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- PATIENTS AND METHODS
We retrospectively investigated the prognostic value of hepatocyte nuclear factor 1 (HNF1) proteins in 159 liver transplant patients with hepatocellular carcinoma (HCC), including 36 (22.6%) exceeding the Milan criteria. The expression of alpha-fetoprotein (AFP), HNF1α, and HNF1β was examined with immunohistochemistry on duplicate tissue microarray slides containing HCC tumor explants. The times to recurrence and cancer death were analyzed with a Cox regression model and were compared according to the expression of markers of interest. We compared risk predictions with area under the receiver operator curves (AUROCs) and C statistics. AFP, HNF1α, and HNF1β were positive in 22.6%, 46.5%, and 61.0% of the tumor immunoprofiles, respectively. Although several variables were associated with the times to recurrence and cancer death in univariate Cox analyses, only AFP expression for the time to recurrence and the Milan criteria and HNF1β expression for the times to recurrence and cancer death remained significant after multivariate adjustments. The expression of HNF1β (but not HNF1α) was related to a serum AFP level ≥ 200 ng/mL, microvascular invasion, and AFP expression (P < 0.05 for all). A subgroup analysis showed that in the group meeting the Milan criteria, recurrence and cancer death rates at 10 years in the HNF1β-negative patients were approximately one-tenth of those in the HNF1β-positive patients, but the difference was not significant in the group exceeding the Milan criteria. The addition of HNF1β expression to the Milan criteria increased the C statistics and AUROCs for both recurrence and mortality (P < 0.05 for all). In conclusion, the immunohistological detection of HNF1β predicts recurrence and HCC-specific death after transplantation and provides an additive benefit in comparison with the Milan selection criteria on their own. Liver Transpl 19:336–345, 2013. © 2013 AASLD.
In patients with hepatocellular carcinoma (HCC), liver transplantation (LT) remains the only therapeutic option that offers an opportunity to cure both the tumor and any underlying cirrhosis.[1, 2] The increasing number of HCC candidates for LT has stimulated discussion about living donor LT, particularly in Asian countries, and has justified the expansion of the tumor burden limit beyond the established criteria (ie, the Milan criteria).[1, 3, 4] However, the increased risk of HCC recurrence after transplantation is a major obstacle to the use of expanded criteria, despite several reports of comparable outcomes.[2, 5] Even though the recurrence of HCC is the main cause of patient death after transplantation, there are no widely accepted parameters predictive of the biological behavior of tumors other than the tumor size and number.
Several studies have suggested that serum alpha-fetoprotein (AFP) is a significant prognostic factor for the risk of posttransplant recurrence.[6-8] In the process of hepatic development and carcinogenesis, the activity of the AFP promoter is tightly regulated by members of the hepatocyte nuclear factor 1 (HNF1) family (more so by HNF1β than HNF1α), and these factors also regulate hepatic gene transcription.[9, 10] In vitro experiments have shown that the morphological dedifferentiation of HCC cells leading to a malignant phenotype is accompanied by reduced levels of HNF1α and increased levels of HNF1β.[10-12] More recently, experiments with an HCC xenograft model found that HNF1α gene therapy led to functional and phenotypic suppression of tumorogenicity.[10, 11, 13]
The consideration of all these findings led us to speculate that the expression of members of the HNF1 family as activators of AFP production might be related to outcomes following LT in patients with HCC. To test this hypothesis, we investigated the relationship between AFP and HNF1 expression in explanted HCC specimens at the protein level, and by means of immunohistochemical analysis using tissue microarrays, we inquired whether the expression of these molecules influenced posttransplant outcomes.
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- PATIENTS AND METHODS
We have shown that after LT in patients with HCC, the expression of HNF1β in tumor tissue is strongly associated with the time-dependent risks of recurrence and HCC-specific death, and this is independent of the Milan criteria. Patients with AFP-positive tumors were also at increased risk for tumor recurrence but not for cancer death. The expression of HNF1β in explants had a more decisive effect on the clinical endpoints in the group meeting the Milan criteria versus the group exceeding the Milan criteria (although the difference may have been due to the small number of cases beyond the Milan criteria); only 1 of 50 patients who were negative for HNF1β experienced recurrence of HCC and died during follow-up. Importantly, the use of HNF1β measurements in combination with the conventional Milan criteria had added benefits in terms of the accuracy of predicting post-LT times to recurrence and cancer death, with significant increases in both C statistics and AUROCs.
LT is one of the best hopes for achieving a cure in select cases of HCC and cirrhosis. It is currently standard practice to select transplantable patients according to the tumor burden, mainly with the Milan criteria, although the details of the selection process differ between countries and even between centers.[1, 4] Although patients within the Milan criteria achieve more than 70% survival 5 years after LT, tumors recur in approximately 20% of patients during the same period.[1, 21] On the other hand, the tumor-based criteria exclude from LT some patients who could potentially benefit from it. It is most important that candidates who are likely to benefit from LT should be accurately selected because of the limited availability of donor livers and the possibility of living donor morbidity and even death in addition to transplant-related complications. Therefore, there is an urgent need for additional predictors of post-LT tumor biology as well as the identification of potential therapeutic targets to improve post-LT outcomes.
Microscopic vascular invasion has emerged as a putative predictor of HCC recurrence in posttransplant patients without gross vascular and distant tumor involvement. Because an increased tumor size and an increased number of tumors have been found to be important predictors of occult vascular invasion, candidacy for LT has usually been limited to patients with early-stage HCC, who are less likely to have microvascular invasion. Moreover, not all patients with microvascular invasion experience postresection tumor recurrence and poor survival, as we also observed in the present study. Accordingly, microvascular invasion may not always be a powerful indicator of a poor prognosis after LT for HCC. Interestingly, the expression of HNF1β was closely associated with microvascular invasion in our transplant cohort. The invasive nature of HNF1β-positive HCC may be responsible for the unfavorable post-LT prognosis.
According to a recent systematic review, patients with high levels of serum AFP before LT have poor outcomes as well as enhanced malignant behavior. In addition, a study by Fujiki et al. found a significant correlation between pre-LT AFP values and the risk of vascular invasion. In agreement with this result, the serological and immunohistological AFP status was also significantly correlated with the microscopic vessel invasion of tumors in our series (data not shown). A previous immunostaining study found that HCC patients whose tissues were positively stained for AFP had a lower rate of recurrence-free survival and displayed a trend toward a lower rate of overall survival after surgical resection. Similarly, our immunoprofiles of the liver explants revealed that AFP-positive tissue was clearly related to the time to recurrence, but the statistical significance of this relationship disappeared after multivariate correction in terms of the time to cancer death, and this corresponded to the pattern of AUROC results. We investigated the status of HNF1β, a critical modulator of AFP promoter activity in the hepatocarcinogenetic process, because this might explain the correlation between the expression of HNF1β in tumor tissue and AFP in the sera and tumor tissues of our cohort of LT recipients. Interestingly, we found that HNF1β played a pivotal role in predicting tumor recurrence and even subsequent death independently of the tissue and serum AFP status. This may indicate that upstream regulation of AFP expression by HNF1β at the transcriptional level operates specifically during the course of HCC progression following recurrence.
In the present study, HNF1α expression was not closely connected to post-LT outcomes. It has been suggested that inactivation of HNF1α may contribute to hepatic carcinogenicity.[13, 25] In contrast, a previous examination by northern blot analysis revealed that HNF1α was up-regulated in HCC samples versus nontumorous liver tissues. These differences in the expression profiling of HNF1α, which may result from multiple regulatory pathways being involved in the development of HCC, may account for its poor predictive power in clinical settings.
The main limitation of this study was that HNF1β expression was assessed only at the tissue level. This biomarker seemed to be useful for selecting candidates for LT per se and also for pre-LT treatment when it was checked before LT. Currently, the utility of pre-LT biopsy for an accurate diagnosis and for further characterization of the tumor is a matter of debate because the risk of seeding and disseminating malignant cells may outweigh the contribution to selecting patients suitable for LT. An examination of HNF1β expression at the plasma level is needed to improve its potential as a useful prognosticator of LT outcomes. Another consideration is that the majority of patients included in this study had HBV-related liver disease. Further studies are needed to assess an extension of the clinical use of HNF1β expression.
In conclusion, we have shown that the hepatic expression of HNF1β is a useful aid for predicting the recurrence of HCC after LT; it evidently influences survival after transplantation and can supplement the standard Milan criteria for the pre-LT selection and post-LT risk stratification of patients. HNF1β protein merits further preclinical study with respect to its role in hepatocarcinogenesis and as a potential novel therapeutic target in HCC. A prospective validation study, including serological measurements of HNF1β, is being planned by our research team.