Anne Fages

A NMR metabonomic approach to explore early biomarkers of Hepatocellular Carcinoma in the European Prospective Investigation into Cancer and Nutrition
Anne Fages

Primary liver cancer (PLC; ranked sixth in incidence and third in mortality worldwide) is a cancer grouping composed of hepatocellular (HCC) and intrahepatic bile duct carcinomas. HCC make up the bulk of PLC and are highly malignant, usually diagnosed at late stages and often have very poor prognosis with limited treatment options. Thus, there is a need to identify early diagnostic biomarkers of HCC risk. Also, it is plausible that metabolic imbalances, reflective of obesity, diabetes, and lifestyle habits play a central role in HCC etiology. Thus, a NMR metabonomic study was undertaken in a case-control study nested within EPIC, a large prospective cohort of over 520,000 subjects from 23 centers in 10 Western European countries. Detailed dietary/lifestyle data and biological samples (stored under liquid nitrogen) were collected at enrolment from the majority of participants.

After an average of 7.6 years of follow-up, 112 first primary incident HCC cases were identified and matched to control subjects (1:2; by age, sex, study centre). Serum samples from matched cases and controls were analyzed by high-field ¹H NMR spectroscopy with the following objectives: (i) to identify early predictive biomarkers of HCC by following a metabonomic approach and (ii) to identify metabolomic profiles representative of distinct dietary or lifestyle patterns. Although the protocols of the EPIC study were not specifically designed with metabolomic analyses in mind, extensive pilot testing has shown that EPIC serum samples are suitable for this type of analysis.

High-resolution one-dimensional ¹H NMR (NOESY and CPMG) spectral profiles were recorded at 800MHz. Additional 2D ¹H-¹H (Jres, TOCSY) and  ¹ H-¹³C (HSQC) data were recorded for metabolite assignments.

We present here the first results of the metabonomic analysis of the EPIC HCC nested casecontrol study that enabled the identification of a metabolomic profile discriminating cases from controls based on serum collected prior to diagnosis. The potential of this study to identify both pre-diagnostic biomarkers as well as those related to exposures associated with disease risk, may have relevant public health impact and enhance understanding of HCC cancer aetiology, particularly in HCC cases arising in the absence of hepatitis B or C
infections.