Early-stage HCC Research Articles

Abstract 5638: Liver preclinical models - acute, chronic and cancer models

Abstract Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death and accounts for >80% of primary liver cancer worldwide. Early stage HCC can be treated by local ablation, surgical resection or liver transplantation. Systemic pharmacological options are limited and only a few available (kinase and immune-checkpoint inhibitors). Most cases of liver cancer occur in the setting of chronic liver diseases. Risk factors include chronic Hepatitis B and C, alcohol addiction and metabolic diseases. HCC is a multistep process comprising chronic liver injury, inflammation, fibrosis/cirrhosis and cancer formation. Therefore, providing palliative and curative options remains a high medical need. In order to better evaluate new preventive and curative treatments of liver cancers we developed complementary and integrated strategies to mimic the liver cancer initiation and progression steps in mouse models. These models involve chemotoxic agents, diet-induced disorders and implantation strategies. We first established an orthotopic syngeneic model using Hepa1.6 mouse liver hepatoma cells. Hepa1.6-derived tumor growth was characterized through liver index, alpha-fetoprotein measurement in serum and liver, and MRI. The response to chemotherapy (Sorafenib) and immunotherapy (anti PD-1) was also assessed. Moreover, a panel of Patient-Derived Xenograft models (PDXs) are available to assess new treatment options in human HCC with regards of the genetic mutations and the variety of etiologies seen in human. During the course of HCC formation, the liver undergoes cycles of inflammation, necrosis with regeneration, fibrosis, cell dysplasia and ultimately HCC. Thus, we developed two models of non- alcoholic steato-hepatitis (NASH); The first model involves chronic administration of hepatotoxic CCL4 associated with a high-fat and high-fructose diet. Mice develop steatohepatitis and fibrosis within 4 to 8 weeks as assessed with biochemistry parameters (AST, ALT…), gene expression levels of inflammatory and fibrotic genes as well as histological scores and MRI. The effect of obeticholic acid was successfully evaluated in this model. The second model is the STAM model induced by a low dose of Streptozotocin and high fat diet regimen. In this model, mice develop metabolic syndrome (increased body weight gain, hyperglycemia and hyperlipidemia), NASH (steatosis, inflammation, fibrosis) within 12 weeks and HCC within 16 weeks. The responses to standard of care and to immunotherapy together with the characterization of the immune cell populations are currently under investigation. Altogether, these results demonstrate the usefulness of this liver diseases development program to discover and identify new treatments that could circumvent the progression of liver cancer. Citation Format: Olivier Duchamp, Gael Krysa, Robin Artus, Hugo Quillery, Maxime Ramelet, Valerie Boullay, Laure Levenez, Adelaide Ferment, Jeremy Odillard, Anne-Benedict Boullay, Peggy Provent, Edwige Nicodeme, Samira Benhamouche-Trouillet, Anne Lazzari, Loic Morgand. Liver preclinical models - acute, chronic and cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5638.

Abstract 5436: Purification and mRNA profiling of extracellular vesicles for early detection of hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the 4th most common cause of cancer-related deaths worldwide. The poor prognosis of HCC is due to the fact that diagnosis is often made at an advanced stage in disease development. It is crucial to develop a non-invasive liquid biopsy-based diagnostic solution for early detection of HCC. In this study, we developed a covalent chemistry-based nanostructured silicon substrate (“EV Click Chip”) for the isolation of HCC extracellular vesicles (EVs). The EV Click Chip leverages specific click chemistry reactions and sensitive multi-marker cocktail antibody identification of the HCC EVs. The EV Click Chip also allows for the subsequent release of the captured HCC EVs and is optimal for the downstream molecular analysis. A well-validated 10 liver-specific genes were quantified using reverse transcription droplet digital PCR (RT-ddPCR) in HCC EVs purified by the optimized EV Click Chips for the detection of HCC. Our EV Click Chip-based HCC-EV Assay is able to differentiate HCC from non-HCC controls (chronic liver diseases, healthy donors and other cancers) and outperformed clinical AFP test for distinguishing early-stage HCC from at-risk cirrhotic patients. Citation Format: Yi-Te Lee, Na Sun, Ryan Y. Zhang, Rueihung Kao, Pin-Jung Chen, Pai-Chi Teng, Jasmine J. Wang, Yingying Yang, Minhyung Kim, Edwin M. Posadas, Sungyong You, Ju Dong Yang, Vatche G. Agopian, Hsian-Rong Tseng, Yazhen Zhu. Purification and mRNA profiling of extracellular vesicles for early detection of hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5436.

Influence of surgical approach and quality of resection on the probability of cure for early-stage HCC occurring in cirrhosis.

Background & AimsThe quality of surgical care of patients with HCC is associated with improved long-term prognosis and may also be influenced by the type of surgical approach. The present study aimed at evaluating the role of the laparoscopic approach on quality of surgical care and long-term prognosis in optimal HCC surgical candidates.MethodsAll consecutive patients undergoing open (OLR) or laparoscopic liver resection (LLR) for early-stage HCC in cirrhosis (METAVIR F4) at 5 French expert hepato-pancreatico-biliary centres between 2010 and 2018 were enrolled. Quality of surgical care was defined by textbook outcome (TO), a combination of 6 criteria representing ideal hospitalisation. Factors associated with TO were determined on multivariate analysis. Comparison between LLR and OLR was performed after propensity score matching (PSM). The primary endpoint was disease-free survival (DFS). Statistical cure was modelled using a non-mixture model.ResultsOverall, 425 patients were included. Median follow-up was 42.0 months. LLR was performed in 267 (62.8%) patients. TO was achieved in 140 (32.9%) patients. LLR was independently associated with TO (odds ratio [OR] 2.81; 95% CI 1.29–6.12; p = 0.009). After PSM, LLR patients cumulated higher number of TO criteria than OLR patients (5 vs. 4; p = 0.012). The 1-, 3-, and 5-year DFS of LLR patients with and without TO were 82.3%, 64.4%, and 62.5%, and 76.9%, 51.4%, and 30.2%, respectively (p = 0.003). On multivariable Cox regression, TO was independently associated with improved DFS (hazard ratio 0.34; p = 0.001). The cure fraction of the whole population was 24.4%. Patients achieving TO had increased cure fraction than patients not achieving TO (32.6% vs. 18.1%).ConclusionsQuality of surgical care improves the prognosis of patients with early-stage HCC and is promoted by the laparoscopic approach.Lay summaryThe overall quality of surgical care, as measured by TO, plays a pivotal role in the prognosis and, in particular, on the probability of statistical cure of patients with resectable early-stage HCC occurring in cirrhosis. By influencing TO, laparoscopy has an indirect impact on the probability of cure and long-term management of these patients. This study strongly supports the promising curative role of mini-invasive treatments for early-stage HCC, such as low-difficulty LLR.

ЗАСТОСУВАННЯ ПЕРКУТАННОЇ ХІМІЧНОЇ АБЛЯЦІЇ В ЛІКУВАННІ ПАЦІЄНТІВ З ГЕПАТОЦЕЛЮЛЯРНОЮ КАРЦИНОМОЮ

Щороку частота виявлення ГЦК невпинно зростає. У період з 1990 по 2015 рік показник вперше діагностованих випадків ГЦК збільшився на 75%. В Україні за 2018 рік спеці- альним лікуванням охоплено лише 18,0% первинних хворих: тільки хірургічним лікуванням - 4,9%, комбінованим та комплексним лікуванням — 2,0 %. Незадовільні результати з охоплення спеціальним лікування пов'язані з особливостями етіопатогенезу ГЦК, асимптоматичним пе- ребігом та недосконалою системою скринінгу пацієнтів групи ризику, що утруднює діагнос- тику захворювання на ранніх стадіях. Вибір тактики лікування ГЦК асоційованою з вірусним гепатитом і цирозом печінки досі залишається складною клінічною проблемою, оскільки “за- стосування радикального лікування на ранній стадії є наріжним каменем для покращення за- гальної виживаності таких пацієнтів. Пацієнтам з ГЦК на ранніх стадіях з цирозом печінки, що не підлягають радикальному хірургічному лікуванню на момент звернення, показано засто- сування методів перкутанної абляції — радіочастотна абляція, хімічна абляція, кріоабляція, мікрохвильова абляція тощо. Основними показаннями для застосування локальної абляції в лікуванні ГЦК — дуже рання та рання стадія (за BCLC – стадія 0 та А), компенсована або субкомпенсована функція печінки при цирозі (Child - Pugh клас A та B), загальносоматичний статус за ECOG 0–1, діаметр пухлинного вогнища до 5,0 см (найбільш оптимально до 3,0 см), вогнищеве вузлове ураження, солітарне або множинне ураження. Перкутанну абляцію застосо- вують у якості “брідж”-терапії з метою локального контролю пухлинного росту у пацієнтів, що відповідають Міланським критеріям, критеріям UCSFC і знаходяться в списку очікуван- ня на трансплантацію. Також дані методи показали свою ефективність як неоад'ювантна “downstaging”-терапія у хворих, що не відповідають критеріям для трансплантації печінки та/або з первинно нерезектабельним ураженням. Протягом багатьох десятиліть перкутан- на хімічна абляція була “золотим” стандартом мініінвазивного лікування ГЦК. Даний метод простий у виконанні, не потребує спеціального обладнання, добре переноситься пацієнтами, безпечний та економічно вигідний. Основні хімічні агенти - етанол та оцтова кислота. Про- цедура перкутанної хімічної абляції передбачає інтратуморальне введення хімічного агенту “real-time” під контролем методів візуалізації (УЗД або КТ). Хімічна абляція - це метод дру- гої лінії і застосовується лише у випадках, наявності протипоказань до виконання радіочас- тотної абляції. Даний метод не супроводжується тяжкими специфічними ускладненнями та летальністю. Ефективність її клінічного застосування безпосередньо залежить від розміру пухлинного вогнища. Оптимальний розмір пухлинного вогнища становить не більше 3,0 см. Інтратуморальне введення етанолу призводить до некротизації 90% -100% пухлинних вузлів ГЦК діаметром ≤ 2,0 см та 70% при пухлинах 2,0 - 3,0 см.

Machine learning-based genome-wide interrogation of somatic copy number aberrations in circulating tumor DNA for early detection of hepatocellular carcinoma.

BackgroundDNAs released from tumor cells into blood (circulating tumor DNAs, ctDNAs) carry tumor-specific genomic aberrations, providing a non-invasive means for cancer detection. In this study, we aimed to leverage somatic copy number aberration (SCNA) in ctDNA to develop assays to detect early-stage HCCs.MethodsWe conducted low-depth whole-genome sequencing (WGS) to profile SCNAs in 384 plasma samples of hepatitis B virus (HBV)-related HCC and cancer-free HBV patients, using one discovery and two validation cohorts. To fully capture the robust signals of WGS data from the complete genome, we developed a machine learning-based statistical model that is focused on detection accuracy in early-stage HCC.FindingsWe built the model using a discovery cohort of 209 patients, achieving an overall area under curve (AUC) of 0.893, with 0.874 for early-stage (Barcelona clinical liver cancer [BCLC] stage 0-A) and 0.933 for advanced-stage (BCLC stage B-D). The performance of the model was then assessed in two validation cohorts (76 and 99 patients) that only consisted of patients with stage 0-A HCC. Our model exhibited a robust predictive performance, with an AUC of 0.920 and 0.812 for the two validation cohorts. Further analyses showed the impact of tumor sample heterogeneity in model training on detecting early-stage tumors, and a refined model addressing the heterogeneity in the discovery cohort significantly increased model performance in validation.InterpretationWe developed an SCNA-based, machine learning-driven model in the non-invasive detection of early-stage HCC in HBV patients and demonstrated its performance through strict independent validations.

LncRNA MAGI2-AS3 is downregulated in the distant recurrence of hepatocellular carcinoma after surgical resection and affects migration and invasion via ROCK2

Introduction and objectivesHepatocellular carcinoma (HCC) is the second most lethal cancer around the world, with poor survival rate and high metastasis rate in patients. Long noncoding RNAs (lncRNAs) have been reported to modulate the initiation and development of liver cancer. We aimed to investigate the role of lncRNA MAGI2-AS3 in HCC and underlying mechanisms. Materials and methodsThe expression levels of MAGI2-AS3 in plasma of HCC patients and the control participants were measured by qPCR. Hep3B and MHCC97-H cells were transfected with MAGI2-AS3 and ROCK2 expression vectors. Cell migration and invasion of HCC cells transfected with the vectors were investigated by transwell assay. In addition, flow cytometry and western blot were performed for apoptosis detection. ResultsWe found that MAGI2-AS3 was downregulated in plasma of early stage HCC patients compared to healthy controls. After surgical resection, the expression levels of MAGI2-AS3 were increased compared to pretreatment levels on the day of discharge. During the follow-up, MAGI2-AS3 was downregulated in patients developed distant recurrence, but not in other patients compared to the levels measured on the day of discharge. In HCC cells, overexpression of MAGI2-AS3 mediated the downregulation of ROCK2. Cell invasion and migration assay showed that overexpression of MAGI2-AS3 mediated the decreased cell invasion and migration rate, while ROCK2 played an opposite role and attenuated the effects of overexpression of MAGI2-AS3. ConclusionOur study indicated that MAGI2-AS3 was downregulated in the distant recurrence of HCC after surgical resection and affected the invasion and migration of HCC cells via ROCK2.

Quantitative Analysis of α-1-Antitrypsin Glycosylation Isoforms in HCC Patients Using LC-HCD-PRM-MS.

The change in glycosylation of serum proteins is often associated with the development of various diseases and thus can be used for diagnosis. In this study, a liquid chromatography-tandem mass spectrometry-based method is used for accurate structural analysis and quantification of site-specific glycoforms of serum α-1-antitrypsin (A1AT) in early-stage HCC and cirrhosis patients. Serum protein A1AT was purified from patient sera by immunoprecipitation with anti-A1AT antibody conjugated agarose beads, and the isolated A1AT protein was digested and analyzed by LC-MS/MS. Two tandem mass spectrometry strategies are integrated in this study: a nontargeted stepped HCD strategy for structural analysis of A1AT glycopeptides and a targeted parallel reaction monitoring (PRM) strategy for quantification of site-specific glycoforms of A1AT in HCC and cirrhosis patient sera. Accordingly, pGlyco2.0 software was used for glycopeptide identification, and Skyline software was used for glycoform quantification using the Y1 ion (peptide+GlcNAc) in MS/MS spectra. Ten site-specific glycopeptides of A1AT were identified with stepped HCD-MS/MS in patient samples, 7 of which were further quantified using HCD-PRM-MS among patient samples. We found that our strategy was able to distinguish isomers of glycopeptides where several isomers showed distinctly different patterns between cirrhosis and HCC patients. We also found that the ratio of different charge states (2+/3+) of one glycopeptide of A1AT can significantly discriminate early-stage HCC from cirrhosis with the area under the receiver operating characteristic curve AUC of 0.9. Further analysis showed that the difference may be related to the sialic acid/galactose linkage of the glycan motif.

Diagnostic value of serum glypican-3 alone and in combination with AFP as an aid in the diagnosis of liver cancer

BackgroundRecently, a series of studies have been published to examine the possible diagnostic and prognostic values of glypican-3 (GPC3) in liver cancer with conflicting results observed. Thus, the present study aimed to assess the values of preoperative serum GPC3 alone and in combination with AFP for the diagnosis of liver cancer. MethodsAn enzyme-linked immunoassay was used to quantify serum GPC3 in hepatocellular carcinoma group (HCC, n = 210), intrahepatic cholangiocarcinoma group (ICC, n = 36), combined hepatocellular cholangiocarcinoma group (cHCC-CC, n = 8), metastatic liver cancer group (MLC, n = 10) and normal controls (NC, n = 134). ResultsThe area under the curve (AUC) of GPC3 for HCC versus NC was 0.879, with a sensitivity of 79.52% at an optimal cutoff value of 0.0414 ng/mL; when GPC3 was combined with AFP, the AUC and sensitivity were increased to 0.925 and 88.10%, respectively. In addition, 43 of 68 AFP-negative patients had elevated GPC3 levels. Furthermore, the positive rate of GPC3 was significantly higher than the that of AFP for HCC in early stage. ConclusionsSerum GPC3 was superior to AFP for the diagnosis of early-stage HCC, and may be complementary to AFP for distinguishing HCC from NC.

Detection of Colorectal Cancer in Circulating Cell-Free DNA by Methylated CpG Tandem Amplification and Sequencing.

Aberrant DNA hypermethylation of CpG islands occurs frequently throughout the genome in human colorectal cancer (CRC). A genome-wide DNA hypermethylation analysis technique using circulating cell-free DNA (cfDNA) is attractive for the noninvasive early detection of CRC and discrimination between CRC and other cancer types. We applied the methylated CpG tandem amplification and sequencing (MCTA-Seq) method, with a fully methylated molecules algorithm, to plasma samples from patients with CRC (n = 147) and controls (n = 136), as well as cancer and adjacent noncancerous tissue samples (n = 66). We also comparatively analyzed plasma samples from patients with hepatocellular carcinoma (HCC; n = 36). Dozens of DNA hypermethylation markers including known (e.g., SEPT9 and IKZF1) and novel (e.g., EMBP1, KCNQ5, CHST11, APBB1IP, and TJP2) genes were identified for effectively detecting CRC in cfDNA. A panel of 80 markers discriminated early-stage CRC patients and controls with a clinical sensitivity of 74% and clinical specificity of 90%. Patients with early-stage CRC and HCC could be discriminated at clinical sensitivities of approximately 70% by another panel of 128 markers. MCTA-Seq is a promising method for the noninvasive detection of CRC.

PD-1 expressions on monocytes from patients with hepatocellular carcinoma differs depending on the progression of the cancer

Abstract Hepatocellular carcinoma (HCC) is the third leading cause of cancer death in the world. Multifocal HCC often arise synchronously or metachronously, leading high mortality rates in HCC patients. Therefore, it is necessary to diagnose the progression for HCC and to appropriately treat it accordingly. In this study, we tried to analyze for immunological characters of monocytes isolated from several stages of HCC patients and examined whether these characters can be used as a new tumor marker for HCC. From 2017 August to 2018 December, 35 patients of advanced-stage HCC and 33 patients of early-stage HCC were enrolled. Monocytes from peripheral blood of these patients were stained for anti-PD-1 antibody, anti-PD-L1 antibody and anti-PD-L2 antibody. These cells were analyzed for flow cytometry. Five healthy donors were used for the control groups. In the results, monocytes from early-stage HCC patients were not express PD-L1 (2.9 ± 1.1%), and express few PD-L2 (43.4 ± 13.5%) and PD-1 (45.2 ± 15.1%). However, monocytes from advanced-stage HCC patients were expressed PD-L1 (75.3 ± 20.7%) and PD-L2 (74.7 ± 15.8%), but not PD-1(1.4 ± 1.1%). As the controls, monocytes from healthy donors were not expressed PD-L1 (21.7 ± 5.5%), PD-L2 (31.7 ± 23.8%) and PD-1 (11.6 ± 6.4%). When CD8− cells from advanced-stage HCC patients were co-cultured with monocytes from syngeneic patients, the turmoricidal activities of these cells were suppressed(15.7±7.5%). In contrast, CD8− cells from early-stage HCC, were co-cultured with syngeneic monocytes, kept turmoricidal activities. In conclusions, PD-L1 expression in monocytes was increased, as HCC progressed and may be using a new marker of the classification for the progression in hepatocellular carcinoma.

Detection of early-stage hepatocellular carcinoma in asymptomatic HBsAg-seropositive individuals by liquid biopsy

Liquid biopsies, based on cell free DNA (cfDNA) and proteins, have shown the potential to detect early stage cancers of diverse tissue types. However, most of these studies were retrospective, using individuals previously diagnosed with cancer as cases and healthy individuals as controls. Here, we developed a liquid biopsy assay, named the hepatocellular carcinoma screen (HCCscreen), to identify HCC from the surface antigen of hepatitis B virus (HBsAg) positive asymptomatic individuals in the community population. The training cohort consisted of individuals who had liver nodules and/or elevated serum α-fetoprotein (AFP) levels, and the assay robustly separated those with HCC from those who were non-HCC with a sensitivity of 85% and a specificity of 93%. We further applied this assay to 331 individuals with normal liver ultrasonography and serum AFP levels. A total of 24 positive cases were identified, and a clinical follow-up for 6-8 mo confirmed four had developed HCC. No HCC cases were diagnosed from the 307 test-negative individuals in the follow-up during the same timescale. Thus, the assay showed 100% sensitivity, 94% specificity, and 17% positive predictive value in the validation cohort. Notably, each of the four HCC cases was at the early stage (<3 cm) when diagnosed. Our study provides evidence that the use of combined detection of cfDNA alterations and protein markers is a feasible approach to identify early stage HCC from asymptomatic community populations with unknown HCC status.

Highlights of This Issue

Many breast cancer–associated SNPs have been discovered. SNP risk information can potentially enhance cancer risk prediction and inform risk-based screening programs, but is more SNP more accurate? Fung and colleagues systematically examined available literature by quantifying the extent of improvement in performance offered by SNPs in breast cancer risk prediction models. SNP information improved model performance (+0.044 in AUC), despite the lack of trend between improvement and number of SNPs. Also, as the majority of risk reclassification occurred in intermediate-risk women, screening could be a two-step process where a questionnaire is first used to identify intermediate-risk individuals, followed by testing for these women only.Using registry data, Withrow and colleagues report stable incidence rates of CNS cancers overall among U.S. children between 1998 and 2013, though statistically significant changes were reported for several subtypes. Glioma and pilocytic astrocytoma incidence rates increased by 0.77 and 0.89% per year, respectively; embryonal cancer rates decreased by 0.88% per year. The investigators estimated these changes resulted in 120 excess gliomas, 94 excess pilocytic astrocytomas, and 72 fewer embryonal CNS tumors in 2013, compared to what would be expected had rates remained stable. These modest trends could be the result of true changes in incidence, or reflect shifts in identification, reporting, and classification.Liver ultrasound is a standard surveillance test for early detection of HCC in high-risk patients. However, liver ultrasound has limited accuracy. The GALAD score is a serum biomarker-based model that predicts the probability of having HCC in patients with chronic liver disease. In the current study, Yang and colleagues showed that the performance of the GALAD score was superior to that of ultrasound. The outstanding performance of the GALAD score was confirmed in an independent multicenter U.S. cohort of early-stage HCC and cirrhosis patients, confirming the utility of the GALAD score as an excellent tool for HCC detection. The study also demonstrates that the combination of GALAD and ultrasound, termed the GALADUS score, further enhances the performance of the GALAD score.Black women with ovarian cancer experience worse survival than white women. Receipt of guideline care improves survival, yet care may vary by race. In this study, Cronin and colleagues evaluated disparities in guideline treatment between black and white women diagnosed with ovarian cancer and ovarian cancer deaths for these women. The authors assessed four patient and provider characteristics associated with receipt of guideline care, with a focus on care from a gynecologic oncologist (GO). Race was not associated with risk of death when guideline care was included in multivariate survival models. However, black patients received less guideline care. GO consultation significantly increased receipt of guideline care. Research is needed to understand treatment perspectives for black patients and their providers to increase the receipt of guideline care and reduce survival disparities.

Plasma GH as a diagnostic and prognostic biomarker in HCC without cirrhosis.

227 Background: The association between the GH/IGF-1 axis and HCC was reported in patients (pt) with underlying cirrhosis. However, there is limited information among HCC pt without (w/o) cirrhosis. We herein investigated the role of GH as a circulating biomarker for HCC diagnosis and prognosis in pt w/o cirrhosis. Methods: Under IRB approval, we prospectively enrolled 1267 newly-diagnosed HCC pt in a case control study at the MD Anderson Cancer Center (2000-2015). Controls were healthy individuals (n = 1104). Plasma GH and AFP were measured 274 HCC pt w/o cirrhosis 200 healthy controls. IGF-1 was measured in 133 and 82 pt, respectively. We classified HCC pt into higher and lower GH values (cutoff for women, 3.7 µg/L; men, &gt; 0.9 µg/L). Results: Most pt (74%) were male, with advanced BCLC staging (C-D, 74%) and 61% were older than 60y. Baseline GH was higher in HCC w/o cirrhosis (mean 3.3 µg/L) than controls (mean 0.4 µg/) (p &lt; .001). ROC curve was plotted to assess diagnostic role. The AUC for AFP was 82.9 (p &lt; .001); for GH 78.2 (p &lt; .001). When only non-cirrhotic HCC pt with early stage (CLIP 0-2) and AFP &lt; 20 ng/m were compared to controls, the GH/IGF-1 ratio had high prediction of early stage HCC - AUC 83 (95% CI 78-89%) (p &lt; .0001). At a specificity of 90%, sensitivity of GH/IGF ratio was 67%. In addition, among HCC w/o cirrhosis, higher GH levels correlated with presence of vascular invasion (p &lt; .001) and thrombosis (p = .004), tumor involvement of &gt; 50% liver (p = .003), and more advanced BCLC (p &lt; .001) and TNM staging (p &lt; .001). Median overall survival (months) of HCC pt w/o cirrhosis with high GH levels was 13.1 (10.8-15.4) compared to 37.4 (19.8-55.1) of pt with lower plasma GH (p &lt; .001). Multivariate cox-regression analysis identified high GH as an independent risk factor for mortality (HR = 1.8; 95% CI, 1.3-2.4; p &lt; .001). Conclusions: Our study demonstrates the diagnostic and prognostic role of plasma GH in non-cirrhotic HCC and identifies the GH/IGF-1 ratio as a promising diagnostic marker for early stage HCC w/o cirrhosis and low AFP; this analysis excludes the confounding effect hepatocyte impaired function by presence of cirrhosis. Further studies are warranted to assess the causes of the observed differences.

Immunohistochemistry Detects Increased Expression of Aldo-Keto Reductase Family 1 Member B10 (AKR1B10) in Early-Stage Hepatocellular Carcinoma.

BackgroundHepatocellular carcinoma (HCC) remains difficult to diagnose at an early stage. Aldo-keto reductase family 1 member B10 (AKR1B10) is an oxidoreductase that is upregulated in some chronic liver diseases. The aim of this study was to use immunohistochemistry to evaluate the expression of AKR1B10 in liver tissue from patients with HCC of different stages.Material/MethodsForty-four patients with a tissue diagnosis of HCC (35 males and 9 females) with 37 control samples of liver tissue containing liver cirrhosis were studied using immunohistochemistry for the expression of AKR1B10. Histological examination determined the grade of HCC; the stage of HCC was determined according to the Barcelona Clinic Liver Cancer (BCLC) staging system. Serum alpha-fetoprotein (AFP) levels were measured and compared between the patients with HCC.ResultsImmunohistochemistry showed increased expression of AKR1B10 in moderately-differentiated HCC compared with well-differentiated HCC, poorly-differentiated HCC, and liver cirrhosis (P<0.05). Sensitivity and specificity of AKR1B10 expression in HCC were high at a cutoff integral optical density (IOD) value of 89.5. A significant increase in AKR1B10 expression was found in early-stage HCC (P<0.05). Serum AFP levels were increased in patients with poorly-differentiated HCC, were increased in intermediate-stage HCC, and were significantly increased in advanced-stage HCC (P<0.05).ConclusionsImmunohistochemistry showed that the expression of AKR1B10 was increased in tumor tissue from patients with early-stage HCC. Further studies are needed to determine the role of AKR1B10 in the early detection of HCC.

Hepatocellular Carcinoma: New multimodal therapy concepts

Hepatocellular carcinoma guidelines are currently under revision. A new edition is expected in 2018. Patients with chronic liver diseases or cirrhosis require HCC screening by ultrasound every 6 months. Surgical resection or liver transplantation are curative treatment options for early stage HCC. A more and more common and well tolerated locoregional therapy approach based on study data is selective internal radio therapy (SIRT), although studies did not show an improvement in outcome comparing SIRT to transarterial chemotherapy (TACE) in BCLC B or sorafenib in BCLC C. Looking at targeted therapies regorafenib is approved for patients under treatment with sorafenib and disease progression as a second line therapy. Positive phase III-study data have been published for lenvatinib as first-line and cabozantinib as second-line therapy. Nivolumab was approved by the FDA as second-line therapy after positive phase I/II-study outcomes. A study on nivolumab versus sorafenib as first-line therapy will be published in 2018.

Abstract B06: A SOX9+ bile duct progenitor as a cell of origin of hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the most frequent form of liver cancer and its associated mortality is increasing. Most HCC patients are diagnosed at an advanced stage of the disease, which is not eligible for curative surgery and for which efficient drugs remain elusive. Consequently, the prognosis of these patients is particularly poor, begging for a revolution in our therapeutic approach of the disease. Our in-depth understanding of HCC etiology suggests that patients at risk could be regularly screened for the early detection and treatment of nascent neoplastic lesions. This strategy requires a detailed knowledge of the early events that initiate HCC since their nature shapes the features of the developing lesions. However, the identity of cells that can serve as a cell of origin for HCC as well as the molecular events that transform them remain unknown, hampering the development of an early-intervention strategy. To address this important gap in our knowledge, we have taken advantage of a tractable model of HCC initiated upon Rb family loss of function, which is an almost universal event in cancer and particularly in HCC. The Cre/Lox-mediated inactivation of Rb family genes in distinct liver cell populations (cholangiocytes, hepatocytes, and various populations of bipotential and committed progenitors) reveals that most tested cell types, while displaying aberrant cell cycle activity, are resistant to transformation upon Rb pathway alteration. However, Rb family inactivation in Sox9-expressing bile duct progenitor cells initiates HCC that recapitulates the human disease. Interestingly, these committed cholangiocyte progenitors rapidly lose their canonical markers to adopt markers that are specific of the hepatocyte lineage, suggesting that Rb gene family inactivation potentiates their cellular plasticity. Importantly, analysis of human HCC array reveals that expansion of Sox9-expressing cells is a common feature of premalignant lesions and early-stage HCC. Despite its almost universal alteration in cancer, our mouse genetic approach demonstrates that only rare cell populations are susceptible to transformation upon Rb pathway inactivation. In addition, our data show that a cholangiocyte-restricted progenitor population can serve as the cell of origin for a hepatocyte-based type of cancer. This model will enable us to deconstruct the molecular mechanisms that initiate and drive early HCC development and ultimately allow us to shift the clinical management of the disease towards its early steps. Citation Format: Patrick Viatour. A SOX9+ bile duct progenitor as a cell of origin of hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B06.

PD-L1 expression of monocytes may be used a new marker of the classification for the cancer progression in hepatocellular carcinoma.

Abstract Hepatocellular carcinoma (HCC) is the fifth common cancer in the world. In previous study, we reported that M2b monocytes which predominately generated in advanced-stage HCC patients were important target for HCC immunotherapy. Recently, an anti-PD-1 antibody has been approved for the treatment against HCC. In this study, we tried to analyzed for immunological characters of monocytes isolated from patients with several stages of HCC. From 2017 August to 2017 December, nine patients diagnosed as advanced-stage HCC patients and five patients diagnosed as early-stage HCC patients were enrolled in this study. Monocytes from peripheral blood of these patients were stained for anti-PD-1 antibody, anti-PD-L1 antibody and anti-PD-L2 antibody. These cells were analyzed for flow cytometry. Furthermore, five healthy donors were used for the control groups. In the results, a majority of monocytes from advanced-stage HCC patients were expressed PD-L1 (81.5 ± 14.7%) and PD-L2 (71.6 ± 18.5%), but not PD-1(20.2 ± 18.6%). And, monocytes from early-stage HCC patients were expressed PD-L1 (9.9 ± 4.1%), PD-L2 (38.74 ± 9.9%) and PD-1 (23.5 ± 9.8%). As the contorls, monocytes from healthy donors were not expressed PD-L1 (21.7 ± 5.5%), PD-L2 (31.7 ± 23.8%) and PD-1 (11.6 ± 6.4%). The PD-L1 expression in monocytes was increased as HCC progressed. These results indicate the PD-L1 expression may be used as a new marker for the classification of the cancer progression. And, PD-1−PD-L1+PD-L2+ monocytes which were predominantly generated in advanced-stage HCC patients, may target for the treatment of anti-PD-L1 antibody.

Downregulation of RFX1 predicts poor prognosis of patients with small hepatocellular carcinoma

ObjectiveRegulatory factor X1 (RFX1) deletion has been reported to be correlated with poor prognosis of some types of cancer. The present study aimed to investigate the prognostic value of RFX1 in HCC, especially in small hepatocellular carcinoma. MethodsImmunohistochemical assay was used to investigate RFX1 expression in 221 HCC tissues and another validation cohort of 71 small HCC samples. We also performed in vitro experiments to investigate if RFX1 regulated invasive capacity of HCC cells and expression of epithelial-mesenchymal transition (EMT) markers. ResultsWe found that RFX1 expression was significantly lower in HCC tissues compared to the corresponding non-tumor tissues. Further survival analysis suggested that the downregulation of RFX1 correlated with poor prognosis and a high recurrence risk in HCC patients, particularly in small HCC patients. Furthermore, another validation cohort of small HCC samples confirmed that downregulation of RFX1 in HCC tissues predicted high recurrence risk and poor prognosis for early stage HCC patients. In vitro studies suggested that knocking down RFX1 facilitated HCC cell invasion, while overexpression of RFX1 reduced the invasion of HCC cells. Western blot assays also indicated that RFX1 regulated expression of some EMT markers. Knocking down RFX1 decreased E-cadherin and increased vimentin expression, while RFX1 overexpression enhanced E-cadherin and decreased vimentin expression. ConclusionsOur study demonstrated that RFX1 downregulation is a new predictive marker of high recurrence risk and poor prognosis of HCC; It has potential to help guide treatment for postoperative HCC patients, especially for small HCC patients.

MOLECULAR EVALUATION OF MIDIKINE (MDK) AS A POTENTIAL BIOMARKER TODETECT HEPATOCELLULAR CARCINOMA

The aim of this study was to assess the efficacy of non-invasive Midkineexpression level as diagnostic biomarkers of Hepatocellular Carcinoma and its relationship with diagnostic parameters. Thirty six subjects were enrolled in the present study, they were divided into three groups: Group (A) included 10 apparently healthy subjects as acontrol group; Group (B) included 14 HCC patients ; Group (C) included 12 HCV patients ; The studied groups under study were age and sex matched. Routine and specific (MDK) laboratory investigations were performed in all included subjects. The median age of the patients was 51 years (rang 44-58 years); where males represented the majority of the groups (69.4%). MDK expression level was detected by quantitative polymerase chain reaction (qRT-PCR). None of the patients were pretreated with local therapies. The MDK level was significantly higher in patient HCC than in the HCV and control groups (p=0.028), whereas MDK level was also higher in HCV patient than healthy patient. Further the correlation between the level of MDK and all laboratory parameters was tested. In this study there is a good positive correlation between MDK levels and SGPT (AST), and SGOT (ALT). The sensitivity and specificity of AFP were 75.7% and 86.4% respectively, while those of MDK were 64.3% and 77.3%, respectively, in early-stage HCC patients but when combinations between two markers lead to higher sensitivity 100% to predict HCC patient.

Abstract 1750: Analysis of non-metastatic HCC patient tumors revealed the significance of cell cycle regulation and tumor immunity in association with overall survival and identified clinically relevant druggable targets

Abstract The lack of efficacious therapies have hindered the progress in improving the survival of HCC patients worldwide. As a disease that associates with the underlying liver injuries caused by various etiologies and remains asymptomatic during most of its decades long progression, HCC presents challenges to accurately depict its biological landscape and determine suitable therapeutic intervention. An approach to tackle this issue is to define significant biological variants that impact patient survival in each disease stage, then attempt to chart paths of potential therapies. To identify significant biological determinants associated with the overall survival (OS) of early stage, non-metastatic HCC patients, we constructed focused Nanostring panels that are represented by 1,164 genes of validated components and transcriptional outputs of multiple signaling pathways, cellular machineries, and tumor immunity. An unbiased analysis of patient survival and gene transcript levels revealed four distinct groups of genes that are significantly prognostic. The first two (Group 1 &amp; 2) are associated with poorer prognosis when expression is high (hazard ratio &amp;gt; 2), whereas the remaining two (Group 3 &amp; 4) correspond to significantly better OS with elevated transcript levels (hazard ratio&amp;lt;0.5). Of note, Gene Set Enrichment Analyses (GSEA) clearly indicated that while Group 2 was substantially enriched in genes positively regulating cell cycle and cell division machinery, Group 4 was overrepresented by genes participating in the positive modulation and feedback mechanisms of adaptive immune response, implicating the survival benefit of acquiring higher immunogenic tumor environment in early stage HCC. Searching for druggable targets associated with poorer prognosis, we identified both EZH2 and CHEK1 as candidates from Group 2. In vitro treatment of three HCC cell lines with EPZ-6438 (EZH2 inhibitor) or GDC-0425 (CHEK1 inhibitor) resulted in significant modulation of H3K27me3 or pCHEK1 levels (s345) respectively. The growth of these cell lines was also dose-dependently suppressed by each inhibitor in colony formation assays . Furthermore, the combination of both inhibitors was able to achieve substantial cell growth inhibition at lower concentrations, suggesting a combinatorial effect of targeting both EZH2 and CHEK1 in HCC cell lines. Xenograft studies are ongoing to assess in vivo efficacy of this combination. In conclusion, utilizing well constructed Nanostring gene panels, we identified significant association of cell cycle machinery and adaptive tumor immunity with HCC patient survival. Our findings not only provided potential rationale for expand testing of cancer immunotherapies to non-metastatic, early stage HCC, but also revealed the potential utility of combining EZH2 and CHEK1 inhibitors to treat HCC. Citation Format: Pei-Zhen Miao, Jeff Cheng, Kwame Okrah, Bonnie Liu, Charlie Sun, Grace Cao, Tony Guo, Shijing Fu, Marie-Claire Wagle, Elizabeth Blackwood, David Shames, Garret Hampton, Lukas Amler, Astrid Kiermaier, Yevgeniya Fridlyand, Mark Lackner, Shih-Min Huang, Jian Zhou, Zhi Dai. Analysis of non-metastatic HCC patient tumors revealed the significance of cell cycle regulation and tumor immunity in association with overall survival and identified clinically relevant druggable targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1750. doi:10.1158/1538-7445.AM2017-1750