Roberts, Lewis R.

Abstract Purpose: Hepatocellular Carcinoma (HCC) is the leading cause of cancer-related mortality in Nigeria. HIV and viral hepatitis B (HBV) and C (HCV) co-infection are common in Nigeria that significantly accelerates liver disease progression including HCC. Aging-related DNA methylation signatures obtained in liquid biopsy, such as circulating cell-free DNA (ccfDNA) extracted from serum/plasma are promising minimally-invasive biomarkers that may inform HIV-associated HCC. We examined the epigenetic age acceleration (EpiAgeAccel) in ccfDNA in HCC patients with HIV. Methods: The study included three groups of participants: a) HIV positive with HCC (n=7); b) HIV positive and cancer-free (n=45); and c) HIV negative with HCC (n=33). Epigenetic age was estimated by Horvath's calculator using genome-wide ccfDNA methylation data profiled by Illumina EPIC array. EpiAgeAccel was computed as the residuals of a linear model of epigenetic age on chronological age, namely the unexplained portion of epigenetic age by chronological age. We used multiple linear regression to compare EpiAgeAccel between HIV/HCC groups, adjusting for sex, age, education, alcohol intake, and HBV/HCV infection. Results: Among HIV positive participants, there was a higher percentage of men (57% vs. 22%, p<0.001), HBV infection (29% vs. 11%, p=0.004), and HCV infection (57% vs. 2%, p<0.001) in the HCC group compared to the cancer-free group. EpiAgeAccel was 4.8 years higher in HIV positive patients with HCC compared to cancer-free HIV positives (p=0.02). Among HCC patients, EpiAgeAccel was 2.1 years higher in HIV positives compared to HIV negative but not statistically significant. Conclusion: Epigenetic age in ccfDNA is accelerated in HIV-positive HCC patients. EpiAgeAccel measured in ccfDNA may be developed into a surrogate biomarker for minimally invasive HCC detection among HIV-infected patients in low- and middle-income countries. Citation Format: Yinan Zheng, Claudia Hawkins, Edith Okeke, Mary Duguru, Emuobor Odeghe, Olufunmilayo Lesi, Yishu Qu, Kyeezu Kim, Brian Joyce, Jun Wang, Lewis Roberts, Demirkan Gursel, Abdulkareem Fatimah, Alani Akanmu, Godwin Imade, Jian-Jun Wei, Masha Kocherginsky, Kwang-Youn Kim, Oluwakemi Odukoya, Wasiu Adeyemo, Firas Wehbe, Chad Achenbach, Atiene Sagay, Folasade Ogunsola, Robert Murphy, Lifang Hou. Blood-Based Circulating Cell-Free DNA Epigenetic Age Is Accelerated Among HIV-Infected Patients with Hepatocellular Carcinoma in Nigeria [abstract]. In: Proceedings of the 9th Annual Symposium on Global Cancer Research; Global Cancer Research and Control: Looking Back and Charting a Path Forward; 2021 Mar 10-11. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2021;30(7 Suppl):Abstract nr 83.

Although hepatitis B virus (HBV) integration into the cellular genome is well known in HCC (hepatocellular carcinoma) patients, its biological role still remains uncertain. This study investigated the patterns of HBV integration and correlated them with TERT (telomerase reverse transcriptase) alterations in paired tumor and non-tumor tissues. Compared to those in non-tumors, tumoral integrations occurred less frequently but with higher read counts and were more preferentially observed in genic regions with significant enrichment of integration into promoters. In HBV-related tumors, TERT promoter was identified as the most frequent site (38.5% (10/26)) of HBV integration. TERT promoter mutation was observed only in tumors (24.2% (8/33)), but not in non-tumors. Only 3.00% (34/1133) of HBV integration sites were shared between tumors and non-tumors. Within the HBV genome, HBV breakpoints were distributed preferentially in the 3’ end of HBx, with more tumoral integrations detected in the preS/S region. The major genes that were recurrently affected by HBV integration included TERT and MLL4 for tumors and FN1 for non-tumors. Functional enrichment analysis of tumoral genes with integrations showed enrichment of cancer-associated genes. The patterns and functions of HBV integration are distinct between tumors and non-tumors. Tumoral integration is often enriched into both human-virus regions with oncogenic regulatory function. The characteristic genomic features of HBV integration together with TERT alteration may dysregulate the affected gene function, thereby contributing to hepatocarcinogenesis.

Background and Aims Intrahepatic cholangiocarcinoma (iCCA) and hepatocellular carcinoma (HCC) are the most common primary liver cancers (PLCs). Differences in their clinical features and outcomes are open for investigation in a large‐scale study. We aim to investigate the differences in clinical features and outcomes between iCCA and HCC. Approach and Results The Surveillance, Epidemiology, and End Results Program 18 Database (2000‐2017) was used to extract demographic and clinical features of HCC and iCCA patients. Logistic regression analysis was performed to identify factors associated with iCCA diagnosis versus HCC. Cox regression analysis was used to assess factors affecting overall survival (OS). There were 13,611 iCCA and 96,151 HCC patients. Half of iCCA (50.7%) and three quarters of HCC (76.3%) patients were male. Diagnosis in recent year, age (<50 or ≥65), female sex, non‐Hispanic White race, higher income, rural area, and higher tumor burden were independently associated with iCCA diagnosis versus HCC. Patients with iCCA had worse OS than those with HCC (9 vs. 13 months; P < 0.001). However, OS was comparable between iCCA and HCC in multivariable analysis (adjusted hazard ratio [aHR] = 1.02; 95% CI = 0.99‐1.05). In subgroup analyses, iCCA was associated with better OS than HCC in patients with tumor ≥5 cm (aHR = 0.83; 95% CI = 0.80‐0.86), lymph node involvement (aHR = 0.76; 95% CI = 0.72‐0.81), distant metastasis (aHR = 0.76; 95% CI = 0.73‐0.79), poorly/undifferentiated tumors (aHR = 0.88; 95% CI = 0.83‐0.94), and those receiving noncurative treatment (aHR = 0.96; 95% CI = 0.93‐0.98). Conclusions We identified the demographic, socioeconomic, and clinical features associated with iCCA diagnosis over HCC among patients with PLC. Although iCCA patients presented at an advanced stage, OS was similar between iCCA and HCC in multivariable analysis. iCCA was associated with longer OS for subgroups with poor prognostic features.

e18521 Background: About 40% of the US population are from minority groups. Minority pts are under-represented in oncology clinical trials, which limits the applicability of results to the general population and perpetuates poor relationships between healthcare systems and minority communities. This assessment investigated underlying causes of this lack of minority representation in clinical trials and proposes plans to promote diversity. Methods: To better understand the limited inclusion of under-represented pts in oncology clinical trials, 10 specialists in cancer care were selected to provide their perspectives. Specialists were chosen because of their experience enrolling minority pts in clinical trials and met virtually in Dec 2020 via Within3, a secure digital communication platform, to discuss obstacles faced in recruiting minority pts and potential strategies to address these concerns. Specificity and alignment in responses were achieved through software analytics and follow-up queries. Results: The 10 specialists identified as Asian (10%), Black (30%), White (50%), and Hispanic (10%), and began practicing medicine in the 1980s-2010s. All are involved in clinical research and treat a range of minority pts in both urban and suburban settings. Most specialists (8/10) reported treating > 20 minority pts with cancer annually. However, few specialists (2/10) reported that > 20% of their minority pts have enrolled in clinical trials. Specialists agreed that minority pts experience barriers to participation in clinical research, including lack of trust in the healthcare system, materials in their native languages, financial support, minority investigators involved in clinical trials, and accessible study sites. The specialists proposed strategies that could be implemented to increase minority enrollment. These included study sites where minority populations live and industry funded outreach and educational efforts in minority communities. If sites are more accessible, this can reduce time and financial pressures associated with study participation. The specialists recommended that studies be designed to be more supportive of minority populations, specifically regarding inclusion and exclusion criteria and reimbursement of costs. They also advised that increased diversity among clinical researchers and allied personnel may increase the ability of the clinical team to connect with pts and assist in building trust in their communities. Finally, they emphasized the importance of providing informed consent forms and study materials in pts’ native languages. Conclusions: While challenges exist to increasing diversity in oncology clinical studies, a broad consortium of clinical specialists agreed that they can be addressed by community outreach and education, making study sites more accessible, availability of study materials in pts’ native languages, and improving diversity of clinical teams.

e16137 Background: Hepatocellular Carcinoma (HCC) is the leading cause of cancer-related mortality in Nigeria. HIV and viral hepatitis B (HBV) and C (HCV) co-infection are common in Nigeria that significantly accelerates liver disease progression including HCC. Aging-related DNA methylation signatures obtained in liquid biopsy, such as circulating cell-free DNA (ccfDNA) extracted from serum/plasma are promising minimally-invasive biomarkers that may inform HIV-associated HCC. We examined the epigenetic age acceleration (EpiAgeAccel) in ccfDNA in HCC patients with HIV. Methods: The study included three groups of participants: a) HIV positive with HCC (n=7); b) HIV positive and cancer-free (n=45); and c) HIV negative with HCC (n=33). Epigenetic age was estimated by Horvath’s calculator using genome-wide ccfDNA methylation data profiled by Illumina EPIC array. EpiAgeAccel was computed as the residuals of a linear model of epigenetic age on chronological age, namely the unexplained portion of epigenetic age by chronological age. We used multiple linear regression to compare EpiAgeAccel between HIV/HCC groups, adjusting for sex, age, education, alcohol intake, and HBV/HCV infection. Results: Among HIV positive participants, there was a higher percentage of men (57% vs. 22%, p<0.001), HBV infection (29% vs. 11%, p=0.004), and HCV infection (57% vs. 2%, p<0.001) in the HCC group compared to the cancer-free group. EpiAgeAccel was 4.8 years higher in HIV positive patients with HCC compared to cancer-free HIV positives (p=0.02). Among HCC patients, EpiAgeAccel was 2.1 years higher in HIV positives compared to HIV negative but not statistically significant. Conclusions: Epigenetic age in ccfDNA is accelerated in HIV-positive HCC patients. EpiAgeAccel measured in ccfDNA may be developed into a surrogate biomarker for minimally invasive HCC detection among HIV-infected patients in low- and middle-income countries.

4100 Background: Checkpoint inhibition with anti-PD1 is able to elicit objective response rates (ORR) of ̃ 20% in patients with advanced hepatocellular carcinoma (aHCC) but molecular biomarkers predicting response remain unknown. Here, we define biomarkers of response and primary resistance to anti-PD1 in aHCC by analyzing molecular features prior to systemic therapy. Methods: Through an international consortium of 13 referral centers, we gathered 111 fresh and archived tumor samples from patients with advanced HCC treated with single agent anti- PD1 therapy. Genomic analysis was performed with whole genome expression arrays, CTNNB1 exon 3 mutation assessment and histological evaluation. Results: Overall, we performed transcriptomic analysis in 83 patients, 28 of which were treated in first-line (ORR: 42.9%) while the remaining 55 patients were treated either in 2nd (41 patients, ORR 29.3%) or 3rd line (14 patients, ORR 7.1%) after prior therapy with tyrosine- kinase inhibitors (TKI) sorafenib or lenvatinib. In patients treated in frontline, response was associated with a significant upregulation in Interferon-γ signaling (IFNγ) and genesets relatedto the antigen presentation machinery (FDR < 0.05). Through differential expression analysis we defined an 11-gene signature that was significantly associated with both major pathways (FDR < 0.01) and was capable of predicting OR as well as progression-free- (PFS) and overall survival (OS) in patients with aHCC. The signature was validated in three independent cohorts of melanoma, lung cancer, and head and neck squamous cell cancer patients were high expression was associated with a significant increase in ORR and longer PFS. In HCC, high expression of the signature was associated with a distinct profile in the immune infiltrate, where an increase in M1 macrophages (p = 0.003), CD4 memory T cell (p < 0.01) and CD4 naïve T cell-infiltration (p = 0.026) was observed. Conversely, low expression of the signature was associated with a marked upregulation of regulatory T cells (p < 0.001). No association was found, however, between either the overall immune infiltrate or CTNNB1 mutations and response. In patients that were treated with TKIs in frontline before receiving subsequent anti-PD1 therapy, the signature was no longer able to predict either OR or PFS/OS, suggesting that TKIs may reshape the microenvironment in a way that renders previously inflamed tumors no longer amenable to anti-PD1. Conclusions: Here, we define an 11-gene signature of response to anti-PD1 in first line aHCC. The signature was validated in patients with other solid cancer types, where it retained its predictive ability. Of note, the signature was not able to identify responders among HCC patients that were treated with TKIs prior to anti-PD1 therapy. The molecular changes induced by different treatment lines would, thus, require fresh biopsies prior to anti-PD1to enable biomarker-driven treatment.

3071 Background: Disparities in cancer screening and outcomes based on factors such as gender, socioeconomic status, and race/ethnicity are well documented.1 The Circulating Cell-free Genome Atlas study (CCGA; NCT02889978) was designed to develop and validate a blood-based multi-cancer early detection (MCED) test analyzing plasma cell-free DNA (cfDNA) to detect cancer signals across multiple cancer types and simultaneously predict cancer signal origin. Findings stratified by race/ethnicity from the third and final CCGA validation sub-study are reported. Methods: CCGA is a prospective, multicenter, case-control, observational study with longitudinal follow-up (overall N = 15,254). In this pre-specified exploratory analysis from the third substudy, key objectives were to evaluate test performance for cancer signal detection (specificity, overall sensitivity, and sensitivity by clinical stage) among racial/ethnic groups. Plasma cfDNA from evaluable samples was analyzed using a targeted methylation bisulfite sequencing assay and a machine learning approach. Overall, 4077 participants comprised the independent validation set with confirmed status (cancer: n = 2823; non-cancer: n = 1254). The groups stratified by race/ethnicity were White Non-Hispanic, Black Non-Hispanic, Other Non-Hispanic (including but not limited to Asian, Native Hawaiian, Pacific Islander, American Indian, Alaska Native), Hispanic (all races), and Other/unknown. The study was not powered to detect statistical differences between groups. Results: Cancer and non-cancer groups were predominantly White (2316/2823, 82.0% and 996/1254, 79.4%, respectively). Across racial/ethnic groups, specificity for cancer signal detection was 99.6% (White Non-Hispanic: 992/996, 95% confidence interval [99.0-99.8%]), 100.0% (Black Non-Hispanic: 85/85 [95.7-100.0%]), 100.0% (Other Non-Hispanic: 33/33 [89.6-100.0%]), 98.1% (Hispanic: 101/103 [93.2-99.5%]), and 100% (Other/unknown: 37/37 [90.6-100.0%]). Despite slight differences in cancer type and staging across racial/ethnic groups, overall sensitivity for cancer signal detection among groups ranged from 43.9% to 63.0% (White Non-Hispanic: 50.5%, 1169/2316 [48.4-52.5%], Black Non-Hispanic: 53.9%, 104/193 [46.8-60.8%], Other Non-Hispanic: 43.9%, 25/57 [31.8-56.7%], Hispanic: 63.0%, 121/192 [56.0-69.5%], and Other/unknown: 52.3%, 34/65 [40.4-64.0%]). For all racial/ethnic groups, sensitivity generally increased with clinical stage (with limited exceptions at Stage IV in some groups with small sample sizes). Conclusions: The MCED test demonstrated consistent specificity and sensitivity across racial/ethnic groups, though results are limited by sample size for some groups. These findings indicate broad applicability and support clinical implementation of this MCED test on a population scale. 1. Zavela et al. Brit J Cancer 2021. Clinical trial information: NCT02889978.

Cholangiocarcinoma (CCA) has poor prognosis due to late‐stage, symptomatic presentation. Altered DNA methylation markers may improve diagnosis of CCA. Reduced‐representation bisulfite sequencing was performed on DNA extracted from frozen CCA tissues and matched to adjacent benign biliary epithelia or liver parenchyma. Methylated DNA markers (MDMs) identified from sequenced differentially methylated regions were selected for biological validation on DNA from independent formalin‐fixed, paraffin‐embedded CCA tumors and adjacent hepatobiliary control tissues using methylation‐specific polymerase chain reaction. Selected MDMs were then blindly assayed on DNA extracted from independent archival biliary brushing specimens, including 12 perihilar cholangiocarcinoma, 4 distal cholangiocarcinoma cases, and 18 controls. Next, MDMs were blindly assayed on plasma DNA from patients with extrahepatic CCA (eCCA), including 54 perihilar CCA and 5 distal CCA cases and 95 healthy and 22 primary sclerosing cholangitis controls, balanced for age and sex. From more than 3,600 MDMs discovered in frozen tissues, 39 were tested in independent samples. In the clinical pilot of 16 MDMs on cytology brushings, methylated EMX1 (empty spiracles homeobox 1) had an area under the curve (AUC) of 0.98 (95% confidence interval [CI], 0.95‐1.0). In the clinical pilot on plasma, a cross‐validated recursive partitioning tree prediction model from nine MDMs was accurate for de novo eCCA (AUC, 0.88 [0.81‐0.95]) but not for primary sclerosing cholangitis–associated eCCA (AUC, 0.54 [0.35‐0.73]). Conclusion : Next‐generation DNA sequencing yielded highly discriminant methylation markers for CCA. Confirmation of these findings in independent tissues, cytology brushings, and plasma supports further development of DNA methylation to augment diagnosis of CCA.

AbstractObjectivesInhibitors to the checkpoint proteins cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4) and programmed cell death protein 1 (PD‐1) are becoming widely used in cancer treatment. However, a lack of understanding of the patient response to treatment limits accurate identification of potential responders to immunotherapy.MethodsIn this study, we assessed the expression of PD‐1 and CTLA‐4 on 19 leucocyte populations in the peripheral blood of 74 cancer patients. A reference data set for PD‐1 and CTLA‐4 was established for 40 healthy volunteers to determine the normal expression patterns for these checkpoint proteins.ResultsUnsupervised hierarchical clustering found four immune profiles shared across the solid tumor types, while chronic lymphocytic leukaemia patients had an immune profile largely unique to them. Furthermore, we measured these leucocyte populations on an additional cohort of 16 cancer patients receiving the PD‐1 inhibitor pembrolizumab in order to identify differences between responders and non‐responders, as well as compared to healthy volunteers (n = 20). We observed that cancer patients had pre‐treatment PD‐1 and CTLA‐4 expression on their leucocyte populations at different levels compared to healthy volunteers and identified two leucocyte populations positive for CTLA‐4 that had not been previously described. We found higher levels of PD‐1+ CD3+ CD4− CD8− cells in patients with progressive disease and have identified it as a potential biomarker of response, as well as identifying other significant differences in phenotypes between responders and non‐responders.ConclusionThese results are suggestive that categorisation of patients based on immune profiles may differentiate responders from non‐responders to immunotherapy for solid tumors.

Background: Direct-acting antivirals (DAA) are highly effective for the treatment of hepatitis C (HCV), although there are limited data on the safety and efficacy of DAA therapy in hepatitis C-positive individuals awaiting liver transplantation for hepatocellular carcinoma (HCC). Methods: We conducted a retrospective cohort study of HCV-positive patients who underwent liver transplantation for HCC at 3 liver transplant centers across the United States from 2014 to 2017 with follow-up to July 2018. Transplant recipients who received DAA before transplant were compared with those who did not (DAA naive) for posttransplant HCC recurrence rate, sustained virological response (SVR), allograft failure, and death using Kaplan-Meier analysis and Cox proportional hazard models. Results: A total of 171 HCV-HCC transplant recipients (99 pretransplant DAA; 72 DAA naive controls) were included, with a median follow-up of 24 months. The overall posttransplant HCC recurrence rate was 9% (15/171). Pretransplant DAA was not associated with HCC recurrence (5% versus 14%; P = 0.07), graft failure (7% versus 3%; P = 0.21), or death (12% versus 19%; P = 0.19) as compared with DAA naive patients. SVR rates were significantly lower (P < 0.01) with pretransplant DAA (75%, 39/52) than posttransplant DAA (97%, 59/61) therapies. Those who received pretransplant DAA and those who did not were not statistically different in age, gender, alpha fetal protein levels, model for end-stage liver disease scores, or transplant wait time. Conclusions: Pretransplant DAA for HCV was not associated with an increased risk of posttransplant HCC recurrence, though pretransplant DAA had lower efficacy than posttransplant DAA in HCV-HCC transplant recipients.