Hepatocellular Carcinoma Biopsies Research Articles

Gene expression signature as a surrogate marker of microvascular invasion on routine hepatocellular carcinoma biopsies

Microvascular invasion (MVI), a major risk factor for tumor recurrence after surgery in hepatocellular carcinoma (HCC), is only detectable by microscopic examination of the surgical specimen. We aimed to define a transcriptomic signature associated with MVI in HCC than can be applied to formalin-fixed paraffin-embedded (FFPE) biopsies for use in clinical practice. To identify a gene expression signature related to MVI by using NanoString technology, we selected a set of 200 genes according to the literature and RNA-sequencing data obtained from a cohort of 150 frozen HCC samples previously published. We used 178 FFPE-archived HCC samples, including 109 surgical samples for the training set and 69 paired pre-operative biopsies for the validation set. In 14 cases of the training set, a paired biopsy was available and was also analyzed. We identified a 6-gene signature (ROS1, UGT2B7, FAS, ANGPTL7, GMNN, MKI67) strongly associated with MVI in the training set of FFPE surgical HCC samples, with 82% accuracy (sensitivity 82%, specificity 81%, AUC 0.82). The NanoString gene expression was highly correlated in 14 paired surgical/biopsy HCC samples (mean R: 0.97). In the validation set of 69 FFPE HCC biopsies, the 6-gene NanoString signature predicted MVI with 74% accuracy (sensitivity 73%, specificity 76%, AUC 0.74). Moreover, on multivariate analysis, the MVI signature was associated with overall survival in both sets (hazard ratio 2.29; 95% CI 1.03-5.07; p= 0.041). We defined a 6-gene signature that can accurately predict MVI in FFPE HCC biopsy samples, which is also associated with overall survival, although its survival impact must be confirmed by extensive study with further clinical data. Microvascular invasion, a major risk factor for tumor recurrence after surgery in hepatocellular carcinoma, is only detectable by microscopic examination of a surgical specimen. In this study, we defined a relevant surrogate signature of microvascular invasion in hepatocellular carcinoma that may be applied in clinical practice with routine tumor biopsy and integrated into the therapeutic strategy.

Abstract No. 560 Quality of large-volume percutaneous core biopsies of hepatocellular carcinoma for research applications

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide. The majority of patients present with incurable disease and will require systemic therapy; however, the development of targeted therapies has been limited by a lack of tissue. As the number of HCC biopsies performed for research in this context increases, there is an increasing need to optimize biopsy quality. Here, we present biopsy quality outcomes from a prospective observational clinical trial profiling HCC.

The Effects of Human Reoviruses on Cancer Cells Derived from Hepatocellular Carcinoma Biopsies

Hepatocellular carcinoma (HCC) is the most common type of liver cancer around the world. Since this cancer is highly resistant to the existing treatments, we used a novel method, which selectively targets HCC cancer cells to improve the treatment process. As normal cells are resistant to reovirus replication, we used oncolytic reoviruses, which can infect, replicate in, and destroy cancer cells. In this study, the effects of oncolytic human reoviruses on cancer cells, derived from HCC biopsies, were investigated. First, reoviruses were purified. Then a plaque assay was performed to estimate the number of viruses and determine the multiplicity of infection (MOI). To evaluate the effects of reoviruses on cancer cells derived from HCC biopsies, replication of reovirus RNA, viral protein production, cytopathic effects (CPE), and cancer cell viability were assessed at different intervals post-infection. Replication of reovirus RNA and viral protein production were detected in cancer cells. Also, different levels of viral protein production, CPE, cytotoxicity, and cancer cell viability were observed at different intervals post-infection with human reoviruses. In contrast, normal human fibroblasts, which were used as negative control, remained unchanged. For the first time, the effects of human reoviruses on HCC biopsies were investigated. The results showed that human reoviruses could replicate in and destroy cancer cells derived from HCC biopsies. Overall, human reoviruses can be potentially used for the treatment of HCC.

Genomic Landscape of Liquid Biopsy for Hepatocellular Carcinoma Personalized Medicine.

Hepatocellular carcinoma (HCC) is the sixth most frequently diagnosed cancer and the third leading cause of cancer-related deaths worldwide. Advanced-stage HCC patients have poor survival rates and this requires the discovery of novel clear biomarkers for HCC early diagnosis and prognosis, identifying risk factors, distinguishing HCC from non-HCC liver diseases, and assessment of treatment response. Liquid biopsy has emerged as a novel minimally invasive approach to enable monitoring tumor progression, metastasis, and recurrence. Since the liquid biopsy analysis has relatively high specificity and low sensitivity in cancer early detection, there is a risk of bias. Next-generation sequencing (NGS) technologies provide accurate and comprehensive gene expression and mutational profiling of liquid biopsies including cell-free circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and genomic components of extracellular vesicles (EVs) including micro-RNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). Since HCC is a highly heterogeneous cancer, HCC patients can display various genomic, epigenomic, and transcriptomic patterns and exhibit varying sensitivity to treatment options. Identification of individual variabilities in genomic signatures in liquid biopsy has the potential to greatly enhance precision oncology capabilities. In this review, we highlight and critically discuss the latest progress in characterizing the genomic landscape of liquid biopsy, which can advance HCC personalized medicine.

Pivotal role of long non-coding ribonucleic acid-X-inactive specific transcript in regulating immune checkpoint programmed death ligand 1 through a shared pathway between miR-194-5p and miR-155-5p in hepatocellular carcinoma.

BACKGROUNDAnti-programmed death therapy has thrust immunotherapy into the spotlight. However, such therapy has a modest response in hepatocellular carcinoma (HCC). Epigenetic immunomodulation is a suggestive combinatorial therapy with immune checkpoint blockade. Non-coding ribonucleic acid (ncRNA) driven regulation is a major mechanism of epigenetic modulation. Given the wide range of ncRNAs that co-opt in programmed cell-death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) regulation, and based on the literature, we hypothesized that miR-155-5p, miR-194-5p and long non-coding RNAs (lncRNAs) X-inactive specific transcript (XIST) and MALAT-1 are involved in a regulatory upstream pathway for PD-1/PD-L1. Recently, nutraceutical therapeutics in cancers have received increasing attention. Thus, it is interesting to study the impact of oleuropein on the respective study key players.AIMTo explore potential upstream regulatory ncRNAs for the immune checkpoint PD-1/PD-L1.METHODSBioinformatics tools including microrna.org and lnCeDB software were adopted to detect targeting of miR-155-5p, miR-194-5p and lncRNAs XIST and MALAT-1 to PD-L1 mRNA, respectively. In addition, Diana tool was used to predict targeting of both aforementioned miRNAs to lncRNAs XIST and MALAT-1. HCC and normal tissue samples were collected for scanning of PD-L1, XIST and MALAT-1 expression. To study the interaction among miR-155-5p, miR-194-5p, lncRNAs XIST and MALAT-1, as well as PD-L1 mRNA, a series of transfections of the Huh-7 cell line was carried out. RESULTSBioinformatics software predicted that miR-155-5p and miR-194-5p can target PD-L1, MALAT-1 and XIST. MALAT-1 and XIST were predicted to target PD-L1 mRNA. PD-L1 and XIST were significantly upregulated in 23 HCC biopsies compared to healthy controls; however, MALAT-1 was barely detected. MiR-194 induced expression elevated the expression of PD-L1, XIST and MALAT-1. However, overexpression of miR-155-5p induced the upregulation of PD-L1 and XIST, while it had a negative impact on MALAT-1 expression. Knockdown of XIST did have an impact on PD-L1 expression; however, following knockdown of the negative regulator of X-inactive specific transcript (TSIX), PD-L1 expression was elevated, and abolished MALAT-1 activity. Upon co-transfection of miR-194-5p with siMALAT-1, PD-L1 expression was elevated. Co-transfection of miR-194-5p with siXIST did not have an impact on PD-L1 expression. Upon co-transfection of miR-194 with siTSIX, PD-L1 expression was upregulated. Interestingly, the same PD-L1 expression pattern was observed following miR-155-5p co-transfections. Oleuropein treatment of Huh-7 cells reduced the expression profile of PD-L1, XIST, and miR-155-5p, upregulated the expression of miR-194-5p and had no significant impact on the MALAT-1 expression profile.CONCLUSIONThis study reported a novel finding revealing that opposing acting miRNAs in HCC, have the same impact on PD-1/PD-L1 immune checkpoint by sharing a common signaling pathway.

Factors and Survival Implications associated with biopsy of hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the few cancers that can be diagnosed based on imaging findings alone. The factors associated with the decision to perform a biopsy and the clinical impact have not been previously studied. MethodsWe collected data of patients diagnosed with HCC between 2004 and 2015 from the National Cancer Database. We assessed associations between biopsy and survival with demographic and clinical factors. ResultsWe included 160,507 patients. The median age was 62 (40–90), 74.1% were male and 74.9% were white. Over the 12-year period, 47.7% (76,524/160,517) underwent a biopsy. Factors associated with a biopsy were black race, older age, presence of metastatic disease, larger tumor size, and treatment at a community cancer center. Factors associated with increased mortality were older age, higher comorbidity index, larger tumor size, presence of metastatic disease, higher AFP and elevated bilirubin. There was a significant decreased use of biopsy over successive years (2007–2015). After adjusting for prognostic factors, biopsy had no significant impact on survival HR 1.01 (95%CI 1.00–1.03. p = 0.07). ConclusionsA significant number of patients underwent a biopsy. Performing a biopsy did not have a significant impact on survival.

Biopsies of hepatocellular carcinoma with no reticulin loss: an important diagnostic pitfall

The reticulin stain is a critical diagnostic aide used to differentiate benign hepatocellular proliferations from well differentiated hepatocellular carcinoma (HCC). Rarely, however, hepatocellular carcinomas do not show definitive loss of reticulin in liver biopsy specimens. To study this group of tumors, 11 HCC with no reticulin loss in 10 patients were collected and studied. Analysis of demographics showed a typical enrichment for men with a typical age for HCC presentation of 69±7 years for adults. The background livers showed advanced fibrosis or cirrhosis in 6 of 6 cases with available information. The tumors were all well differentiated. Cytological atypia was mild and consisted of very mild nuclear atypia (8 cases), mild increase in N:C ratio (3 cases), and pseudorosette formation (4 cases). The cytological/architectural atypia was insufficient in isolation to diagnose HCC. Additional studies, however, showed an increased Ki-67 proliferative rate (N=10/10 stained cases). The Ki-67 proliferative rate was estimated to be between 5 and 10% in all tested cases and was clearly increased from adjacent liver at low power. Glypican 3 positivity (4 tumors) and alpha fetoprotein (AFP) (1/8 stained cases) positivity also helped make the diagnosis of HCC. Morphologically, the HCC had conventional morphology with five showing steatosis/steatohepatitic features and one showing intratumoral fibrosis. A control group of macroregenerative/dysplastic nodules showed no increase in Ki-67 proliferation and no staining for glypican 3. These findings highlight an important diagnostic pitfall: rare HCC show no reticulin loss on biopsy. In these challenging cases, additional findings are useful to make a diagnosis of HCC: increased Ki-67 and positive staining for aberrant expression of proteins such as glypican 3 or AFP.

Neurotrophin 3 hinders the growth and metastasis of hepatocellular carcinoma cells*

Abstract Objective Neurotrophin 3 (NTF3) is involved in numerous biological processes; however, its role in hepatocellular carcinoma (HCC) is not well studied. This study investigated NTF3 function in HCC progression and revealed its underlying molecular mechanisms. Methods The prognostic relevance of NTF3 was determined through a bioinformatical analysis of publicly available TCGA data. Immunohistochemistry of HCC biopsies was performed to explore the expression of NTF3. Cell growth and proliferation were analyzed using a Cell Counting Kit-8 (CCK-8) assay. Cell invasion and migration were analyzed using Boyden Transwell and wound healing assays. Protein expression and mRNA levels were evaluated through immunoblotting and quantitative polymerase chain reaction (qPCR). Cell apoptosis was evaluated with flow cytometry. Results NTF3 expression was significantly lower in HCC tissues than in adjacent non-tumor tissues. Low NTF3 expression was significantly associated with decreased patient survival and specific clinicopathological features. NTF3 overexpression reduced the proliferation, migration, and invasion abilities of HCC cell lines. Conclusion Decreased expression of NTF3 is associated with poor prognosis in HCC patients, likely due to its action in promoting HCC cell proliferation, migration, and invasion. Our findings provide a novel understanding into the pathogenesis of HCC and the role of NTF3 in tumor progression, suggesting that targeting NTF3 has potential therapeutic and diagnostic value for HCC.

Somatic copy number profiling from hepatocellular carcinoma circulating tumor cells

Somatic copy number alterations (SCNAs) are important genetic drivers of many cancers. We investigated the feasibility of obtaining SCNA profiles from circulating tumor cells (CTCs) as a molecular liquid biopsy for hepatocellular carcinoma (HCC). CTCs from ten HCC patients underwent SCNA profiling. The Cancer Genome Atlas (TCGA) SCNA data were used to develop a cancer origin classification model, which was then evaluated for classifying 44 CTCs from multiple cancer types. Sequencing of 18 CTC samples (median: 4 CTCs/sample) from 10 HCC patients using a low-resolution whole-genome sequencing strategy (median: 0.88 million reads/sample) revealed frequent SCNAs in previously reported HCC regions such as 8q amplifications and 17p deletions. SCNA profiling revealed that CTCs share a median of 80% concordance with the primary tumor. CTCs had SCNAs not seen in the primary tumor, some with prognostic implications. Using a SCNA profiling model, the tissue of origin was correctly identified for 32/44 (73%) CTCs from 12/16 (75%) patients with different cancer types.

Usefulness of Tumor Tissue Biopsy for Predicting the Biological Behavior of Hepatocellular Carcinoma.

Assessment of the biological behavior of tumors is important for choosing an appropriate cancer therapy. In hepatocellular carcinoma (HCC), the biological behaviour can be assessed by tumor morphology and molecular biology. This study investigated the usefulness of tumor tissue biopsy for predicting the biological behavior of HCC. We studied 43 patients who underwent hepatectomy and preoperative liver tumor biopsy for HCC. We performed clinicopathological and immunohistochemical (IHC) analyses. The expression of the following molecules was examined: regulator of G-protein signaling 5 (RGS5), glypican-3 (GPC3), keratin 19 (K19), epithelial cell adhesion molecule (EpCAM), protein induced by vitamin K absence or antagonist-II (PIVKA-II), β-Catenin, and p53. There was an overall 83.7% agreement regarding tumor differentiation between the preoperative biopsy specimens and the resected specimens. The accuracy of IHC analysis was more than 70% for all molecules between the preoperative biopsy specimens and the resected specimens. The RGS5-positive biopsy cases had higher serum α-fetoprotein levels (p=0.04), a higher rate of moderately or poorly differentiated tumors (p=0.02) and portal vein invasion (p=0.0003) than the RGS5-negative biopsy cases. The GPC3-positive biopsy cases were younger (p=0.04), had higher serum PIVKA-II levels (p=0.01), and a higher rate of portal vein invasion (p=0.03) than the GPC3-negative biopsy cases. The PIVKA-II-positive biopsy cases had significantly higher serum PIVKA-II levels than the PIVKA-II-negative biopsy cases (p=0.02). The other molecular markers showed no significantly different clinical findings between the positive and negative cases. In HCC, there was a high agreement rate of both the histopathological and IHC findings between preoperative biopsy specimens and resected specimens. In the biopsy specimens of HCC, RGS5 and GPC3 expression were useful molecular makers for predicting portal vein invasion. Liver tumor biopsy is useful for predicting the biological behavior of HCC through histopathological and immunohistochemical findings.

Complement factor H-deficient mice develop spontaneous hepatic tumors.

Hepatocellular carcinoma (HCC) is difficult to detect, carries a poor prognosis, and is one of few cancers with an increasing yearly incidence. Molecular defects in complement factor H (CFH), a critical regulatory protein of the complement alternative pathway (AP), are typically associated with inflammatory diseases of the eye and kidney. Little is known regarding the role of CFH in controlling complement activation within the liver. While studying aging CFH-deficient (fH-/-) mice, we observed spontaneous hepatic tumor formation in more than 50% of aged fH-/- males. Examination of fH-/- livers (3-24 months) for evidence of complement-mediated inflammation revealed widespread deposition of complement-activation fragments throughout the sinusoids, elevated transaminase levels, increased hepatic CD8+ and F4/80+ cells, overexpression of hepatic mRNA associated with inflammatory signaling pathways, steatosis, and increased collagen deposition. Immunostaining of human HCC biopsies revealed extensive deposition of complement fragments within the tumors. Investigating the Cancer Genome Atlas also revealed that increased CFH mRNA expression is associated with improved survival in patients with HCC, whereas mutations are associated with worse survival. These results indicate that CFH is critical for controlling complement activation in the liver, and in its absence, AP activation leads to chronic inflammation and promotes hepatic carcinogenesis.

3:18 PM Abstract No. 201 Percutaneous biopsy of hepatocellular carcinoma in the setting of endovascular locoregional therapy

Percutaneous biopsy for the characterization of intermediate and advanced stage hepatocellular carcinoma (HCC) is projected to increase in the era of precision medicine. Given its controversial role in management, the purpose of this study was to profile the safety and efficacy of acquiring high-volume HCC core biopsies. Between April 2016 and September 2019, 30 patients with embolotherapy-naïve advanced HCC, were enrolled in this IRB-approved prospective trial. Multiple co-axial 18G core-biopsies of a targetable HCC were obtained during transarterial chemoembolization (TACE) or radioembolization (TARE). Cores were sent to pathology, the Center for Personalized Diagnostics (CPD) for solid-tumor next generation sequencing, or prepared for custom sequencing using an HCC panel developed in our lab. From 30 patients, 33 LR4 (1) or LR5 (32) HCCs were biopsied. A median of 7 core biopsies were obtained during the initial treatment. Nine tumors were biopsied a second time (post-TACE), and 2 were biopsied a third time (recurrence). Of 44 biopsy encounters, 1 (2.3%) minor (SIR class A or B) and 0 major biopsy-attributable adverse events were reported during the postprocedural period. Three (9.1%) biopsies revealed discordant pathology of cholangiocarcinoma resulting in a change in management. Four (12%) biopsies revealed poorly differentiated carcinoma, ultimately favored to represent HCC over cholangiocarcinoma. Five (15%) biopsies were negative for tumor, only 1 of which was later confirmed as sampling error. Of 12 cores sent to CPD, 9 (75%) were adequate for analysis. In custom sequenced samples, TERT, CTNNB1, and ALB were the most frequent variant alleles in a markedly heterogeneous pool. No changes in genetic mutations were identified in post-TACE samples when compared to pre-TACE biopsies. Obtaining a high-volume of co-axial percutaneous core biopsies during HCC treatment by TACE or TARE is safe, providing desperately needed tissue for the molecular characterization of intermediate and advanced stage HCC. Biopsy results may change management in imaging discordant tumors.

Immune checkpoint blockade (ICB) response evaluation with MRI/MR elastography (MRE) in surgical and nonsurgical patients with HCC.

480 Background: Currently, there is a lack of imaging biomarkers of immunotherapy outcome in hepatocellular carcinoma (HCC). The study aim was to determine if HCC enhancement on MRI and stiffness change measured by magnetic resonance elastography (MRE) can predict immunotherapy response. Methods: This was a prospective, Institutional Review Board approved study of 38 patients with HCC treated with immune checkpoint blockade (ICB) therapy. All patients had liver MRI/MRE and HCC biopsy at baseline, and MRI/MRE with biopsy or resection after 6 weeks therapy. HCC stiffness (kPa) was measured on MRE elastograms (liver stiffness maps). HCC enhancement and change in stiffness were compared with treatment response to ICB in 1) non-surgical patients (pembrolizumab), and 2) surgical patients (nivolumab +/- ipilimumab). For non-surgical patients, treatment response was defined as overall survival >1 year. For surgical patients, treatment response was defined as <50% viable tumor at time of resection. Analysis was performed using descriptive statistics and Spearman correlation; p-value <0.05 was considered statistically significant. Results: Twenty-five patients were evaluable. Median age was 67 years (32, 78). Etiology of liver disease was NASH (n=8), HCV (n=8), HBV (n=2) and unknown (n=7). Treatment response occurred in 11/25 (44%) patients. Median HCC size and change in size were 4.7 cm (1.2, 14.0) and –0.32 cm, respectively. Median baseline HCC stiffness and change in stiffness were 5 kPa (2.2, 12.4) and –0.1 kPa (–2.2, 1.5), respectively. Median change in HCC size for responders and non-responders was –1.2 cm (–4.8, 0.4) and 0 cm (–1.5, 1.1), respectively (p = 0.02). Treatment response was associated with absence of portal venous phase capsular enhancement and increase in HCC stiffness, (p<0.001). Conclusions: Capsular enhancement and MRE stiffness change may be useful biomarkers of immune cell activated response to ICB therapy.

Biopsy for hepatocellular carcinoma: only occasionally.

We would like to highlight that the non-invasive criteria for HCC diagnosis, defined by the presence of hyperenhancement in the arterial phase (wash-in) followed by hypoenhancement (wash-out) in venous phases on MRI and/or CT, have been validated by multiple cohort studies and meta-analysis. These criteria are only applicable when the pre-test probability is high (lesions > 1 cm in patients diagnosed of liver cirrhosis or chronic hepatitis B). In addition, for an adequate use of these criteria, other processes such as active (or past) neoplasms must be ruled out. In this scenario, non-invasive criteria have repeatedly demonstrated a diagnostic specificity and positive predictive value close to 100 %. Moreover, we would like to remark the transversality in the consensus of the non-invasive diagnosis of HCC, since not only the hepatology societies recommend its use, but also the oncology and radiology ones.

Methylation of the ataxia telangiectasia mutated gene (ATM) promoter as a radiotherapy outcome biomarker in patients with hepatocellular carcinoma.

The goal of this study was to evaluate the contribution of ataxia telangiectasia mutated (ATM) gene promoter methylation to hepatocellular carcinoma (HCC) and the predictive value of radiotherapy outcome. ATM promoter methylation status was detected using methylation-specific PCR in 118 HCC, 50 adjacent liver, and 20 normal liver samples. PCR products were verified by bisulfite sequencing PCR. ATM expression was detected by quantitative PCR (qPCR) and immunohistochemistry (IHC) in 50 paired HCC and adjacent normal tissues and 68 locally advanced HCC biopsy tissues. Furthermore, radiotherapy outcomes in 68 locally advanced HCC patients were determined using European Association for the Study of Liver criteria and survival analysis. The results revealed that the methylation frequency of the ATM promoter was significantly higher in HCC tissues than in normal liver tissues (χ2 = 16.830, P < .001). Quantitative PCR (qPCR) and IHC results showed a significant association between ATM promoter methylation and ATM expression in HCC (χ2 = 10.510, P < .001), and methylated ATM was correlated with lower ATM expression compared with unmethylated ATM (r = 0.356, P < .001). Furthermore, methylation of the ATM promoter was significantly associated with superior outcomes in patients with locally advanced HCC who initially received radiotherapy. Together, these results indicate that ATM promoter methylation might increase the risk of HCC by regulating ATM expression, and thus may function as a potential biomarker for predicting radiotherapy outcomes in HCC patients.

Functional and Phenotypic Characterization of Tumor-Infiltrating Leukocyte Subsets and Their Contribution to the Pathogenesis of Hepatocellular Carcinoma and Cholangiocarcinoma

Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) represent the most common primary liver malignancies whose outcome is influenced by the immune response. In the present study, we evaluated the tumor-infiltrating leukocyte (TIL) populations in 21 HCC patients and 8 CCA patients by flow cytometry immediately after the surgical procedure. Moreover, CD4+ T cells, CD8+ T cells, monocytes, and macrophages were purified by cell sorting for further analysis of gene expression by quantitative reverse-transcription polymerase chain reaction. Regarding tumor-infiltrating macrophages, we observed a significantly higher expression of markers associated with M2 phenotype and a higher expression of PD-L1 in patients with HCC in comparison to CCA. In addition, for HCC patients, we found a significant increase in the expression of CD200R in macrophages from tumors that were in grade G3-G4 as compared to tumors in grade G1-G2. Besides, a significantly higher frequency of tumor-infiltrating lymphocytes, CD8+CD56+ T cells, and natural killer cells was detected in HCC biopsies in comparison to CCA. In summary, this study has revealed functional and phenotypic differences in TIL cell subpopulations between CCA and HCC, as well as among different histopathological grades and tumor aggressiveness degrees, and it has provided evidence to better understand the tumor immune microenvironment of CCA and HCC.

NOTCH Signaling via WNT Regulates the Proliferation of Alternative, CCR2-Independent Tumor-Associated Macrophages in Hepatocellular Carcinoma

Tumor-associated macrophages (TAM) play pivotal roles in tumor progression and metastasis, but the contribution and regulation of different macrophage populations remain unclear. Here we show that Notch signaling plays distinct roles in regulating different TAM subsets in hepatocellular carcinoma (HCC). Myeloid-specific NOTCH blockade by conditional disruption of recombination signal binding protein Jκ (RBPj cKO) significantly delayed the growth of subcutaneously inoculated Lewis lung carcinoma (LLC), but accelerated orthotopically inoculated hepatic Hepa1-6 tumors in mice. In contrast to subcutaneous LLC, RBPj cKO significantly increased the number of TAMs in hepatic Hepa1-6 tumors despite impeded differentiation of monocyte-derived TAMs (moTAM). The dominating TAMs in orthotopic HCC manifested properties of Kupffer cells (KC) and hence are tentatively named KC-like TAMs (kclTAM). The increased proliferation of RBPj cKO kclTAMs was maintained even in Ccr2 -/- mice, in which moTAMs were genetically blocked. NOTCH signaling blockade accelerated proliferation of kclTAMs via enhanced β-catenin-dependent WNT signaling, which also downregulated IL12 and upregulated IL10 expression by kclTAMs likely through c-MYC. In addition, myeloid-specific RBPj cKO facilitated hepatic metastasis of colorectal cancer but suppressed lung metastasis in mice, suggesting that the phenotype of RBPj cKO in promoting tumor growth was liver-specific. In patient-derived HCC biopsies, NOTCH signaling negatively correlated with WNT activation in CD68+ macrophages, which positively correlated with advanced HCC stages. Therefore, NOTCH blockade impedes the differentiation of moTAMs, but upregulates Wnt/β-catenin signaling to promote the proliferation and protumor cytokine production of kclTAMs, facilitating HCC progression and hepatic metastasis of colorectal cancer. SIGNIFICANCE: These findings highlight the role of NOTCH and WNT signaling in regulating TAMs in hepatocellular carcinoma.

Establishment of hepatocellular carcinoma patient-derived xenografts from image-guided percutaneous biopsies

While patient-derived xenograft (PDX) models of hepatocellular carcinoma (HCC) have been successfully generated from resected tissues, no reliable methods have been reported for the generation of PDXs from patients who are not candidates for resection and represent the vast majority of patients with HCC. Here we compare two methods for the creation of PDXs from HCC biopsies and find that implantation of whole biopsy samples without the addition of basement membrane matrix favors the formation of PDX tumors that resemble Epstein-Barr virus (EBV)-driven B-cell lymphomas rather than HCC tumors. In contrast, implantation with Matrigel supports growth of HCC cells and leads to a high rate of HCC tumor formation from these biopsies. We validate the resulting PDXs, confirm their fidelity to the patients’ disease and conclude that minimally invasive percutaneous liver biopsies can be used with relatively high efficiency to generate PDXs of HCC.

Immunologic Features of Patients With Advanced Hepatocellular Carcinoma Before and During Sorafenib or Anti-programmed Death-1/Programmed Death-L1 Treatment.

INTRODUCTION:Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Today, a promising treatment strategy is focused on the enhancement of antitumor immune responses by immune checkpoint modification. However, as only 20% of patients with HCC are responders, identification of predictive factors is urgently required. Therefore, for the first time, the features of the intrahepatic and circulating immune system in patients with advanced-stage HCC, before and during the treatment, were analyzed.METHODS:We collected fresh HCC biopsies, along with adjacent tumor-free liver tissues and peripheral blood samples, from 21 patients with advanced HCC. Furthermore, we performed an extensive immunomonitoring of patients with HCC treated with sorafenib or programmed death (PD)-1/PD-L1 pathway blockade using multiparametric flow cytometry.RESULTS:We observed that regardless of the treatment, low baseline intratumoral CD4+/CD8+ T-cell ratio was associated with better overall survival (P = 0.0002). The baseline frequency of intratumoral PD-1high CD8+ T cells was significantly lower in patients responding to sorafenib treatment than in the nonresponders (P = 0.0117), and the frequency of circulating PD-1high T cells increased with tumor progression (P = 0.0329). By contrast, responders to PD-1/PD-L1 pathway blockade showed a trend of high baseline frequency of intratumoral PD-1high CD8+ T cells. Moreover, we observed a trend of LAG3 and TIM3 upregulation on circulating T cells in nonresponding patients to PD-1/PD-L1 pathway blockade.DISCUSSION:Immunosuppressive state, characterized by an enhanced intratumoral CD4+/CD8+ T-cell ratio, was associated with poor prognosis. Additionally, our results suggest that the frequency of intratumoral PD-1high CD8+ T cells may serve as a biomarker to identify which individuals will benefit from which treatment and support the use of combination strategies.

Hepatocellular Carcinoma Xenografts Established From Needle Biopsies Preserve the Characteristics of the Originating Tumors.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer‐related deaths worldwide. Treatment options for patients with advanced‐stage disease are limited. A major obstacle in drug development is the lack of an in vivo model that accurately reflects the broad spectrum of human HCC. Patient‐derived xenograft (PDX) tumor mouse models could overcome the limitations of cancer cell lines. PDX tumors maintain the genetic and histologic heterogeneity of the originating tumors and are used for preclinical drug development in various cancers. Controversy exists about their genetic and molecular stability through serial passaging in mice. We aimed to establish PDX models from human HCC biopsies and to characterize their histologic and molecular stability during serial passaging. A total of 54 human HCC needle biopsies that were derived from patients with various underlying liver diseases and tumor stages were transplanted subcutaneously into immunodeficient, nonobese, diabetic/severe combined immunodeficiency gamma‐c mice; 11 successfully engrafted. All successfully transplanted HCCs were Edmondson grade III or IV. HCC PDX tumors retained the histopathologic, transcriptomic, and genomic characteristics of the original HCC biopsies over 6 generations of retransplantation. These characteristics included Edmondson grade, expression of tumor markers, tumor gene signature, tumor‐associated mutations, and copy number alterations. Conclusion: PDX mouse models can be established from undifferentiated HCCs, with an overall success rate of approximately 20%. The transplanted tumors represent the entire spectrum of the molecular landscape of HCCs and preserve the characteristics of the originating tumors through serial passaging. HCC PDX models are a promising tool for preclinical personalized drug development.