The phenotypic and histopathological characterization of malignancies is increasingly applied to tailor personalized medicine in regards to the management of treatment decision-making. Adjuvant treatment in patients with HCC is still not established because positive phase 3 clinical trials are lacking, probably due to an insufficient patient selection in recent studies. It is believed that selected patients could benefit from approved treatment modalities, e.g. transarterial chemoembolization (TACE), tyrosine kinase inhibitors, immune checkpoint inhibitors, in an adjuvant setting under consideration of stringent selection criteria. Similar to colorectal cancer, high or low risk features for early metastases-dissemination and local aggressiveness could be key for adjuvant treatment after curative-intended resection. To this respect, CSC-features, in particular EpCAM-expression, are therefore a highly attractive surrogate marker in HCC. However, to what extent intratumoural heterogeneity of EpCAM expression is present in HCC nodules and how it might affect clinical outcome of patients has not yet been evaluated in a large enough patient cohort. The proportion of intratumoural CSC-features as well as the intensity of expression-levels in each HCC nodule remains uncertain, and could provide an ideal marker for stringent patient selection in an adjuvant treatment trial setting.
To our knowledge, this is the largest cohort of HCC patients to have been investigated precisely in terms of presence or absence of intratumoural CSC heterogeneity. Herein, we could verify that CSC-features are substantially heterogeneously distributed within HCC nodules. Homogeneous EpCAM staining was present in only 1 out of 10 patients whereas EpCAM heterogeneity was found in every other patient. We showed, that homogeneous distribution of CSC-features was leading to earlier dissemination, recurrence and/or death after curative-intended resection with 5 months RFS for E+/+ patients compared to 14 months for E-/+. Based on our findings, we postulate that only homogeneous distribution of CSC-features lead to more aggressive tumours, alongside with a higher risk of satellite nodules (i.e. local metastases).
Moreover, homogeneous distribution of CSC features was significantly associated with higher serum AFP-levels (p = 0.048) and the number of intrahepatic satellite HCC lesions (p = 0.026) compared to heterogeneous EpCAM expression. Again: homogeneous presence of CSC features was associated with local aggressiveness and consecutively lead to shorter TTR and RFS. Of note, all E+/- and E-/- cases had similar clinical outcomes, suggesting the same biological behaviour in HCC with none or low expression pattern of CSC features.
Hepatic stem and progenitor cells can be the source of tumour initiation and CSC can drive hepatocarcinogenesis11,12. We here highlight, that only a full-scale CSC milieu within HCC nodules was of clinical significance in terms of prognosis. A subpopulation of cells with CSC features is responsible for sustainment of many different solid tumours13. However, a minor CSC cluster or single CSC appear not to be sufficient enough to influence tumour proliferation and metastases-dissemination.
Whether CSC features cause HCC aggressiveness, has to be further studied by functional experiments.
Substantial EpCAM expression is believed to be the result of a distinct underlying molecular and mutational profile as introduced by molecular classifications14. Herein, HCC can be categorized in a proliferation and non-proliferation class14. EpCAM positivity and abundant CSC features are associated with the G1/S2/iCluster 1, termed the progenitor group. This group is characterized by RPS6KA3, TP53, and AXIN1 mutations as well as IGF1R, AKT/mTOR signaling14. Thus, it will be crucial to identify the underlying mutational profile, eventually leading to HCC with homogeneous or heterogeneous CSC distribution. WNT-ß-catenin activation via transcription factor TCF-4 is also associated with transcriptional activation of EpCAM expression15. A Japanese study demonstrated in two human HCC samples inactivating TP53 mutations (c.844C > T; c.767C > T) in EpCAM positive HCC12. In HCC, activating CTNNB1 mutations and inactivating TP53 mutations are major oncogenic events with frequencies of up to 37% and 24%, respectively, and could be in part reasonable drivers in homogeneous distribution in EpCAM expression16. Due to methodological limitations of the present study, analysis of the mutational background was not within the scope of this work, but upcoming studies will need to address the molecular and genetic impact on homogeneous or heterogeneous CSC patterns. Longitudinal sampling, focusing on clonal evolution, might also help clarifying this issue. Ultimately, it will be necessary to study CSC heterogeneity within a large patient cohort at a clonal level through single cell analysis. Nevertheless, with our comprehensive data we demonstrate that clinical parameters, mainly risk factors and presence of underlying chronic liver disease, are not involved in determining HCC with a homogeneous or heterogeneous CSC expression pattern.
We believe that accurate histopathological investigation of resected HCC lesions harbour important information to stratify patient management. With respect to intensity and proportion of CSC features, a simple EpCAM score could estimate the risk of recurrence. Subsequently, the score could be used to establish a clinical trial, which investigates adjuvant treatment options. Herein, patients could be stratified by the E+/+ status versus E+/- (and/or E-/-) status, followed by an adjuvant treatment.
Several trials in HCC, predominantly focusing on combinatory treatments with checkpoint inhibition, are currently on-going. It is well known that PD-1 or PD-L1 expression within the tumour lesion and microenvironment in general are not sufficient surrogate markers for treatment response in patients receiving checkpoint inhibition in HCC14. Recently, several study groups emphasized that mesenchymal stem cells suppress inflammation and inhibit the immune response by the PD-1/PD-L1 axis17,18. To our knowledge there is only minor or no clinical evidence in large HCC cohorts to support the efficacy of immune checkpoint inhibitors in CSC-predominant subtypes of HCC. It could be elucidating to investigate the PD-1 and PD-L1 status in HCC nodules harbouring homogeneous CSC-distribution and its response to checkpoint inhibitors. Eventually, patients under mono-therapy of immune checkpoint inhibition need to be co-tested for EpCAM, PD-1, and PD-L1 expression in order to obtain the predictive value towards treatment response.
Our data set underlines again the presence and impact of intratumoural heterogeneity in HCC. We already know from other gastrointestinal cancer types that intratumoural heterogeneity can play a major role for treatment decision-making, e.g. HER2/neu expression in gastric cancer19. In their study, sampling procedures contained a significant risk to generate a false-negative Her2/neu status. 25% of patients with gastric cancer and Her2/neu overexpression have been neglected due to sampling error, leading to withholding of effective treatment with trastuzumab. Additionally, a minor false-positive rate of 6% has been reported19.
We addressed the issue of representative sampling, taking into account the EpCAM expression patterns of a single spot compared to all spots of one HCC nodule and its significance towards the EpCAM score. We discovered that every other patient with a positive EpCAM result harbours in fact heterogeneous EpCAM expression, and therefore has the same prognosis of a patient with an EpCAM negative HCC nodule. In conclusion, one single biopsy does not seem to be sufficient to predict patient outcome, regarding SCS-features and its impact on recurrence. The indication for performing biopsies in patients with late stage disease will be increasing in the future. Our results were generated from patients with early tumour stages. Thus, our conclusions cannot be transferred to patients with advanced tumour stages. However, we believe the subject of sampling error has to be addressed in patients with later stages, because cancers with CSC features are more resistant to systemic chemotherapy20. The results of the present study and its impact on clinical outcome suggest that more than one biopsy is required to minimize the risk of false negative or positive predictive histopathological staining.