Borderline Hepatocellular Adenomas: A Practical Diagnostic Approach Based on Pathologic and Molecular Features.

Borderline hepatocellular adenomas (BL-HCA) are characterized by focal architectural/cytologic atypia and reticulin loss, features that are insufficient for a definitive diagnosis of hepatocellular carcinoma (HCC). The diagnosis and management of BL-HCA are challenging as their biological behavior, especially in terms of malignant potential, is still debated. We aimed to compare the clinicopathologic and molecular features of BL-HCA with those of typical HCA (T-HCA), HCA with malignant transformation (HCC on HCA), and HCC to assess the risk of malignancy. One hundred six liver resection specimens were retrospectively selected from 2 reference centers, including 39 BL-HCA, 42 T-HCA, 12 HCC on HCA, and 13 HCC specimens. Somatic mutations, including TERT promoter mutations associated with HCA malignant transformation and the gene expression levels of 96 genes, were investigated in 93 frozen samples. Additionally, TERT promoter mutations were investigated in 44 formalin-fixed, paraffin-embedded samples. The clinical features of patients with BL-HCA were similar to those of patients with T-HCA, patients being mainly women (69%) with a median age of 37 years. The median tumor size was 7.5 cm, 64% of patients had a single nodule, and no recurrence was observed. Compared with T-HCA, BL-HCA was significantly enriched in ß-catenin-mutated HCA in exon 3 (41% vs 6%; P < .001). Unsupervised statistical analysis based on gene expression showed that BL-HCA overlapped with T-HCA and HCC on HCA, favoring a molecular continuum of the tumors. TERT promoter mutations were observed only in HCC on HCA (42%) and in HCC (38%). In conclusion, these results suggest that despite their worrisome morphologic features, the clinicopathologic and molecular features of BL-HCA are much closer to those of T-HCA than those of HCC on HCA or HCC. This strongly supports the usefulness of combining morphologic and molecular analyses in a practical diagnostic approach for guiding the management of BL-HCA.

Common genetic variation in alcohol-related hepatocellular carcinoma: a case-control genome-wide association study.

BACKGROUND: Hepatocellular carcinoma is a frequent consequence of alcohol-related liver disease, with variable incidence among heavy drinkers. We did a genome-wide association study (GWAS) to identify common genetic variants for alcohol-related hepatocellular carcinoma. METHODS: We conducted a two-stage case-control GWAS in a discovery cohort of 2107 unrelated European patients with alcohol-related liver disease aged 20-92 years recruited between Oct 22, 1993, and March 12, 2017. Cases were patients with alcohol-related hepatocellular carcinoma diagnosed by imaging or histology. Controls were patients with alcohol-related liver disease without hepatocellular carcinoma. We used an additive logistic regression model adjusted for the first ten principal components to assess genetic variants associated with alcohol-related hepatocellular carcinoma. We did another analysis with adjustment for age, sex, and liver fibrosis. New candidate associations (p<1 × 10-6) and variants previously associated with alcohol-related hepatocellular carcinoma were evaluated in a validation cohort of 1933 patients with alcohol-related liver disease aged 29-92 years recruited between July 21, 1995, and May 2, 2019. We did a meta-analysis of the two case-control cohorts. FINDINGS: The discovery cohort included 775 cases and 1332 controls. Of 7 962 325 variants assessed, we identified WNT3A-WNT9A (rs708113; p=1·11 × 10-8) and found support for previously reported regions associated with alcohol-related hepatocellular carcinoma risk at TM6SF2 (rs58542926; p=6·02 × 10-10), PNPLA3 (rs738409; p=9·29 × 10-7), and HSD17B13 (rs72613567; p=2·49 × 10-4). The validation cohort included 874 cases and 1059 controls and three variants were replicated: WNT3A-WNT9A (rs708113; p=1·17 × 10-3), TM6SF2 (rs58542926; p=4·06 × 10-5), and PNPLA3 (rs738409; p=1·17 × 10-4). All three variants reached GWAS significance in the meta-analysis: WNT3A-WNT9A (odds ratio 0·73, 95% CI 0·66-0·81; p=3·93 × 10-10), TM6SF2 (1·77, 1·52-2·07; p=3·84×10-13), PNPLA3 (1·34, 1·22-1·47; p=7·30 × 10-10). Adjustment for clinical covariates yielded similar results. We observed an additive effect of at-risk alleles on alcohol-related hepatocellular carcinoma. WNT3A-WNT9A rs708113 was not associated with liver fibrosis. INTERPRETATION: WNT3A-WNT9A is a susceptibility locus for alcohol-related hepatocellular carcinoma, suggesting an early role of the Wnt-ß-catenin pathway in alcohol-related hepatocellular carcinoma carcinogenesis. FUNDING: Ligue Nationale contre le Cancer, Bpifrance, INSERM, AFEF, CARPEM, Labex OncoImmunology, and Agence Nationale de la Recherche.

Molecular Subtypes and Gene Expression Signatures as Prognostic Features in Fully Resected Clear Cell Renal Cell Carcinoma: A Tailored Approach to Adjuvant Trials.

BACKGROUND: Trials with adjuvant vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGFR-TKIs) failed to demonstrate meaningful benefit in clinically high-risk, fully resected clear cell renal cell carcinoma (ccRCC). We evaluated whether the ccrcc1-4 molecular subtypes and gene expression signatures (GES) are associated with outcomes in this setting. MATERIALS AND METHODS: We determined molecular subtypes as well as angiogenesis- and immune-related GES through RNA sequencing of 75 fresh frozen (FF) and 62 formalin-fixed, paraffin-embedded (FFPE) tumor samples. We studied disease-free (DFS) and overall survival (OS) and determined correlations among GES and Leibovich score. RESULTS: Angiogenesis-related GES and molecular subtypes were associated with longer DFS and OS across both cohorts, whereas immune-related GES were not. In the FF cohort, molecular subtypes (ccrcc2 & 3 vs. ccrcc1 & 4) were associated with DFS and OS, on bivariable analysis with Leibovich score (HR 0.62, 95%CI 0.39-0.98, P = .04 and HR 0.35, 95%CI 0.19-0.64, P < .001). In the FFPE cohort, molecular subtypes (ccrcc2 & 3 vs. ccrcc1&4) were also associated with DFS (HR 0.53, 95%CI 0.31-0.93, P = .03), but not OS (HR 0.59, 95%CI 0.31-1.13, P = .11) on bivariable analysis with Leibovich score. Leibovich score was significantly inversely correlated with all angiogenesis-related GES (all P < .01), but not correlated with immune-related GES. CONCLUSIONS: Molecular subtypes and angiogenesis-related GES are prognostic for DFS and OS in fully resected, localized ccRCC. Favorable ccrcc2 & 3 molecular subtypes with high angiogenesis-related GES, which respond best to VEGFR-TKIs, are at lower risk of relapse but were probably underrepresented in the adjuvant VEGFR-TKI trials since they inversely correlate with Leibovich score. Conversely, immune-related GES are not correlated with Leibovich score and clinically high-risk tumors can display both high and low immune-related GES. Therefore, molecular characterization could guide patient selection for adjuvant treatment.

Molecular underpinnings of glandular tropism in metastatic clear cell renal cell carcinoma: therapeutic implications.

BACKGROUND: Glandular metastases (GM) have been associated with improved survival in metastatic clear cell renal cell carcinoma (m-ccRCC). We aimed to molecularly characterize m-ccRCC with GM. MATERIAL AND METHODS: We performed a retrospective cohort study on all m-ccRCC patients with available tissue at our institution, diagnosed with metastatic disease from 2000 to 2019. We determined previously described angiogenesis- and immune-related gene expression signatures (GES) and ccrcc molecular subtypes through whole transcriptome RNA sequencing of primary tumors and metastases. We tested differences in GES and molecular subtypes across groups and studied overall (OS) and progression-free survival (PFS) using Kaplan-Meier survival analysis and Cox regression models. RESULTS: Primary tumors of patients who developed GM (n = 55) had higher IMmotion Angio (p < 0.001) and JAVELIN Angio (p = 0.003) GES as well as a higher proportion of angiogenic ccrcc2 molecular subtypes (p = 0.008) than primary tumors of patients with non-GM (n = 128). Metastatic lesions in glandular organs (n = 32) also had higher IMmotion Angio (p = 0.008) and JAVELIN Angio (p = 0.02) GES and were more frequently of the ccrcc2 molecular subtype (p = 0.03), compared to metastatic lesions in non-glandular organs in patients who did not develop any GM (n = 231), but not compared to metastatic lesions in non-glandular organs in patients who also developed GM (n = 18). Patients with GM had better OS (HR 0.49, p < 0.001) and PFS on first-line vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGFR-TKIs) (HR 0.64, p = 0.045) than patients with non-GM. PFS on first- or any-line immuno-oncology (IO) was not different. IMmotion Angio, JAVELIN Angio GES, and ccrcc2 molecular subtype were associated with better OS and PFS on first-line VEGFR-TKIs, but not PFS on first or any-line IO. CONCLUSIONS: Patients with m-ccRCC who develop GM are molecularly characterized by heightened angiogenesis, translating into better prognosis and better outcomes on VEGFR-TKIs, but not IO. Based on these findings, VEGFR-TKIs should be included in the first-line treatment of m-ccRCC patients with GM.

MicroRNAs Targeting HIF-2α, VEGFR1 and/or VEGFR2 as Potential Predictive Biomarkers for VEGFR Tyrosine Kinase and HIF-2α Inhibitors in Metastatic Clear-Cell Renal Cell Carcinoma.

Metastatic clear-cell renal cell carcinoma (m-ccRCC) is characterized by increased hypoxia-induced factor (HIF)-2α and vascular endothelial growth factor receptor (VEGFR)-dependent angiogenesis through loss of function of the von Hippel-Lindau protein. VEGFR tyrosine kinase inhibitors (VEGFR-TKIs) are a cornerstone of m-ccRCC treatment, and new treatments targeting HIF-2α are currently under investigation. However, predictive biomarkers for these treatments are lacking. In this retrospective cohort study including 109 patients treated with VEGFR-targeted therapies as first-line treatment, we aimed to study the possible predictive function of microRNAs (miRNAs) targeting HIF-2α, VEGFR1 and VEGFR2. We selected miRNAs inversely correlated with HIF-2α, VEGFR1 and/or VEGFR2 expression and with predicted target sites in the respective genes and subsequently studied their impact on therapeutic outcomes. We identified four miRNAs (miR-34c-5p, miR-221-3p, miR-222-3p and miR-3529-3p) inversely correlated with VEGFR1 and/or VEGFR2 expression and associated with tumor shrinkage and progression-free survival (PFS) upon treatment with VEGFR-TKIs, highlighting the potential predictive value of these miRNAs. Moreover, we identified three miRNAs (miR-185-5p, miR-223-3p and miR-3529-3p) inversely correlated with HIF-2α expression and associated with tumor shrinkage and PFS upon treatment with VEGFR-TKIs. These three miRNAs can have a predictive value not only upon treatment with VEGFR-TKIs but possibly also upon treatment with the upcoming HIF-2α inhibitor belzutifan.

MicroRNAs Possibly Involved in the Development of Bone Metastasis in Clear-Cell Renal Cell Carcinoma.

Bone metastasis in clear-cell renal cell carcinoma (ccRCC) leads to substantial morbidity through skeletal related adverse events and implicates worse clinical outcomes. MicroRNAs (miRNA) are small non-protein coding RNA molecules with important regulatory functions in cancer development and metastasis. In this retrospective analysis we present dysregulated miRNA in ccRCC, which are associated with bone metastasis. In particular, miR-23a-3p, miR-27a-3p, miR-20a-5p, and miR-335-3p specifically correlated with the earlier appearance of bone metastasis, compared to metastasis in other organs. In contrast, miR-30b-3p and miR-139-3p were correlated with less occurrence of bone metastasis. These miRNAs are potential biomarkers and attractive targets for miRNA inhibitors or mimics, which could lead to novel therapeutic possibilities for bone targeted treatment in metastatic ccRCC.

DNA Methylation Signatures Reveal the Diversity of Processes Remodeling Hepatocellular Carcinoma Methylomes.

BACKGROUND AND AIMS: DNA methylation patterns are highly rearranged in HCCs. However, diverse sources of variation are intermingled in cancer methylomes, precluding the precise characterization of underlying molecular mechanisms. We developed a computational framework (methylation signature analysis with independent component analysis [MethICA]) leveraging independent component analysis to disentangle the diverse processes contributing to DNA methylation changes in tumors. APPROACH AND RESULTS: Applied to a collection of 738 HCCs, MethICA unraveled 13 stable methylation components preferentially active in specific chromatin states, sequence contexts, and replication timings. These included signatures of general processes associated with sex and age but also signatures related to specific driver events and molecular subgroups. Catenin beta 1 mutations were major modulators of methylation patterns in HCC, characterized by a targeted hypomethylation of transcription factor 7-bound enhancers in the vicinity of Wnt target genes as well as a widespread hypomethylation of late-replicated partially methylated domains. By contrast, demethylation of early replicated highly methylated domains was a signature of replication stress, leading to an extensive hypomethylator phenotype in cyclin-activated HCC. Inactivating mutations of the chromatin remodeler AT-rich interactive domain-containing protein 1A were associated with epigenetic silencing of differentiation-promoting transcriptional networks, also detectable in cirrhotic liver. Finally, a hypermethylation signature targeting polycomb-repressed chromatin domains was identified in the G1 molecular subgroup with progenitor features. CONCLUSIONS: This study elucidates the diversity of processes remodeling HCC methylomes and reveals the epigenetic and transcriptional impact of driver alterations.

MicroRNA expression profiles in molecular subtypes of clear-cell renal cell carcinoma are associated with clinical outcome and repression of specific mRNA targets.

Clear-cell renal cell carcinomas (ccRCC) can be divided into four transcriptomic subtypes, two of which have a favorable and two an unfavorable prognosis. To assess mechanisms driving these subtypes, we investigated their miRNA expression patterns. miRNAs are master regulators of mRNAs, that are widely deregulated in cancer. Unsupervised clustering in our dataset (n = 128) and The Cancer Genome Atlas (TCGA) validation set identified two distinct miRNA clusters that overlapped with the transcriptomic subtypes, underscoring the validity of these subtypes on a multi-omics level and suggesting a driving role for miRNAs. Discriminatory miRNAs for the favorable subtypes repressed epithelial-to-mesenchymal transition, based on gene set enrichment analysis and target-mRNA expression levels. Strikingly, throughout the entire dataset, miRNAs associated with favorable subtypes were also associated with longer overall survival after diagnosis, and miRNAs associated with unfavorable subtypes with shorter overall survival (Pearson r = -0.54, p<0.0001). These findings indicate a general shift in miRNA expression between more and less aggressive tumors. This adds to current literature, which usually suggests only a small subset of miRNAs as markers of aggressive disease. In conclusion, this study reveals distinct mRNA expression patterns underlying transcriptomic ccRCC-subtypes, whereby miRNAs associated with favorable subtypes counteract epithelial-to-mesenchymal transition. There is a general shift in miRNA expression in ccRCC, between more and less aggressive tumors.

Recurrent chromosomal rearrangements of ROS1, FRK and IL6 activating JAK/STAT pathway in inflammatory hepatocellular adenomas.

BACKGROUND: Inflammatory hepatocellular adenomas (IHCAs) are benign liver tumours characterised by an activation of the janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway caused by oncogenic activating mutations. However, a subset of IHCA lacks of identified mutation explaining the inflammatory phenotype. METHODS: 657 hepatocellular adenomas developed in 504 patients were analysed for gene expression of 17 genes and for mutations in seven genes by sequencing. 22 non-mutated IHCAs were analysed by whole-exome and/or RNA sequencing. RESULTS: We identified 296 IHCA (45%), 81% of them were mutated in either IL6ST (61%), FRK (8%), STAT3 (5%), GNAS (3%) or JAK1 (2%). Among non-mutated IHCA, RNA sequencing identified recurrent chromosome rearrangement involving ROS1, FRK or IL6 genes. ROS1 fusions were identified in 8 IHCA, involving C-terminal part of genes highly expressed in the liver (PLG, RBP4, APOB) fused with exon 33-35 to 43 of ROS1 including the tyrosine kinase domain. In two cases a truncated ROS1 transcript from exon 36 to 43 was identified. ROS1 rearrangements were validated by fluorescence in situ hybridisation (FISH) and led to ROS1 overexpression. Among the 5 IHCA with FRK rearrangements, 5 different partners were identified (MIA3, MIA2, LMO7, PLEKHA5, SEC16B) fused to a common region in FRK that included exon 3-8. No overexpression of FRK transcript was detected but the predicted chimeric proteins lacked the auto-inhibitory SH2-SH3 domains. In two IHCA, we identified truncated 3'UTR of IL6 associated with overexpression of the transcript. CONCLUSION: Recurrent chromosomal alterations involving ROS1, FRK or IL6 genes lead to activation of the JAK/STAT pathway in IHCAs.

Polyploidy spectrum: a new marker in HCC classification.

OBJECTIVES: Polyploidy is a fascinating characteristic of liver parenchyma. Hepatocyte polyploidy depends on the DNA content of each nucleus (nuclear ploidy) and the number of nuclei per cell (cellular ploidy). Which role can be assigned to polyploidy during human hepatocellular carcinoma (HCC) development is still an open question. Here, we investigated whether a specific ploidy spectrum is associated with clinical and molecular features of HCC. DESIGN: Ploidy spectra were determined on surgically resected tissues from patients with HCC as well as healthy control tissues. To define ploidy profiles, a quantitative and qualitative in situ imaging approach was used on paraffin tissue liver sections. RESULTS: We first demonstrated that polyploid hepatocytes are the major components of human liver parenchyma, polyploidy being mainly cellular (binuclear hepatocytes). Across liver lobules, polyploid hepatocytes do not exhibit a specific zonation pattern. During liver tumorigenesis, cellular ploidy is drastically reduced; binuclear polyploid hepatocytes are barely present in HCC tumours. Remarkably, nuclear ploidy is specifically amplified in HCC tumours. In fact, nuclear ploidy is amplified in HCCs harbouring a low degree of differentiation and TP53 mutations. Finally, our results demonstrated that highly polyploid tumours are associated with a poor prognosis. CONCLUSIONS: Our results underline the importance of quantification of cellular and nuclear ploidy spectra during HCC tumorigenesis.

Clinical Impact of Genomic Diversity From Early to Advanced Hepatocellular Carcinoma.

To date, genomic analyses of hepatocellular carcinoma (HCC) have been limited to early stages obtained from liver resection. We aim to describe the genomic profiling of HCC from early to advanced stages. We analyzed 801 HCC from 720 patients (410 resections, 137 transplantations, 122 percutaneous ablations, and 52 noncurative) for 190 gene expressions and for 31 gene mutations. Forty-one advanced HCC and 156 whole exome of Barcelona Clinic Liver Cancer (BCLC) 0/A were analyzed by whole-exome sequencing. Genomic profiling was correlated with tumor stages, clinical features, and survival. Our cohort included patients classified in BCLC stage 0 (9.4%), A (59.5%), B (16.2%), and C (14.9%). Among the overall 801 HCC, the most frequently mutated genes were telomerase reverse transcriptase (TERT) (58.1%), catenin beta 1 (CTNNB1) (30.7%), tumor protein 53 (TP53; 18.7%), AT-rich interaction domain 1A (ARID1A) (13%), albumin (11.4%), apolipoprotein B (APOB) (9.4%), and AXIN1 (9.2%). Advanced-stage HCC (BCLC B/C) showed higher frequencies of splicing factor 3b subunit 1 (SF3B1) (P = 0.0003), TP53 (P = 0.0006), and RB Transcriptional Corepressor 1 mutations (P = 0.03). G1-G6 transcriptomic classification and the molecular prognostic 5-gene score showed different distributions according to the stage of the disease and the type of treatment with an enrichment of G3 (P < 0.0001), poor prognostic score (P < 0.0001), and increased proliferation and dedifferentiation at the transcriptomic level in advanced HCC. The 5-gene score predicted survival in patients treated by resection (P < 0.0001) and ablation (P = 0.01) and in advanced HCC (P = 0.04). Twenty-two percent of advanced HCC harbored potentially druggable genetic alterations, and MET amplification was associated with complete tumor response in patients with advanced HCC treated by a specific MET inhibitor. Conclusion: Genomic analysis across the different stages of HCC revealed the mechanisms of tumor progression and helped to identify biomarkers of response to targeted therapies.

PTEN expression and mutations in TSC1, TSC2 and MTOR are associated with response to rapalogs in patients with renal cell carcinoma.

The mammalian target of rapamycin (mTOR) pathway inhibitors are key drugs for the treatment of many tumor types, however, there are no predictive biomarkers in clinical use. Here, we performed a molecular and immunohistochemical characterization of key mTOR pathway components in a series of 105 renal cell carcinoma patients treated with rapalogs, aimed at identifying markers of treatment response. Mutational analysis in MTOR, TSC1 and TSC2 was performed through targeted next-generation sequencing (NGS), and immunohistochemistry (IHC) was performed for PTEN, pAKT, pS6K1, pS6 and p21. Among patients with NGS data, 11 of 87 (13%) had mTOR pathway mutations (8 in MTOR, 1 in TSC1 and 2 in TSC2). When comparing the molecular data to the response of the patients, we found that partial response was more frequent in cases with mTOR pathway mutations than in those without mutations (odds ratio [OR] = 0.08, 95% confidence interval [CI] = 0.008-0.79, p = 0.030 univariate; p = 0.038 multivariable). Regarding IHC, negative PTEN staining was detected in 58% of the tumors, and it was more frequent in rapalog responder patients (OR = 0.24, 95% CI = 0.065-0.86, p = 0.029 univariate; p = 0.029 multivariable). Mutations and PTEN IHC were not mutually exclusive events and its combination improved response prediction (OR = 0.16, 95% CI = 0.04-0.62, p = 0.008 univariate; p = 0.013 multivariable). The staining of other proteins did not show and association with response and no association with PFS was observed in unselected patients. In conclusion, our findings suggest that mTOR pathway mutations, negative PTEN IHC and their combination are potential markers of rapalog response.

BAP1 mutations define a homogeneous subgroup of hepatocellular carcinoma with fibrolamellar-like features and activated PKA.

BACKGROUND & AIMS: DNAJB1-PRKACA fusion is a specific driver event in fibrolamellar carcinoma (FLC), a rare subtype of hepatocellular carcinoma (HCC) that occurs in adolescents and young adults. In older patients, molecular determinants of HCC with mixed histological features of HCC and FLC (mixed-FLC/HCC) remain to be discovered. METHODS: A series of 151 liver tumors including 126 HCC, 15 FLC, and 10 mixed-FLC/HCC were analyzed by RNAseq and whole-genome- or whole-exome sequencing. Western blots were performed to validate genomic discoveries. Results were validated using the TCGA database. RESULTS: Most of the mixed-FLC/HCC RNAseq clustered in a robust subgroup of 17 tumors, which all had mutations or translocations inactivating BAP1, the gene encoding BRCA1-associated protein-1. Like FLC, BAP1-HCC were significantly enriched in females, patients with a lack of chronic liver disease, and fibrotic tumors compared to non-BAP1 HCC. However, patients were older and had a poorer prognosis than those with FLC. BAP1 tumors were immune hot, showed progenitor features and did not show DNAJB1-PRKACA fusion, while almost none of these tumors had mutations in CTNNB1, TP53 and TERT promoter. In contrast, 80% of the BAP1 tumors showed a chromosome gain of PRKACA at 19p13, combined with a loss of PRKAR2A (coding for the inhibitory regulatory subunit of PKA) at 3p21, leading to a high PRKACA/PRKAR2A ratio at the mRNA and protein levels. CONCLUSION: We have characterized a subgroup of BAP1-driven HCC with fibrolamellar-like features and a dysregulation of the PKA pathway, which could be at the root of the clinical and histological similarities between BAP1 tumors and DNAJB1-PRKACA FLCs. LAY SUMMARY: Herein, we have defined a homogeneous subgroup of hepatocellular carcinomas in which the BAP1 gene is inactivated. This leads to the development of cancers with features similar to those of fibrolamellar carcinoma. These tumors more frequently develop in females without chronic liver disease or cirrhosis. The presence of PKA activation and T cell infiltrates suggest that these tumors could be treated with PKA inhibitors or immunomodulators.

Clear-cell Renal Cell Carcinoma: Molecular Characterization of IMDC Risk Groups and Sarcomatoid Tumors.

INTRODUCTION: Recent trials have suggested predictive biomarkers in advanced clear-cell renal cell carcinoma (accRCC): International Metastatic RCC Database Consortium (IMDC) good risk or angiogenic gene signature for sunitinib and IMDC intermediate/poor risk for ipilimumab-nivolumab and T-effector cell signature or sarcomatoid dedifferentiation for atezolizumab-bevacizumab. We hypothesized that earlier described molecular subtypes, ccrcc1 to ccrcc4, could provide similar information as a single generic biomarker and molecularly characterize the heterogeneous intermediate-risk group. PATIENTS AND METHODS: Patients with accRCC treated with systemic therapies were included. We assessed associations between the 5 biomarkers and their impact on progression-free survival (PFS) and response rate (RR) on first-line sunitinib or pazopanib. The cutoff percentage of sarcomatoid dedifferentiation with optimal discriminative value was determined. RESULTS: In total, 430 patients were included (163 with molecular data). The molecular ccrcc2 subtype identified tumors with higher angiogenic gene expression across IMDC risk groups: prevalence was high in IMDC good risk and low in IMDC poor risk (P < .001). Molecular subtype, IMDC, and angiogenic gene expression had comparable C-indices to predict PFS and RR (range, 60%-66%). The ccrcc2 subtype and angiogenic gene expression were positive predictors of PFS in IMDC intermediate-risk patients (P = .006; P = .04). Immune signature did not differ between IMDC groups, but was strongly correlated with molecular subtype (P = .8 and P = .0007). A cutoff value of 25% sarcomatoid differentiation discriminated tumors with distinct molecular characteristics and therapeutic sensitivity. CONCLUSION: In accRCC, molecular subtypes can explain differences in IMDC risk group, expression of angiogenesis and immune response genes, and sarcomatoid dedifferentiation. They can identify molecularly different patient populations within the heterogeneous IMDC intermediate group and select patients for systemic therapies.

Analysis of Liver Cancer Cell Lines Identifies Agents With Likely Efficacy Against Hepatocellular Carcinoma and Markers of Response.

BACKGROUND AND AIMS: Hepatocellular carcinomas (HCCs) are heterogeneous aggressive tumors with low rates of response to treatment at advanced stages. We screened a large panel of liver cancer cell lines (LCCLs) to identify agents that might be effective against HCC and markers of therapeutic response. METHODS: We performed whole-exome RNA and microRNA sequencing and quantification of 126 proteins in 34 LCCLs. We screened 31 anticancer agents for their ability to decrease cell viability. We compared genetic, RNA, and protein profiles of LCCLs with those of primary HCC samples and searched for markers of response. RESULTS: The protein, RNA and mutational signatures of the LCCLs were similar to those of the proliferation class of HCC, which is the most aggressive tumor type. Cell lines with alterations in genes encoding members of the Ras-MAPK signaling pathway and that required fibroblast growth factor (FGF)19 signaling via FGF receptor 4 for survival were more sensitive to trametinib than to FGF receptor 4 inhibitors. Amplification of FGF19 resulted in increased activity of FGF19 only in tumor cells that kept a gene expression pattern of hepatocyte differentiation. We identified single agents and combinations of agents that reduced viability of cells with features of the progenitor subclass of HCC. LCCLs with inactivating mutations in TSC1 and TSC2 were sensitive to the mammalian target of rapamycin inhibitor rapamycin, and cells with inactivating mutations in TP53 were sensitive to the Aurora kinase A inhibitor alisertib. Amplification of MET was associated with hypersensitivity to cabozantinib and the combination of sorafenib and inhibitors of MAP kinase 1 and MAP kinase2 had a synergistic antiproliferative effect. CONCLUSION: LCCLs can be screened for drugs and agents that might be effective for treatment of HCC. We identified genetic alterations and gene expression patterns associated with response to these agents. This information might be used to select patients for clinical trials.

Dynamics and predicted drug response of a gene network linking dedifferentiation with beta-catenin dysfunction in hepatocellular carcinoma.

BACKGROUND & AIMS: Alterations of individual genes variably affect the development of hepatocellular carcinoma (HCC). Thus, we aimed to characterize the function of tumor-promoting genes in the context of gene regulatory networks (GRNs). METHODS: Using data from The Cancer Genome Atlas, from the LIRI-JP (Liver Cancer - RIKEN, JP project), and from our transcriptomic, transfection and mouse transgenic experiments, we identify a GRN which functionally links LIN28B-dependent dedifferentiation with dysfunction of ß-catenin (CTNNB1). We further generated and validated a quantitative mathematical model of the GRN using human cell lines and in vivo expression data. RESULTS: We found that LIN28B and CTNNB1 form a GRN with SMARCA4, Let-7b (MIRLET7B), SOX9, TP53 and MYC. GRN functionality is detected in HCC and gastrointestinal cancers, but not in other cancer types. GRN status negatively correlates with HCC prognosis, and positively correlates with hyperproliferation, dedifferentiation and HGF/MET pathway activation, suggesting that it contributes to a transcriptomic profile typical of the proliferative class of HCC. The mathematical model predicts how the expression of GRN components changes when the expression of another GRN member varies or is inhibited by a pharmacological drug. The dynamics of GRN component expression reveal distinct cell states that can switch reversibly in normal conditions, and irreversibly in HCC. The mathematical model is available via a web-based tool which can evaluate the GRN status of HCC samples and predict the impact of therapeutic agents on the GRN. CONCLUSIONS: We conclude that identification and modelling of the GRN provide insights into the prognosis of HCC and the mechanisms by which tumor-promoting genes impact on HCC development. LAY SUMMARY: Hepatocellular carcinoma (HCC) is a heterogeneous disease driven by the concomitant deregulation of several genes functionally organized as networks. Here, we identified a gene regulatory network involved in a subset of HCCs. This subset is characterized by increased proliferation and poor prognosis. We developed a mathematical model which uncovers the dynamics of the network and allows us to predict the impact of a therapeutic agent, not only on its specific target but on all the genes belonging to the network.

Lect2 Controls Inflammatory Monocytes to Constrain the Growth and Progression of Hepatocellular Carcinoma.

Leukocyte cell-derived chemotaxin-2 (LECT2) was originally identified as a hepatocyte-secreted chemokine-like factor and a positive target of ß-catenin signaling. Here, we dissected out the mechanisms by which LECT2 modulates hepatocellular carcinoma (HCC) development using both HCC mouse models and human HCC samples. We have demonstrated that LECT2 exhibits dual abilities as it has profound repercussions on the tumor phenotype itself and the immune microenvironment. Its absence confers Ctnnb-1-mutated tumor hepatocytes a stronger ability to undergo epithelial to mesenchymal transition and fosters the accumulation of pejorative inflammatory monocytes harboring immunosuppressive properties and strong tumor-promoting potential. Consistent with our HCC mouse model, a low level of LECT2 in human HCC is strongly associated with high tumor grade and the presence of inflammatory infiltrates, emphasizing the clinical value of LECT2 in human liver tumorigenesis. Conclusion: Our findings have demonstrated that LECT2 is a key player in liver tumorigenesis because its absence reshapes the tumor microenvironment and the tumor phenotype, revealing LECT2 as a promising immunotherapeutic option for HCC.

Fibroblast Growth Factor Receptor-2 Polymorphism rs2981582 is Correlated With Progression-free Survival and Overall Survival in Patients With Metastatic Clear-cell Renal Cell Carcinoma Treated With Sunitinib.

BACKGROUND: There are no validated markers that predict response or resistance in patients with metastatic clear-cell renal cell carcinoma (mccRCC) treated with vascular endothelial growth factor receptor tyrosine kinase inhibitors such as sunitinib and pazopanib. Recently, single nucleotide polymorphism (SNP) rs2981582 in Fibroblast Growth Factor Receptor 2 (FGFR2) was found to be associated with clinical outcome in patients with mccRCC treated with pazopanib and sunitinib. We aimed to validate these findings in patients treated with sunitinib. MATERIALS AND METHODS: Germline DNA was collected in patients with mccRCC starting first-line systemic therapy with sunitinib. SNP rs2981582 in FGFR2 C>T was genotyped. Association of the genotype with response rate, tumor shrinkage, median progression-free survival (mPFS), and median overall survival (mOS) was studied. RESULTS: We collected clinical data from 154 patients with available germline DNA. Baseline prognostic markers were well-balanced between both subgroups. Patients with the TT genotype had a poorer outcome compared with patients with the CT/CC genotype. The median shrinkage of selected tumor target lesions during treatment with sunitinib was -16% versus -31% (P = .002), mPFS was 8 versus 15 months (P = .0007), and mOS was 22 versus 33 months (P = .04), respectively. On multivariate analysis, rs2981582 remained an independent predictor of PFS (hazard ratio, 2.858; 95% confidence interval, 1.659-4.923; P < .0001) and OS (hazard ratio, 1.795; 95% confidence interval, 1.003-3.212; P = .049). CONCLUSION: Polymorphism rs2981582 in FGFR2 is correlated to PFS and OS in patients with mccRCC treated with sunitinib. Prospective validation of the impact of this SNP is warranted.