. 2023 Jan;164(1):72-88.e18.

doi: 10.1053/j.gastro.2022.09.005. Epub 2022 Sep 12.

Molecular Markers of Response to Anti-PD1 Therapy in Advanced Hepatocellular Carcinoma

Philipp K Haber¹, Florian Castet², Miguel Torres-Martin², Carmen Andreu-Oller³, Marc Puigvehí⁴, Maeda Miho³, Pompilia Radu⁵, Jean-Francois Dufour⁶, Chris Verslype⁷, Carolin Zimpel⁸, Jens U Marquardt⁸, Peter R Galle⁹, Arndt Vogel¹⁰, Melanie Bathon¹⁰, Tim Meyer¹¹, Ismail Labgaa¹², Antonia Digklia¹³, Lewis R Roberts¹⁴, Mohamed A Mohamed Ali¹⁴, Beatriz Mínguez¹⁵, Davide Citterio¹⁶, Vincenzo Mazzaferro¹⁶, Fabian Finkelmeier¹⁷, Jörg Trojan¹⁷, Burcin Özdirik¹⁸, Tobias Müller¹⁸, Moritz Schmelzle¹⁹, Anthony Bejjani²⁰, Max W Sung³, Myron E Schwartz³, Richard S Finn²⁰, Swan Thung³, Augusto Villanueva³, Daniela Sia³, Josep M Llovet²¹

Affiliations

¹ Mount Sinai Liver Cancer Program, Division of Liver Diseases, Tisch Cancer Institute at the Icahn School of Medicine at Mount Sinai, New York, New York, Division of Hematology and Medical Oncology, Icahn School of Medicine at Mount Sinai, New York, New York, Department of Surgery, Recanti/Miller Transplant Institute at the Icahn School of Medicine at Mount Sinai, New York, New York, Department of Pathology, Molecular and Cell-Based Medicine, Icahn School of Medicine at Mount Sinai, New York, New York; Department of Surgery, Campus Charité Mitte and Campus Virchow Klinikum, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health, Berlin, Germany.
² Translational Research in Hepatic Oncology, Liver Unit, IDIBAPS, Hospital Clinic, University of Barcelona, Catalonia, Spain.
³ Mount Sinai Liver Cancer Program, Division of Liver Diseases, Tisch Cancer Institute at the Icahn School of Medicine at Mount Sinai, New York, New York, Division of Hematology and Medical Oncology, Icahn School of Medicine at Mount Sinai, New York, New York, Department of Surgery, Recanti/Miller Transplant Institute at the Icahn School of Medicine at Mount Sinai, New York, New York, Department of Pathology, Molecular and Cell-Based Medicine, Icahn School of Medicine at Mount Sinai, New York, New York.
⁴ Mount Sinai Liver Cancer Program, Division of Liver Diseases, Tisch Cancer Institute at the Icahn School of Medicine at Mount Sinai, New York, New York, Division of Hematology and Medical Oncology, Icahn School of Medicine at Mount Sinai, New York, New York, Department of Surgery, Recanti/Miller Transplant Institute at the Icahn School of Medicine at Mount Sinai, New York, New York, Department of Pathology, Molecular and Cell-Based Medicine, Icahn School of Medicine at Mount Sinai, New York, New York; Hepatology Section, Gastroenterology Department, Parc de Salut Mar, IMIM (Hospital del Mar Medical Research Institute), Universitat Autònoma de Barcelona, Barcelona, Spain.
⁵ University Clinic for Visceral Surgery and Medicine, University of Bern, Inselspital, Bern, Switzerland.
⁶ University Clinic for Visceral Surgery and Medicine, University of Bern, Inselspital, Bern, Switzerland; Hepatology, Department of Clinical Research, University of Bern, Bern, Switzerland.
⁷ Department of Gastroenterology and Hepatology, KU Leuven, Leuven, Belgium.
⁸ Department of Medicine I, University Medical Center of the Johannes-Gutenberg University, Mainz, Germany; Department of Medicine I, University of Lübeck, UKSH - Campus Lübeck, Lübeck, Germany.
⁹ Department of Medicine I, University Medical Center of the Johannes-Gutenberg University, Mainz, Germany.
¹⁰ Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany.
¹¹ Department of Oncology, University College London Cancer Institute, London, United Kingdom.
¹² Department of Visceral Surgery, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland.
¹³ Department of Oncology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland.
¹⁴ Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine and Science, Rochester, Minnesota.
¹⁵ Liver Unit, Hospital Universitari Vall d'Hebron, Vall d'Hebron Barcelona Hospital Campus, Barcelona, Spain, Liver Diseases Research Group, Vall d'Hebron Institute of Research (VHIR), Vall d'Hebron Barcelona Hospital Campus, Barcelona, Spain, CIBERehd, Universitat Autònoma de Barcelona, Barcelona, Spain.
¹⁶ Gastrointestinal Surgery and Liver Transplantation Unit, National Cancer Institute, Department of Oncology, University of Milan, Milan, Italy.
¹⁷ Department of Gastroenterology, University Liver and Cancer Centre, Frankfurt, Germany.
¹⁸ Department of Hepatology and Gastroenterology, Campus Virchow Klinikum and Campus Charité Mitte, Charité University Medicine Berlin, Berlin, Germany.
¹⁹ Department of Surgery, Campus Charité Mitte and Campus Virchow Klinikum, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health, Berlin, Germany; Department of General, Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany.
²⁰ Division of Hematology/Oncology, Geffen School of Medicine at UCLA, Los Angeles, California.
²¹ Mount Sinai Liver Cancer Program, Division of Liver Diseases, Tisch Cancer Institute at the Icahn School of Medicine at Mount Sinai, New York, New York, Division of Hematology and Medical Oncology, Icahn School of Medicine at Mount Sinai, New York, New York, Department of Surgery, Recanti/Miller Transplant Institute at the Icahn School of Medicine at Mount Sinai, New York, New York, Department of Pathology, Molecular and Cell-Based Medicine, Icahn School of Medicine at Mount Sinai, New York, New York; Translational Research in Hepatic Oncology, Liver Unit, IDIBAPS, Hospital Clinic, University of Barcelona, Catalonia, Spain; Institució Catalana de Recerca i Estudis Avançats, Barcelona, Catalonia, Spain. Electronic address: josep.llovet@mssm.edu.

PMID: 36108710
PMCID: PMC12182972
DOI: 10.1053/j.gastro.2022.09.005

Molecular Markers of Response to Anti-PD1 Therapy in Advanced Hepatocellular Carcinoma

Philipp K Haber et al. Gastroenterology. 2023 Jan.

. 2023 Jan;164(1):72-88.e18.

doi: 10.1053/j.gastro.2022.09.005. Epub 2022 Sep 12.

Authors

Affiliations

¹ Mount Sinai Liver Cancer Program, Division of Liver Diseases, Tisch Cancer Institute at the Icahn School of Medicine at Mount Sinai, New York, New York, Division of Hematology and Medical Oncology, Icahn School of Medicine at Mount Sinai, New York, New York, Department of Surgery, Recanti/Miller Transplant Institute at the Icahn School of Medicine at Mount Sinai, New York, New York, Department of Pathology, Molecular and Cell-Based Medicine, Icahn School of Medicine at Mount Sinai, New York, New York; Department of Surgery, Campus Charité Mitte and Campus Virchow Klinikum, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health, Berlin, Germany.
² Translational Research in Hepatic Oncology, Liver Unit, IDIBAPS, Hospital Clinic, University of Barcelona, Catalonia, Spain.
³ Mount Sinai Liver Cancer Program, Division of Liver Diseases, Tisch Cancer Institute at the Icahn School of Medicine at Mount Sinai, New York, New York, Division of Hematology and Medical Oncology, Icahn School of Medicine at Mount Sinai, New York, New York, Department of Surgery, Recanti/Miller Transplant Institute at the Icahn School of Medicine at Mount Sinai, New York, New York, Department of Pathology, Molecular and Cell-Based Medicine, Icahn School of Medicine at Mount Sinai, New York, New York.
⁴ Mount Sinai Liver Cancer Program, Division of Liver Diseases, Tisch Cancer Institute at the Icahn School of Medicine at Mount Sinai, New York, New York, Division of Hematology and Medical Oncology, Icahn School of Medicine at Mount Sinai, New York, New York, Department of Surgery, Recanti/Miller Transplant Institute at the Icahn School of Medicine at Mount Sinai, New York, New York, Department of Pathology, Molecular and Cell-Based Medicine, Icahn School of Medicine at Mount Sinai, New York, New York; Hepatology Section, Gastroenterology Department, Parc de Salut Mar, IMIM (Hospital del Mar Medical Research Institute), Universitat Autònoma de Barcelona, Barcelona, Spain.
⁵ University Clinic for Visceral Surgery and Medicine, University of Bern, Inselspital, Bern, Switzerland.
⁶ University Clinic for Visceral Surgery and Medicine, University of Bern, Inselspital, Bern, Switzerland; Hepatology, Department of Clinical Research, University of Bern, Bern, Switzerland.
⁷ Department of Gastroenterology and Hepatology, KU Leuven, Leuven, Belgium.
⁸ Department of Medicine I, University Medical Center of the Johannes-Gutenberg University, Mainz, Germany; Department of Medicine I, University of Lübeck, UKSH - Campus Lübeck, Lübeck, Germany.
⁹ Department of Medicine I, University Medical Center of the Johannes-Gutenberg University, Mainz, Germany.
¹⁰ Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany.
¹¹ Department of Oncology, University College London Cancer Institute, London, United Kingdom.
¹² Department of Visceral Surgery, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland.
¹³ Department of Oncology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland.
¹⁴ Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine and Science, Rochester, Minnesota.
¹⁵ Liver Unit, Hospital Universitari Vall d'Hebron, Vall d'Hebron Barcelona Hospital Campus, Barcelona, Spain, Liver Diseases Research Group, Vall d'Hebron Institute of Research (VHIR), Vall d'Hebron Barcelona Hospital Campus, Barcelona, Spain, CIBERehd, Universitat Autònoma de Barcelona, Barcelona, Spain.
¹⁶ Gastrointestinal Surgery and Liver Transplantation Unit, National Cancer Institute, Department of Oncology, University of Milan, Milan, Italy.
¹⁷ Department of Gastroenterology, University Liver and Cancer Centre, Frankfurt, Germany.
¹⁸ Department of Hepatology and Gastroenterology, Campus Virchow Klinikum and Campus Charité Mitte, Charité University Medicine Berlin, Berlin, Germany.
¹⁹ Department of Surgery, Campus Charité Mitte and Campus Virchow Klinikum, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health, Berlin, Germany; Department of General, Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany.
²⁰ Division of Hematology/Oncology, Geffen School of Medicine at UCLA, Los Angeles, California.
²¹ Mount Sinai Liver Cancer Program, Division of Liver Diseases, Tisch Cancer Institute at the Icahn School of Medicine at Mount Sinai, New York, New York, Division of Hematology and Medical Oncology, Icahn School of Medicine at Mount Sinai, New York, New York, Department of Surgery, Recanti/Miller Transplant Institute at the Icahn School of Medicine at Mount Sinai, New York, New York, Department of Pathology, Molecular and Cell-Based Medicine, Icahn School of Medicine at Mount Sinai, New York, New York; Translational Research in Hepatic Oncology, Liver Unit, IDIBAPS, Hospital Clinic, University of Barcelona, Catalonia, Spain; Institució Catalana de Recerca i Estudis Avançats, Barcelona, Catalonia, Spain. Electronic address: josep.llovet@mssm.edu.

PMID: 36108710
PMCID: PMC12182972
DOI: 10.1053/j.gastro.2022.09.005

Abstract

Background & aims: Single-agent anti-PD1 checkpoint inhibitors convey outstanding clinical benefits in a small fraction (∼20%) of patients with advanced hepatocellular carcinoma (aHCC) but the molecular mechanisms determining response are unknown. To fill this gap, we herein analyze the molecular and immune traits of aHCC in patients treated with anti-PD1.

Methods: Overall, 111 tumor samples from patients with aHCC were obtained from 13 centers before systemic therapies. We performed molecular analysis and immune deconvolution using whole-genome expression data (n = 83), mutational analysis (n = 72), and histologic evaluation with an endpoint of objective response.

Results: Among 83 patients with transcriptomic data, 28 were treated in frontline, whereas 55 patients were treated after tyrosine kinase inhibitors (TKI) either in second or third line. Responders treated in frontline showed upregulated interferon-γ signaling and major histocompatibility complex II-related antigen presentation. We generated an 11-gene signature (IFNAP), capturing these molecular features, which predicts response and survival in patients treated with anti-PD1 in frontline. The signature was validated in a separate cohort of aHCC and >240 patients with other solid cancer types where it also predicted response and survival. Of note, the same signature was unable to predict response in archival tissue of patients treated with frontline TKIs, highlighting the need for fresh biopsies before immunotherapy.

Conclusion: Interferon signaling and major histocompatibility complex-related genes are key molecular features of HCCs responding to anti-PD1. A novel 11-gene signature predicts response in frontline aHCC, but not in patients pretreated with TKIs. These results must be confirmed in prospective studies and highlights the need for biopsies before immunotherapy to identify biomarkers of response.

Keywords: Biomarkers; Hepatocellular Carcinoma; Immunotherapy; Predictors of Response.

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Conflict of interest statement

Conflicts of Interest

These authors disclose the following: Jean-Francois Dufour has received consulting fees from AbbVie, Bayer Healthcare, Bristol-Myers Squibb, Falk, Genfit, Genkyotex, Gilead Sciences, HepaRegenix, Intercept, Eli Lilly, Merck, Novartis, Roche. Jens U. Marquardt received honoraria from Roche, Bayer, Ipsen, Merz, AstraZeneca, MSD, and Leap-Tx, Eisai. Peter R. Galle is receiving honoraria from Adaptimmune, Bayer, BMS, AstraZeneca, Sirtex, MSD, Eisai, Ipsen, Roche, Lilly, and Guerbet. Arndt Vogel has received consulting fees and honoraria from AstraZeneca, Bayer, BMS, Eisai, Incyte, Ipsen, Janssen, Lilly, Merck, MSD, Novartis, Pierre Fabre, Roche, and Sanofi. Tim Meyer reports consulting fees from Ipsen, AstraZeneca, Roche, Bayer Healthcare, Adaptimmune, Boston Scientific, and Eisai. Lewis R. Roberts has received grant funding from Bayer, BTG International, Exact Sciences, Gilead Sciences, GlycoTest, Redhill, TARGET PharmaSolutions, and FUJIFILM Medical Systems, and has consulted for AstraZeneca, Bayer, Exact Sciences, Gilead Sciences, GRAIL, QED Therapeutics, and TAVEC. Beatriz Mínguez received consultancy fees from Bayer-Shering Pharma and Eisai-Merck, lectures/speaker fees from Eisai, MSD, and Roche, and a research grant from Laboratorios Viñas S.L. Moritz Schmelzle is receiving honoraria from ERBE, Amgen, Merck, and Bayer Healthcare. Max W. Sung is receiving consulting fees from Bayer, EISAI, and Exelixis. Richard S. Finn has received consulting fees from AstraZeneca, Bayer Healthcare, Eisai, CStone, Bristol-Myers Squibb, Eli Lilly, Pfizer, Merck, Roche/Genenetech, and Exelixis. Augusto Villanueva has received consulting fees from Guidepoint, Fujifilm, Boehringer Ingelheim, FirstWord, and MHLife. Josep M. Llovet is receiving research support from Bayer HealthCare Pharmaceuticals, Eisai Inc, Bristol-Myers Squibb, Boehringer Ingelheim, and Ipsen, and consulting fees from Eli Lilly, Bayer HealthCare Pharmaceuticals, Bristol-Myers Squibb, Eisai Inc, Celsion Corporation, Exelixis, Merck, Ipsen, Genentech, Roche, Glycotest, Nucleix, Sirtex, Mina Alpha Ltd and AstraZeneca. The remaining authors disclose no conflicts.

Figures

**Figure 1.**
Cohort overview and outcomes. (A) Study flowchart: Of the 111 samples collected for this study, 83 cases, treated with anti-PD1 in either frontline or after exposure to TKIs, were eventually included in the transcriptomic analysis. (B) Alluvial plot showing response patterns based on treatment line. The numbers in the boxes represent the number of patients with that specific response (*C, D*) Kaplan-Meier (KM) estimates of all patients included in the transcriptomic analysis are shown for OS (C) and PFS (D) based on whether patients exhibited OR, SD, or PD. P values in KM curves represent log-rank tests.

**Figure 2.**
Upregulation of inflammation and antigen-presentation associated genes in responders. (*A, B*) Kaplan-Meier (KM) estimates for OS (A) and PFS for all 28 patients treated with anti-PD1 in frontline based on whether patients exhibited OR or NR. (C) Heatmap of gene expression analysis based on observed response types. Each signature or individual gene is significantly upregulated in one response subgroup relative to the others, whereas no differences were observed regarding PD-L1 by immunohistochemistry. (D) Volcano plot showing differentially expressed genes in responders compared with nonresponders. Genes differentially expressed at P < .05 are depicted in red, all others in orange. (E) Gene Ontology gene set enrichment analysis of differentially expressed genes using the *biological processes* classification. P values in KM curves represent log-rank tests.

**Figure 3.**
Association of previously reported gene signatures and HCC molecular classes with response and resistance to anti-PD1. (A) Circular classification plot integrating response types with molecular classes of HCC. Each sector represents 1 patient. Significant enrichment of the *S1/2* classes and the *Inflamed HCC* subgroup is observed in responders. (B) Boxplot comparison for the expression of previously reported gene signatures based on observed response. (C–J) Kaplan-Meier (KM) estimates for PFS (C–F) and OS (G–J) based on expression of previously reported signatures. P values in boxplot comparison represent Mann-Whitney test, and those in the KM curves represent log-rank tests.

**Figure 4.**
Generation and validation of expression signature associated with response. (A) Heatmap of genes incorporated in the IFNAP signature. (*B, C*) Kaplan-Meier (KM) estimates for PFS (B) and OS (C) are shown based on expression of IFNAP. (D) Receiver operating characteristic (ROC) curve is shown for IFNAP and previously characterized signatures of response. AUC, area under the curve. (*E, F*) Validation of IFNAP in 2 independent datasets of anti-PD1/anti-PDL1-treated patients with melanoma, non–small-cell lung cancer (NSCLC) and head and neck squamous cell cancer (*E, G*) and NSCLC (*F, H*), respectively. Patients with response showed marked enrichment in IFNAP (*E, F*) which was associated with longer PFS (*G, H*). P values for KM analysis derive from log-rank test whereas those in the barplots represent 2-sided χ² test.

**Figure 5.**
Characterization of IFNAP and correlates of resistance to anti-PD1. (A) Histologic assessment of the immune infiltrate, applying a previously characterized semi-quantitative score, in the intratumoral compartment and at the invasive margin. (B) Boxplot representation of virtual microdissection with CIBERSORTx based on IFNAP expression. (C) Correlation of IFNAP expression with previously characterized resistance markers. (D) Correlation heatmap with unsupervised clustering of factors associated with response and resistance to anti-PD1 therapy. (E) Heatmap of patients treated in frontline with anti-PD1 ordered by response and CTNNB1 mutational status. No differences in response rates were observed, whereas a trend toward increased inflammatory signaling in responders with CTNNB1 mutations compared with nonresponders with mutations was noted. P values in boxplot comparison represents Mann-Whitney test, and those in the correlation plots represent Pearson tests.

**Figure 6.**
TKI therapy compromises predictive potential of response signatures. (*A, B*) Kaplan-Meier estimates for PFS and OS in patients treated with anti-PD1 in second/third line. (C) Heatmap of patients treated with anti-PD1 in second and third line highlights inability of previously characterized markers to capture responders to anti-PD1 after TKI therapy. (*D, E*) Forest plots showing log hazard ratios from a Cox regression model for PFS defines high infiltration of Tregs (Top 20%) as a poor prognostic marker in patients treated with anti-PD1 both in frontline (D) and after exposure to TKIs (E). P values in Kaplan-Meier curves represent a log-rank test.

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Comment in

Have We Found the "Holy Grail" That May Predict Response to Immunotherapy in Hepatocellular Carcinoma?
Brandi G, Tovoli F, Tavolari S. Brandi G, et al. Gastroenterology. 2023 Jan;164(1):15-18. doi: 10.1053/j.gastro.2022.10.030. Epub 2022 Nov 9. Gastroenterology. 2023. PMID: 36334819 No abstract available.

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Molecular Markers of Response to Anti-PD1 Therapy in Advanced Hepatocellular Carcinoma

Affiliations

Molecular Markers of Response to Anti-PD1 Therapy in Advanced Hepatocellular Carcinoma

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

Comment in

References

Publication types

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Grants and funding

LinkOut - more resources

Full Text Sources

Medical

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