluation Criteria In Solid Tumors version 1.1) for the expansion phase. This study is registered with ClinicalTrials.gov, number NCT01658878. FINDINGS: Between Nov 26, 2012, and Aug 8, 2016, 262 eligible patients were treated (48 patients in the dose-escalation phase and 214 in the dose-expansion phase). 202 (77%) of 262 patients have completed treatment and follow-up is ongoing. During dose escalation, nivolumab showed a manageable safety profile, including acceptable tolerability. In this phase, 46 (96%) of 48 patients discontinued treatment, 42 (88%) due to disease progression. Incidence of treatment-related adverse events did not seem to be associated with dose and no maximum tolerated dose was reached. 12 (25%) of 48 patients had grade 3/4 treatment-related adverse events. Three (6%) patients had treatment-related serious adverse events (pemphigoid, adrenal insufficiency, liver disorder). 30 (63%) of 48 patients in the dose-escalation phase died (not determined to be related to nivolumab therapy). Nivolumab 3 mg/kg was chosen for dose expansion. The objective response rate was 20% (95% CI 15-26) in patients treated with nivolumab 3 mg/kg in the dose-expansion phase and 15% (95% CI 6-28) in the dose-escalation phase. INTERPRETATION: Nivolumab had a manageable safety profile and no new signals were observed in patients with advanced hepatocellular carcinoma. Durable objective responses show the potential of nivolumab for treatment of advanced hepatocellular carcinoma. FUNDING: Bristol-Myers Squibb.