HOXD8 hypermethylation as a fully sensitive and specific biomarker for biliary tract cancer detectable in tissue and bile samples

Eleonora Loi,María Rullán,Jesús Urmán ,Ana Florencia Vega-Benedetti,Loredana Moi,Pina Ziranu,Eleonora Lai,Mario Scartozzi,María Arechederra ,Claudia Sabato,Elisabetta Ferretti,Carmen Berasain,Sergio Alonso,Cesare Zavattari,Matteo Canale,Luca Faloppi,Juan Carrascosa,Marco Puzzoni,Agnese Pò ,Matı́as A Avila ,Alessandro Tommasi,Irene Amat,Andrea Casadei‐Gardini ,Patrizia Zavattari

doi:10.1038/s41416-022-01738-1

Abstract

BackgroundBiliary tract cancers (BTC) are rare but highly aggressive tumours with poor prognosis, usually detected at advanced stages. Herein, we aimed at identifying BTC-specific DNA methylation alterations.MethodsStudy design included statistical power and sample size estimation. A genome-wide methylation study of an explorative cohort (50 BTC and ten matched non-tumoral tissue samples) has been performed. BTC-specific altered CpG islands were validated in over 180 samples (174 BTCs and 13 non-tumoral controls). The final biomarkers, selected by a machine-learning approach, were validated in independent tissue (18 BTCs, 14 matched non-tumoral samples) and bile (24 BTCs, five non-tumoral samples) replication series, using droplet digital PCR.ResultsWe identified and successfully validated BTC-specific DNA methylation alterations in over 200 BTC samples. The two-biomarker panel, selected by an in-house algorithm, showed an AUC > 0.97. The best-performing biomarker (chr2:176993479-176995557), associated with HOXD8, a pivotal gene in cancer-related pathways, achieved 100% sensitivity and specificity in a new series of tissue and bile samples.ConclusionsWe identified a novel fully efficient BTC biomarker, associated with HOXD8 gene, detectable both in tissue and bile by a standardised assay ready-to-use in clinical trials also including samples from non-invasive matrices.

Highlights

Biliary tract cancers (BTC) are rare but highly aggressive tumours with poor prognosis, usually detected at advanced stages
The experimental strategy to identify highly specific and sensitive BTC methylation-based biomarkers consisted of: (1) global DNA methylation analysis of the Discovery cohort samples; (2) validation of the results in The Cancer Genome Atlas (TCGA)-CHOL cohort and GSE89803 cohort samples; (3) selection of methylation alterations not shared with other gastrointestinal tumours, those with high incidence, i.e. colon cancer (TCGA-COAD), rectal cancer (TCGAREAD) and gastric cancer (TCGA-STAD); (4) application of a machine-learning approach to select the minimum number of the most specific and sensitive alterations; (5) identification of the best-performing biomarker, tested in bile samples
No statistically significant differentially methylated CpG islands (CGIs) was detected after P value correction for multi-hypothesis testing

Summary

Introduction

Biliary tract cancers (BTC) are rare but highly aggressive tumours with poor prognosis, usually detected at advanced stages. We aimed at identifying BTC-specific DNA methylation alterations. A genome-wide methylation study of an explorative cohort (50 BTC and ten matched non-tumoral tissue samples) has been performed. RESULTS: We identified and successfully validated BTC-specific DNA methylation alterations in over 200 BTC samples. The best-performing biomarker (chr2:176993479176995557), associated with HOXD8, a pivotal gene in cancer-related pathways, achieved 100% sensitivity and specificity in a new series of tissue and bile samples. CONCLUSIONS: We identified a novel fully efficient BTC biomarker, associated with HOXD8 gene, detectable both in tissue and bile by a standardised assay ready-to-use in clinical trials including samples from non-invasive matrices. Endoscopic retrograde cholangiopancreatography (ERCP) combined with biliary brush cytology and cyto-histological analysis of tumour tissue could be performed to confirm a suspected case of BTC [5]

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