Gene expression in fixed tissues and outcome in hepatocellular carcinoma

Abstract

Background: It is a challenge to identify patients who, after undergoing potentially curative treatment for hepatocellular carcinoma, are at greatest risk for recurrence. Such high-risk patients could receive novel interventional measures. An obstacle to the development of genome-based predictors of outcome in patients with hepatocellular carcinoma has been the lack of a means to carry out genomewide expression profiling of fixed, as opposed to frozen, tissue.

Methods: We aimed to demonstrate the feasibility of gene-expression profiling of more than 6000 human genes in formalin-fixed, paraffin-embedded tissues. We applied the method to tissues from 307 patients with hepatocellular carcinoma, from four series of patients, to discover and validate a gene-expression signature associated with survival.

Results: The expression-profiling method for formalin-fixed, paraffin-embedded tissue was highly effective: samples from 90% of the patients yielded data of high quality, including samples that had been archived for more than 24 years. Gene-expression profiles of tumor tissue failed to yield a significant association with survival. In contrast, profiles of the surrounding nontumoral liver tissue were highly correlated with survival in a training set of tissue samples from 82 Japanese patients, and the signature was validated in tissues from an independent group of 225 patients from the United States and Europe (P=0.04).

Conclusions: We have demonstrated the feasibility of genomewide expression profiling of formalin-fixed, paraffin-embedded tissues and have shown that a reproducible gene-expression signature correlated with survival is present in liver tissue adjacent to the tumor in patients with hepatocellular carcinoma.

Figure 1. Study Design

In the training set, tumor tissue and liver tissue adjacent to the tumor were profiled separately, and each was used to generate an outcome model. The model based on adjacent liver tissue was validated with the use of an independent validation set.

Figure 2. Survival Signatures and Survival Curves in the Training Set

Curves are shown for survival according to the association of the gene signature with survival, based on leave-one-out cross-validation testing (Panel A), and for overall survival according to the level of expression of the 186 signature genes (Panel B); of these, 113 were associated with a good prognosis and 73 with a poor prognosis. Panel C shows the expression pattern of the survival signature (comprising 186 genes). The 20 genes most closely associated with a poor prognosis are listed on the left, and the 20 most closely associated with a good prognosis on the right. Red indicates high expression; blue indicates low expression. Panel D shows representative photomicrographs of sections of liver tissue adjacent to tumor that were profiled in this study; there were no histologic correlates with survival. Staining was with hematoxylin and eosin.

Figure 3. Survival Signatures and Survival Curves in the Validation Set

Panel A shows the expression pattern of the 186-gene survival signature. Red indicates a poor prognosis; blue indicates a good prognosis. Survival curves are shown for overall survival according to the level of expression of the 186 signature genes among all 225 patients whose tissue samples constituted the validation set (Panel B) and among the 168 patients with a longer duration of follow-up (treated no later than 2004) (Panel C). Panel D shows the probability of late recurrence according to the level of expression of the late-recurrence gene signature. Data are missing for one patient in Panel D.

Figure 4. Hazard Ratios for Poor Survival and Late Recurrence in Selected Subgroups of Patients in the Validation Set

The hazard ratio was for poor survival among patients with the poor-prognosis gene signature (Panel A) or for late recurrence among patients with the late-recurrence gene signature (Panel B), as compared with those without the signature. BCLC denotes Barcelona Clinic Liver Cancer staging system, which ranks hepatocellular carcinoma in five stages, ranging from 0 (very early stage) to D (terminal stage). Data are missing for one patient in Panel B.