Baseline parameters represent a cohort, typical of western countries
Male patients were predominant [n=53 (76.8%)]. The median age was 66 years, ranging from 18 to 84 years. Of the cases, 39 (63.9%) were classified BCLC stage A. BCLC stage B [n=18 (29.5%)] was less frequent, and a few cases with stage C [n=4 (6.6%)] were included. The majority of patients (98.5%) were fully active or at least able to carry out work of a light or sedentary nature according to the Eastern Cooperative Oncology Group (ECOG 0 and 1). Chronic liver disease was common as underlying morbidity in our patients. Within our cohort, 41.9% of patients had liver cirrhosis (Child-Pugh A 88.5% and B 11.5%) and 24.2% of patients were treated without any histological signs of fibrosis. The remaining patients revealed fibrosis (33.9%). Known aetiologies for HCC were equally distributed across the cohort with alcohol abuse [22 (26.2%)], chronic hepatitis C [15 (17.9%)] and B [13 (15.5%)], non-alcoholic steatohepatitis (NASH) [11 (13.1%)], and rare causes or no known underlying risk factor [23 (27.4%)]. More than two thirds of the patients (73%) underwent resection for a single lesion; only a minority presented 4 or more HCC-nodules (11.1%). Pathological reviewing classified 76.8% of patients T1 or T2, 98.5% exhibited N0, 98.5% L0, and 91.3% R0. 62.3% of patients revealed neither microscopic nor macroscopic vascular invasion. The majority (71%) of tumors were moderately differentiated (G2), 15.9% well differentiated (G1), and 13% poorly differentiated (G3). Overall, our patients represented a typical cohort undergoing curative resection in Western countries. Clinical baseline and general histopathological tumor characteristics are summarized in Table 1.
Spatial heterogeneity of EpCAM was markedly present
We confirmed the presence of spatial heterogeneity of CSC-features, measured by intensity and proportion of EpCAM-expression within the same tumor nodule. 29 patients (42%) did not exhibit any EpCAM-expression (E-/-), whereas 7 patients (10.1%) stood out with homogenous EpCAM-expression throughout all tumor spots (E+/+). 33 patients (47.8%) contained tumor spots with and without EpCAM-expression of diverse quality, confirming the presence of spatial heterogeneity of CSC-features in nearly half of patients with HCC (E-/+) (Fig 2A). EpCAM expression of control tissues of chronic liver diseases did not correlate with aetiology or HCC recurrence (Fig 2B). Figure 2 illustrates the results of EpCAM-staining in intensity and proportion in all 314 tumor spots with regard to EpCAM-classification score [negative (E-/-), heterogeneous (E-/+), homogeneous (E+/+)] and in correlation with baseline features.
Only homogeneous EpCAM-expression correlated significantly with early recurrence
Based upon the EpCAM-score [negative (E-/-), heterogeneous (E-/+), homogeneous (E+/+)], we were interested in the outcome of the patients in order to understand whether EpCAM-expression determined a tumor subgroup characterized by poor prognosis.
First, we checked for group comparability with regard to the baseline characteristics (Table 2). We did not find any significant differences in the demographic data, performance status, risk factors, presence or absence of underlying chronic liver disease, liver function (bilirubin, albumin, INR estimation), or staging according to the BCLC classification between the EpCAM-classification groups. Likewise, there were no significant differences between the three groups with regard to the L-, V-, N-, R- or G-stage. Overall, we did not identify any confounders between the three groups for our final outcome analysis.
To test for the clinical impact of EpCAM expression in HCC, we determined the TTR and the RFS for early recurrence (i.e. within 24 months). Recurrence within the first 24 months after curative resection is considered more likely to be true recurrence instead of de novo tumors. As highlighted in Figure 3, TTR is significantly shorter at 5 months in patients with E+/+ compared to patients with E+/- at 19 months (p=0.022). The median TTR was not reached for patients scored E-/-. The hazard ratio for recurrence between E+/+ and E-/- was 3.8 (95% CI, 1.32 – 11.2, p=0.014). Results for RFS, depicted in Figure 4, were also significantly different at 5 months for E+/+ compared to 14 months for E-/+, and 21 for E-/- (p=0.016). The hazard ratio was 3.6 (95% CI, 1.39 – 9.5, p=0.009). Interestingly, in both endpoints, TTR and RFS, we found a similar outcome in E+/- and E-/- groups (Figures 3 and 4). Recurrence-free survival rates at 24 months were 57.2% and 35.6% for the E-/- and E+/- groups (p=n.s.), respectively. No patient in the E+/+ group remained without recurrence or death in the 24 months after resection. In summary, we demonstrated a significantly worse prognosis for patients with homogeneous EpCAM-expression, and also showed that heterogeneous EpCAM and negative EpCAM-expressing tumors exhibited a similar outcome, highlighting the clinical impact of thorough assessment of spatial EpCAM-expression in HCC nodules.
Homogeneous EpCAM-expression was indicative for local aggressiveness and tumor dissemination
We observed that the group of E+/+ patients separated from E+/- and E-/- patients in the context of tumor aggressiveness (serum AFP-levels (</≥400 ng/mL) and number of HCC nodules). Remarkably, E+/- and E-/- demonstrated not only similar clinical behaviour, but also similar serum AFP-levels, and number of HCC nodules. So we wondered whether these surrogate parameters also statistically discriminated the group of homogeneous EpCAM-expressing HCC from the negative and heterogeneous expressing tumors, as described for EpCAM-expressing tumors in general by Yamashita et al..[11] Therefore, we assessed AFP serum levels prior to resection, as well as the number of satellite lesions. In HCC patients, an AFP concentration higher than 400 ng/mL has been consistently associated with poor prognosis in several treatment settings and used as the cut-off for several clinical trials such as REACH and REACH-2.[20] AFP levels were significantly higher in the E+/+ group compared to the other groups. In detail, 57% of patients with E+/+ presented AFP levels >400 ng/mL compared to 13.8% in E+/- and 11.5% in E-/-) (p=0.031). Correspondingly, patients with E-/- and E+/- status harboured significantly less satellite tumor lesions at the time of resection compared to the E+/+ group (p=0.006) (Table 3.)
In summary, homogeneous EpCAM-expression was significantly indicative for higher local aggressiveness and earlier tumor dissemination compared to heterogeneous EpCAM-expression. EpCAM-negative tumor nodules demonstrated similar low aggressiveness in tumor features like EpCAM-heterogeneous tumor nodules.
The positive predictive value of a single biopsy to diagnose HCC with homogeneous EpCAM-expression was only 45%
Since only homogeneous EpCAM-expression was associated with more aggressive tumor behaviour and subsequently, a poorer outcome, we were interested in the predictive value of a single biopsy towards the overall EpCAM-score (E+/+, E+/-, or E-/-). Therefore, we assessed the positive and negative prediction values, and the sensitivity and specificity of the EpCAM expression pattern of a single spot. We discovered that the sensitivity for a single spot was 100%, the specificity was 87%, the negative predictive value was 100%, but the positive predictive value was only 45%. Moreover, the positive predictive value of a single spot for early recurrence within 24 months after resection was 58%, the negative predictive value was 58.5%, the sensitivity was 27%, and the specificity was 84%.