MOLECULAR & CELLULAR ONCOLOGY 2016, VOL. 3, NO. 4, e1057316 (3 pages) http://dx.doi.org/10.1080/23723556.2015.1057316
AUTHOR’S VIEW
Translating the molecular diversity of hepatocellular carcinoma into clinical practice Kornelius Schulzea,b,c,d and Jessica Zucman-Rossia,b,c,d,e Inserm, UMR-1162, Genomique fonctionnelle des Tumeurs solides, Equipe Labellisee Ligue Contre le Cancer, Institut Universitaire d’Hematologie, Paris, France; bUniversite Paris Descartes, Labex Immuno-Oncology, Sorbonne Paris Cite, Faculte de Medecine, Paris, France; cUniversite Paris 13, opital Europeen Georges Pompidou, Paris, France Bobigny, France; dUniversite Paris Diderot, Paris, France; ; eAssistance Publique-H^opitaux de Paris, H^ a
ABSTRACT
ARTICLE HISTORY
Deciphering genomic diversity could improve clinical care for patients with hepatocellular carcinoma. Recently, our study group identified 161 putative driver genes and 2 new mutational signatures, and demonstrated that 28% of patients harbor targetable alterations. This could be the first promising step in the development of genome-based clinical trials.
Received 26 May 2015 Revised 26 May 2015 Accepted 27 May 2015 KEYWORDS
Aflatoxin B1; ARID1A (AT rich interactive domain 1A); AXIN1 (axin 1); cancer driver genes; CTNNB1 (catenin ?); disease stage; exome sequencing; genomics; genome-based clinical trials; hepatocellular carcinoma (HCC); mutational signatures; overall survival; risk factors; TERT (telomerase reverse transcriptase); TP53 (tumor protein p53)
Introduction In spite of the increasing incidence of hepatocellular carcinoma (HCC) worldwide (782,000 new cases in 2012) treatment options for patients are limited, particularly for those with progressed disease, and HCC is consequently the second leading cause of cancer-related death.1,2 Clinically, HCC is highly heterogeneous with respect to various risk factors, the level of underlying liver disease, and different stages of tumor progression. This clinical diversity is linked to genomic diversity. Thus, translating this molecular diversity into identification of potential biomarkers for improvement of clinical care is pivotal to the prediction of HCC occurrence, diagnosis, prognosis, and optimized personalized treatment. Our research group recently reported the identification of new mutational signatures and genetic alterations that are potentially targetable by Food and Drug Administration (FDA)-certified drugs.3 Identification of candidate driver genes and associated oncogenic pathways For this study we performed exome sequencing of 236 HCC samples and corresponding non-tumor liver tissue and identified 18,479 non-silent mutations and 1,724 copy-number chromosome alterations in 11,287 genes. To identify candidate driver genes we performed a bioinformatics pipeline analysis, integrating focal amplifications, homozygous deletions, and mutations with gene expression, and CONTACT Jessica Zucman-Rossi © 2016 Taylor & Francis Group, LLC
[email protected]
identified 161 putative cancer drivers operating in HCC including TERT (telomerase reverse transcriptase), CTNNB1 (catenin b 1), TP53 (tumor protein p53), AXIN1 (axin 1), ALB (albumin), ARID1A (AT rich interactive domain 1A), ARID2 (AT rich interactive domain 2), ACVR2A (activin receptor, type IIA), NFE2L2 (nuclear factor, erythroid 2-like 2), RPS6KA3 (ribosomal protein S6 kinase, polypeptide 3), KEAP1 (kelch-like ECH-associated protein 1), RPL22 (ribosomal protein L22), CDKN2A (cyclin-dependent kinase inhibitor 2A), CDKN1A (cyclin-dependent kinase inhibitor 1A), and RB1 (retinoblastoma 1) at the top of the list. Using the Gene Ontology database, we manually curated the precise role of each gene and its cellular function, resulting in 11 pathways that were altered in more than 5% of our HCC series. Activation of telomerase expression was the most frequent event, with 60% of cases showing TERT promoter mutations. The following additional events were found, listed from the most to the least frequent rates of alteration: Wnt (wingless-type MMTV integration site family)/b-catenin signaling (54%), PI3K (phosphatidylinositol4,5-bisphosphate 3-kinase)/AKT (V-Akt murine thymoma viral oncogene homolog)/mTOR (mechanistic target of rapamycin) signaling (51%), TP53/cell cycle (49%), MAP (mitogen-activated protein) kinase signaling (43%), hepatic differentiation (34%), epigenetic regulation (32%), chromatin remodeling (28%), oxidative stress (12%), IL6
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K. SCHULZE AND J. ZUCMAN-ROSSI
(interleukin 6)/JAK (Janus kinase)/STAT (signal transducer and activator of transcription) pathway (9%), and transforming growth factor, b (TGFb) signaling (5%). By analyzing the most frequent associations of gene alterations, we identified 3 major clusters of HCC centered on alterations of CTNNB1 (linked to TERT, APOB [apolipoprotein B], KMT2D [lysine K-specific methyltransferase 2D], NFE2L2, and ARID2), AXIN1 (linked to ARID1A and RPS6KA3), and TP53 (linked to KEAP1, TSC2 [tuberous sclerosis 2], and CCND1 [cyclin D1]). Interestingly, alterations of genes that were part of the same pathway were frequently distributed among different clusters. These results could be considered to represent cooperation, functional redundancy, or lethality of gene combinations. Gene signatures and risk factors We were also interested in gene signatures associated with different risk factors. Mutations in TERT, CTNNB1, ARID1A, SMARCA2 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2), HGF (hepatocyte growth factor), and homozygous deletions in CDKN2A were significantly enriched in alcohol-related cases. TP53 mutations were associated with hepatitis B virus infection, and IL6ST alterations and significantly fewer TERT promoter mutations were exclusively found in HCC cases without known etiologies. Next, we investigated nucleotide signatures that have recently been introduced in a pan-cancer study of signatures of mutational processes in human cancer by Alexandrov et al.4 Overall, we found 8 nucleotide signatures that occur in at least one HCC in our series. Of these, 6 signatures have been described previously in liver cancer: signatures 1B and 1A (associated with age); signature 4 (associated with tobacco); signature 5, signature 6 (associated with DNA mismatch repair); and signature 16 (exclusively found in liver cancer). We identified 2 new mutational signatures in our series: signature 23, found in a hypermutated HCC (>6,000 mutations) of a female patient in a non-cirrhotic liver but with black pigment disposition resulting from an unknown mutagenic cause, and signature 24, found in 5 patients originating from Africa and associated with aflatoxin B1-linked R249S mutations in TP53. The remaining risk factor nucleotide signatures need to be deciphered in future studies. Gene signatures and tumor progression In this exome sequencing study we selected HCC of progressive disease stages, including precancerous dysplastic macronodules, early, small, and progressed, classic, and poor prognosis HCC. By taking into account this sequence of HCC progression, we identified TERT promoter mutations as the earliest alterations occurring during malignant transformation. TERT alterations had already appeared in precancerous macronodules, whereas alterations in other cancer driver genes, such as CTNNB1, TP53, ARID1A, and FGF3/4/19 (fibroblast growth factor 3/4/19), occurred only in more advanced HCC.3,5,6 Additionally, inactivating homozygous deletions of CDKN2A and focal amplifications of the FGF locus were associated with poorer overall survival.
Targeted treatment Systemic treatment options are limited in HCC, especially for patients with progressed disease who constitute the major patient population. To date, sorafenib is the only agent shown to improve overall survival. Promising clinical trials testing targeted drugs have failed, probably mainly because of nonselective patient enrollment. Within our dataset we identified several druggable gene alterations. Unfortunately, most targetable alterations are found in the AKT/mTOR pathway/MAP kinase signaling pathway and only occur at a low frequency. On the other hand, approved drugs do not target frequently altered genes in HCC, such as TERT, CTNNB1, TP53, ARID1A, or AXIN1. Nevertheless, thinking toward a genome-based clinical trial design, FDA-approved drugs could target 28% of HCC patients. Moreover, drugs that are currently being studied in phase I to phase III clinical trials could target up to 86% of HCC patients.
Conclusion In conclusion, in our recently reported study we defined 161 putative drivers belonging to 11 cellular pathways operative in HCC. Additionally, we identified 2 new mutational signatures in HCC—signature 23, associated with a new mutagenic mechanism, and signature 24, associated with aflatoxin B1—and demonstrated that 28% of HCCs harbor at least one potentially targetable alteration. Although the functionality of alterations needs to be further defined in future experimental studies, this could be the first promising step in the development of genome-based clinical trials.
Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.
Funding K.S. is supported by the Deutsche Forschungsgemeinschaft (DFG Grant Number: SCHU 2893/2-1).
References 1. Mortality GBD, Causes of Death C. Global, regional, and national agesex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet 2015; 385:117-71; PMID:25530442; http://dx. doi.org/10.1016/S0140-6736(14)61682-2 2. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 2015; 136:E359-86; PMID:25220842; http://dx.doi.org/10.1002/ ijc.29210 3. Schulze K, Imbeaud S, Letouze E, Alexandrov LB, Calderaro J, Rebouissou S, Couchy G, Meiller C, Shinde J, Soysouvanh F, et al. Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets. Nat Genet 2015; 47:505-11; PMID:25822088; http://dx.doi.org/10.1038/ng.3252 4. Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SA, Behjati S, Biankin AV, Bignell GR, Bolli N, Borg A, Børresen-Dale AL, et al. Signatures of mutational processes in human cancer. Nature 2013; 500:41521; PMID:23945592; http://dx.doi.org/10.1038/nature12477
MOLECULAR & CELLULAR ONCOLOGY
5. Nault JC, Mallet M, Pilati C, Calderaro J, Bioulac-Sage P, Laurent C, Laurent A, Cherqui D, Balabaud C, Zucman-Rossi J. High frequency of telomerase reverse-transcriptase promoter somatic mutations in hepatocellular carcinoma and preneoplastic lesions. Nat Commun 2013; 4:2218; PMID:23887712; http://dx.doi.org/10.1038/ncomms3218
e1057316-3
6. Nault JC, Calderaro J, Di Tommaso L, Balabaud C, Zafrani ES, Bioulac-Sage P, Roncalli M, Zucman-Rossi J. Telomerase reverse transcriptase promoter mutation is an early somatic genetic alteration in the transformation of premalignant nodules in hepatocellular carcinoma on cirrhosis. Hepatology 2014; 60:1983-92; PMID:25123086; http://dx.doi.org/10.1002/hep.27372
AUTHOR’S VIEW
Translating the molecular diversity of hepatocellular carcinoma into clinical practice Kornelius Schulzea,b,c,d and Jessica Zucman-Rossia,b,c,d,e Inserm, UMR-1162, Genomique fonctionnelle des Tumeurs solides, Equipe Labellisee Ligue Contre le Cancer, Institut Universitaire d’Hematologie, Paris, France; bUniversite Paris Descartes, Labex Immuno-Oncology, Sorbonne Paris Cite, Faculte de Medecine, Paris, France; cUniversite Paris 13, opital Europeen Georges Pompidou, Paris, France Bobigny, France; dUniversite Paris Diderot, Paris, France; ; eAssistance Publique-H^opitaux de Paris, H^ a
ABSTRACT
ARTICLE HISTORY
Deciphering genomic diversity could improve clinical care for patients with hepatocellular carcinoma. Recently, our study group identified 161 putative driver genes and 2 new mutational signatures, and demonstrated that 28% of patients harbor targetable alterations. This could be the first promising step in the development of genome-based clinical trials.
Received 26 May 2015 Revised 26 May 2015 Accepted 27 May 2015 KEYWORDS
Aflatoxin B1; ARID1A (AT rich interactive domain 1A); AXIN1 (axin 1); cancer driver genes; CTNNB1 (catenin ?); disease stage; exome sequencing; genomics; genome-based clinical trials; hepatocellular carcinoma (HCC); mutational signatures; overall survival; risk factors; TERT (telomerase reverse transcriptase); TP53 (tumor protein p53)
Introduction In spite of the increasing incidence of hepatocellular carcinoma (HCC) worldwide (782,000 new cases in 2012) treatment options for patients are limited, particularly for those with progressed disease, and HCC is consequently the second leading cause of cancer-related death.1,2 Clinically, HCC is highly heterogeneous with respect to various risk factors, the level of underlying liver disease, and different stages of tumor progression. This clinical diversity is linked to genomic diversity. Thus, translating this molecular diversity into identification of potential biomarkers for improvement of clinical care is pivotal to the prediction of HCC occurrence, diagnosis, prognosis, and optimized personalized treatment. Our research group recently reported the identification of new mutational signatures and genetic alterations that are potentially targetable by Food and Drug Administration (FDA)-certified drugs.3 Identification of candidate driver genes and associated oncogenic pathways For this study we performed exome sequencing of 236 HCC samples and corresponding non-tumor liver tissue and identified 18,479 non-silent mutations and 1,724 copy-number chromosome alterations in 11,287 genes. To identify candidate driver genes we performed a bioinformatics pipeline analysis, integrating focal amplifications, homozygous deletions, and mutations with gene expression, and CONTACT Jessica Zucman-Rossi © 2016 Taylor & Francis Group, LLC
[email protected]
identified 161 putative cancer drivers operating in HCC including TERT (telomerase reverse transcriptase), CTNNB1 (catenin b 1), TP53 (tumor protein p53), AXIN1 (axin 1), ALB (albumin), ARID1A (AT rich interactive domain 1A), ARID2 (AT rich interactive domain 2), ACVR2A (activin receptor, type IIA), NFE2L2 (nuclear factor, erythroid 2-like 2), RPS6KA3 (ribosomal protein S6 kinase, polypeptide 3), KEAP1 (kelch-like ECH-associated protein 1), RPL22 (ribosomal protein L22), CDKN2A (cyclin-dependent kinase inhibitor 2A), CDKN1A (cyclin-dependent kinase inhibitor 1A), and RB1 (retinoblastoma 1) at the top of the list. Using the Gene Ontology database, we manually curated the precise role of each gene and its cellular function, resulting in 11 pathways that were altered in more than 5% of our HCC series. Activation of telomerase expression was the most frequent event, with 60% of cases showing TERT promoter mutations. The following additional events were found, listed from the most to the least frequent rates of alteration: Wnt (wingless-type MMTV integration site family)/b-catenin signaling (54%), PI3K (phosphatidylinositol4,5-bisphosphate 3-kinase)/AKT (V-Akt murine thymoma viral oncogene homolog)/mTOR (mechanistic target of rapamycin) signaling (51%), TP53/cell cycle (49%), MAP (mitogen-activated protein) kinase signaling (43%), hepatic differentiation (34%), epigenetic regulation (32%), chromatin remodeling (28%), oxidative stress (12%), IL6
e1057316-2
K. SCHULZE AND J. ZUCMAN-ROSSI
(interleukin 6)/JAK (Janus kinase)/STAT (signal transducer and activator of transcription) pathway (9%), and transforming growth factor, b (TGFb) signaling (5%). By analyzing the most frequent associations of gene alterations, we identified 3 major clusters of HCC centered on alterations of CTNNB1 (linked to TERT, APOB [apolipoprotein B], KMT2D [lysine K-specific methyltransferase 2D], NFE2L2, and ARID2), AXIN1 (linked to ARID1A and RPS6KA3), and TP53 (linked to KEAP1, TSC2 [tuberous sclerosis 2], and CCND1 [cyclin D1]). Interestingly, alterations of genes that were part of the same pathway were frequently distributed among different clusters. These results could be considered to represent cooperation, functional redundancy, or lethality of gene combinations. Gene signatures and risk factors We were also interested in gene signatures associated with different risk factors. Mutations in TERT, CTNNB1, ARID1A, SMARCA2 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2), HGF (hepatocyte growth factor), and homozygous deletions in CDKN2A were significantly enriched in alcohol-related cases. TP53 mutations were associated with hepatitis B virus infection, and IL6ST alterations and significantly fewer TERT promoter mutations were exclusively found in HCC cases without known etiologies. Next, we investigated nucleotide signatures that have recently been introduced in a pan-cancer study of signatures of mutational processes in human cancer by Alexandrov et al.4 Overall, we found 8 nucleotide signatures that occur in at least one HCC in our series. Of these, 6 signatures have been described previously in liver cancer: signatures 1B and 1A (associated with age); signature 4 (associated with tobacco); signature 5, signature 6 (associated with DNA mismatch repair); and signature 16 (exclusively found in liver cancer). We identified 2 new mutational signatures in our series: signature 23, found in a hypermutated HCC (>6,000 mutations) of a female patient in a non-cirrhotic liver but with black pigment disposition resulting from an unknown mutagenic cause, and signature 24, found in 5 patients originating from Africa and associated with aflatoxin B1-linked R249S mutations in TP53. The remaining risk factor nucleotide signatures need to be deciphered in future studies. Gene signatures and tumor progression In this exome sequencing study we selected HCC of progressive disease stages, including precancerous dysplastic macronodules, early, small, and progressed, classic, and poor prognosis HCC. By taking into account this sequence of HCC progression, we identified TERT promoter mutations as the earliest alterations occurring during malignant transformation. TERT alterations had already appeared in precancerous macronodules, whereas alterations in other cancer driver genes, such as CTNNB1, TP53, ARID1A, and FGF3/4/19 (fibroblast growth factor 3/4/19), occurred only in more advanced HCC.3,5,6 Additionally, inactivating homozygous deletions of CDKN2A and focal amplifications of the FGF locus were associated with poorer overall survival.
Targeted treatment Systemic treatment options are limited in HCC, especially for patients with progressed disease who constitute the major patient population. To date, sorafenib is the only agent shown to improve overall survival. Promising clinical trials testing targeted drugs have failed, probably mainly because of nonselective patient enrollment. Within our dataset we identified several druggable gene alterations. Unfortunately, most targetable alterations are found in the AKT/mTOR pathway/MAP kinase signaling pathway and only occur at a low frequency. On the other hand, approved drugs do not target frequently altered genes in HCC, such as TERT, CTNNB1, TP53, ARID1A, or AXIN1. Nevertheless, thinking toward a genome-based clinical trial design, FDA-approved drugs could target 28% of HCC patients. Moreover, drugs that are currently being studied in phase I to phase III clinical trials could target up to 86% of HCC patients.
Conclusion In conclusion, in our recently reported study we defined 161 putative drivers belonging to 11 cellular pathways operative in HCC. Additionally, we identified 2 new mutational signatures in HCC—signature 23, associated with a new mutagenic mechanism, and signature 24, associated with aflatoxin B1—and demonstrated that 28% of HCCs harbor at least one potentially targetable alteration. Although the functionality of alterations needs to be further defined in future experimental studies, this could be the first promising step in the development of genome-based clinical trials.
Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.
Funding K.S. is supported by the Deutsche Forschungsgemeinschaft (DFG Grant Number: SCHU 2893/2-1).
References 1. Mortality GBD, Causes of Death C. Global, regional, and national agesex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet 2015; 385:117-71; PMID:25530442; http://dx. doi.org/10.1016/S0140-6736(14)61682-2 2. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 2015; 136:E359-86; PMID:25220842; http://dx.doi.org/10.1002/ ijc.29210 3. Schulze K, Imbeaud S, Letouze E, Alexandrov LB, Calderaro J, Rebouissou S, Couchy G, Meiller C, Shinde J, Soysouvanh F, et al. Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets. Nat Genet 2015; 47:505-11; PMID:25822088; http://dx.doi.org/10.1038/ng.3252 4. Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SA, Behjati S, Biankin AV, Bignell GR, Bolli N, Borg A, Børresen-Dale AL, et al. Signatures of mutational processes in human cancer. Nature 2013; 500:41521; PMID:23945592; http://dx.doi.org/10.1038/nature12477
MOLECULAR & CELLULAR ONCOLOGY
5. Nault JC, Mallet M, Pilati C, Calderaro J, Bioulac-Sage P, Laurent C, Laurent A, Cherqui D, Balabaud C, Zucman-Rossi J. High frequency of telomerase reverse-transcriptase promoter somatic mutations in hepatocellular carcinoma and preneoplastic lesions. Nat Commun 2013; 4:2218; PMID:23887712; http://dx.doi.org/10.1038/ncomms3218
e1057316-3
6. Nault JC, Calderaro J, Di Tommaso L, Balabaud C, Zafrani ES, Bioulac-Sage P, Roncalli M, Zucman-Rossi J. Telomerase reverse transcriptase promoter mutation is an early somatic genetic alteration in the transformation of premalignant nodules in hepatocellular carcinoma on cirrhosis. Hepatology 2014; 60:1983-92; PMID:25123086; http://dx.doi.org/10.1002/hep.27372
