Complex phenotypic heterogeneity of combined hepatocellular-cholangiocarcinoma with a homogenous TERT promoter mutation

Abstract

To clarify the mechanism underlying the development and poor prognosis of combined hepatocellular-cholangiocarcinoma (cHCC-CCA), we characterized liver cancer driver mutations and poor prognostic markers in both the HCC and intrahepatic CCA (iCCA) components of a cHCC-CCA tumor. The telomerase reverse transcriptase (TERT) promoter mutation C228T was quantified by digital polymerase chain reaction using DNA from multiple microdissected cancer components of a single cHCC-CCA nodule. The protein expression of cancer-related markers, including TERT, was examined by serial thin-section immunohistochemistry and double-staining immunofluorescence. TERT promoter mutation and TERT protein expression were detected in all cancer components but not in noncancer regions. TERT promoter mutation frequencies were similar among components; those of TERT protein-positive cancer cells were higher in iCCA and mixed components than in HCC. The frequencies of Ki67- and p53-positive cells were similarly higher in iCCA and mixed components than in HCC. However, double-positive cells for the three proteins were unexpectedly rare; single-positive cells dominated, indicating phenotypic microheterogeneity in cancer cells within a component. Interestingly, HCC and CCA marker protein immunohistochemistry suggested dedifferentiation of HCC and transdifferentiation from HCC to iCCA in HCC and iCCA components, respectively. Such phenotypic intercomponent heterogeneity and intracomponent microheterogeneity were detected in a tumor nodule of cHCC-CCA uniformly carrying the early HCC driver mutation. Moreover, poor prognostic markers were randomly expressed without a regular pattern, consistent with the poor prognosis.

Introduction

Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare tumor that accounts for 2% to 5% of primary liver cancers [1]; the actual incidence is likely higher due to underestimation of cHCC-CCA without biopsy [2]. Regardless, this tumor is aggressive and has a poor prognosis; in fact, its prognosis is worse than that of HCC and similar to that of intrahepatic cholangiocarcinoma (iCCA) [1,2]. To understand the genesis of characteristic cHCC-CCA and identify significant clinical indicators, it is necessary to thoroughly investigate biologic behavior, such as the simultaneous development of two different tumors and aggressive progression. Regarding genesis, there are two theories involved in the synchronous development of the HCC and iCCA components of cHCC-CCA: multicentric origin and monoclonal origin. Current studies on sequence-based mutational profiles of individual cancer components of cHCC-CCA have shown that most cases, especially cHCC-CCA defined according to international consensus and the World Health Organization (WHO) [3,4], are of monoclonal origin [5-7]. Monoclonal origin includes two scenarios: hepatic progenitor cell origin [5] and transdifferentiation of HCC to iCCA [6,8]. Indeed, we demonstrated the latter, i.e., transdifferentiation of partial HCC to iCCA, using clinical samples obtained from a case of metachronous liver cancer development from HCC to cHCC-CCA over a three-year interval [8]. Although the mechanism of transdifferentiation remains unclear, the process itself or its environmental background could be factors leading to cancer progression with poor prognosis.

With respect to aggressive progression, three cancer-related factors were investigated in this study: telomerase reverse transcriptase (TERT) promoter mutation, TERT protein expression, and Ki67 protein and p53 protein expression in multiple components of a cHCC-CCA tumor. TERT reactivation occurs in most cancers and contributes to cancer formation and progression through telomere extension. TERT promoter mutation is key for many regulatory mechanisms involved in telomerase reactivation [9] and is closely associated with poor prognosis in terms of both disease-free survival and overall survival in HCC [10,11]. Overexpression of Ki67 and p53 is also a cancer-associated phenotype that plays a role in the development and progression of cancers, including HCC. Ki67 is a proliferation marker widely employed for human tumor diagnostics. p53 is also frequently overexpressed in human tumors, including HCC. In general, immunohistochemistry (IHC)-has shown that p53 overexpression correlates with p53 gene mutations in HCC patients (82.9% positive in mutant-p53 HCC), and p53 genetic alterations are associated with aggressive malignant behavior and poor survival in HCC [12]. Although the above three proteins are related to cancer malignancy, the positive expression rate of these malignant markers in multiple cancer components of cHCC-CCA is unclear, and it is unknown whether their expression occurs in the same cancer cell. In this study, we examined the correlation of TERT promoter mutation frequency and TERT protein expression in multiple components of a single tumor nodule of cHCC-CCA and the relationship between the expression levels of the proteins TERT, Ki67, and p53. The findings obtained are helpful for understanding the biological behaviors of these cancer cells in relation to tumor development and progression.

Materials and methods

The patient in this case was male, 73 years old, and positive for anti-hepatitis C virus antibody. He underwent partial liver resection (5×3.5×3.5 cm) as curative treatment for a liver tumor (1.5×1.5×1.2 cm) in liver segment 8 (Figure 1A). Formalin-fixed, paraffin-embedded (FFPE) liver tissue blocks were prepared from the resected liver: #1, #2a, #2b, #3 and #4 (Figure 1A). The tumor was nodular-type and milky white; it was histologically diagnosed as cHCC-CCA. This study was approved by the Ethics Committee of Nihon University School of Medicine (approval no. 237-1). Informed consent was obtained from the patient prior to the start of the study. Detailed materials and methods such as liver specimens, DNA extraction, digital polymerase chain reaction (dPCR) of the TERT promoter mutation C228T, immunohistochemistry, immunofluorescence double staining for TERT/Ki67 and TERT/p53 and statistical analysis are described in Supplementary Materials (Materials and methods).

Figure 1.

Gross and histopathologic findings of cHCC-CCA. (A) Gross cross-sections and histologic specimens of a curatively resected liver. The liver cancer cHCC-CCA was observed in sections #2, #3 and #4. Red indicates the cutting lines for the specimens. The block arrows in #1 and #4 indicate the direction of thin slice sections. The bar indicates 10 mm. Histological macrofeatures of HCC (blue) and iCCA (yellow) components, including mixed HCC-iCCA components (green), are shown in each thin slice section stained with H&E. (B) Representative IHC images of cHCC-CCA. Serial thin sections of the #2b tissue shown in (A) were subjected to H&E staining and IHC staining for HepPar-1, CK7, CK19 and CA19-9. Representative images of four regions, mixed HCC-iCCA, HCC, iCCA and nontumorous liver (N), are shown. Bars indicate 50 µm. Particular IHC images of HCC and iCCA components are shown in the lower panel. HCC negative for HepPar-1 and CK7; iCCA cells positive for HepPar-1 and CK7. Bars indicate 100 µm. (C) Representative IHC images of HCC cells positive for CA19-9 in the mixed HCC-iCCA component (blue lines). High-magnification images are shown in the left (blue solid line) and middle (blue dotted line) panels (bar, 50 µm), and CA19-9(+)/HepPar-1(+)/CK7(-) HCC cells in areas of the HCC components (red dotted line) are shown by high magnification in the right panel (bar, 100 µm). Black dotted lines indicate a border between cancer and noncancerous regions.

Results

Histological and immunohistochemical findings for cHCC-CCA

A single tumor nodule of cHCC-CCA was regionally separated histologically (Figure 1A). The HCC component was major and observed in blocks #2a, #2b, #3 and #4. The iCCA component was minor and observed in blocks #2b and #3. A component with a mixture of HCC and iCCA was also observed in blocks #2b and #3 in the middle of the tumor nodule (mixed HCC-iCCA) (Figure 1A). These components shown by hematoxylin and eosin (H&E) staining were confirmed by IHC for HCC (HepPar-1) and CCA (CK7, CK19, CA19-9) markers (Figure 1B). Interestingly, the HCC component occasionally contained HepPar-1-negative HCC cells (Figure 1B, lower), whereas HepPar-1 was specific for all hepatocytes in the noncancer region and HCC (Figure 1B, upper). This HCC component was also negative for CK7 (Figure 1B, lower). In contrast, the iCCA component occasionally contained rare iCCA cells positive for HepPar-1 (Figure 1B, lower). Thus, a variety of cancer cells were observed as minor components with dedifferentiated and transdifferentiated statuses. Second, CA19-9 was not only specific for iCCA but also positive in the iCCA-containing stroma (Figure 1B, upper). In particular, HCC adjacent to iCCA in the mixed HCC-iCCA region was strongly positive for CA19-9 (Figure 1C, for example, blue-marked areas-upper and lower- shown in the left and middle panels by high magnification, respectively), whereas most of the HCC component was negative for this marker (Figure 1B and 1C). However, the HCC component contained rare HCC cells positive for CA19-9 (Figure 1C, a red-dotted area shown in the right panel by high magnification).

Frequency of the TERT promoter mutation C228T in multiple components of cHCC-CCA

The TERT promoter mutation is one of the driver mutations of HCC, distinct from iCCA [13,14]. We found the monoclonal origin of cHCC-CCA using mutational profiles including the TERT promoter mutation C228T [8]. To examine the clonality and heterogeneity of cHCC-CCA, the mutation frequency of the TERT promoter mutation C228T was determined in multiple components of cHCC-CCA. Digital PCR for the TERT promoter mutation C228T was performed using DNA extracted from the whole tumor nodule (mixture of HCC and iCCA), three HCC components, an iCCA component, and two noncancer regions (N) in four independent experiments: Exp. 1, Exp. 2, Exp. 3 and Exp. 4 (Figure 2A). The mutation was detected in all tumor samples (Figure 2B); the mutation frequency was 19.3% to 34.9% in the HCC components, 36.2% in the iCCA component and 33.1% in the mixture (HCC, iCCA) (Figure 2A). Thus, in the present case of cHCC-CCA, all cancer components were positive for the examined TERT promoter mutation, with small variation in frequency, which was probably due to the varied stromal content within the microdissected components. The origin of HCC and iCCA might be the same, as shown in most cases of cHCC-CCA [5-8]. The liver cirrhotic lesions (the N1, N2) mostly showed no mutations in this case (Figure 2).

Figure 2.

Frequency of the TERT promoter mutation C228T determined by dPCR. (A) Macroscopic and representative histologic images of seven liver tissue samples used for DNA extraction. Sampling areas are shown by colored dotted lines in four experiments using sections #2b and #3. In Exp. 1, cHCC-CCA containing components of mixed HCC-iCCA, HCC and iCCA (mixture in red) and noncancerous lesions (N1 in white) were macroscopically dissected from #2b tissue thin sections. In Exp. 2, the HCC component was laser-capture microdissected (HCC1 in blue). In Exp. 3, components of HCC (HCC2 in blue), iCCA (iCCA in yellow) and noncancerous lesions (N2 in white) were microscopically or macroscopically dissected from #2b. In Exp. 4, the HCC component was laser-capture microdissected from #3 (HCC3 in blue). The frequency of the TERT promoter mutation C228T determined by dPCR is shown under each panel. Bars indicate 100 µm. (B) Scatter plots of dPCR results for the C228T TERT promoter mutation. The seven DNA samples shown in (A) were subjected to dPCR. Blue (FAM), mutant allele; red (VIC), wild-type allele; green (FAM-VIC), both alleles; yellow, undetermined.

Protein expression of TERT, Ki67, and p53 in multiple components of cHCC-CCA

The TERT promoter mutation C228T elevates the level of TERT mRNA by creating the ETS active element sequence for the transcription factor GABP. To examine TERT expression at the protein level, we performed IHC (Figure 3A). TERT protein expression was positive in the nuclei of cancer cells but not all cells; the positive rate varied from 10% to 20% in each cancer component (Figure 3B). Noncancer regions were mostly negative for the TERT protein (Figure 3B). Thus, not all cancer cells carrying the TERT promoter mutation were positive for the TERT protein in our IHC system.

Figure 3.

IHC of TERT, Ki67 and p53 proteins in various components of cHCC-CCA. A. Representative images of serial thin sections from #2b and #3 with H&E staining and IHC for TERT. Bars indicate 50 µm. B. Frequency of cells positive for TERT protein in mixed HCC-iCCA, HCC, iCCA, HCC (#3) and N. Only cancer components of HCC, iCCA and HCC (#3) were subjected to statistical analysis (*, P<0.05; **, P<0.01 by Mann-Whitney U test). C. Representative images of serial thin sections from #2b and #3 with H&E staining and IHC for Ki67 and p53. Bars indicate 50 µm. D. Frequency of cells positive for Ki67 and p53 in mixed HCC-iCCA, HCC, iCCA, HCC (#3) and N. Only cancer components of HCC, iCCA and HCC (#3) were subjected to statistical analysis (**, P<0.01; ***, P<0.001 by Mann-Whitney U test). E. Distribution of three types of cancer cells (Ki67+, p53+, Ki67+/p53+) in mixed HCC-iCCA, HCC and iCCA components. DP, double-positive cells (Ki67+/p53+).

To examine the relationship of TERT-positive cells with markers of a poor prognosis, the proteins Ki67 and p53, we performed IHC using serial thin sections (Figure 3C). The Ki67 and p53 proteins were positive in the nuclei of cancer cells, and the proportion of positive cells was different in different components of cancer. HCC components had lower positive rates for both markers than iCCA (P<0.01, P<0.001 Mann-Whitney U test), but the positivity rates for two markers were similar within each cancer component (Figure 3D). Moreover, the frequency of cancer cells double-positive for Ki67 and p53 (KI67+/p53+) in each cancer component was unexpectedly low, at only 9.1%-24.5% of any positive cancer cell type (Figure 3E). In addition, the distribution of three types of cancer cells (Ki67+, p53+ and Ki67+/p53+) between HCC and iCCA components significantly differed (P<0.05, chi-square test), with Ki67+/p53+ cancer cells having a significantly higher rate in iCCA than in HCC (P<0.01, Mann-Whitney U test) (Figure 3E).

Relationship of TERT-positive cells with Ki67-positive and p53-positive cells

To precisely determine the relationship of TERT-positive cells to Ki67-positive cells or p53-positive cells, immunofluorescence double staining for TERT/Ki67 and TERT/p53 was performed in the mixed HCC-iCCA component (Figure 4A). TERT+/Ki67+ double-positive cells comprised 9.7% of any positive cell type, and TERT+/p53+ double-positive cells comprised 13.3%; the frequency was similarly low and not significantly different between the two (P=0.658, chi-square test) (Figure 4B). Positive cytoplasmic staining of TERT was demonstrated by immunofluorescence staining (Figure 4A), and the cytoplasmic TERT staining rate was significantly lower among TERT+/Ki67+ double-positive cells than among TERT+ single-positive cells (P<0.05, Kruskal-Wallis test) (Figure 4C).

Figure 4.

Immunofluorescence double staining of TERT/Ki67 and TERT/p53 proteins in the mixed HCC-iCCA component. A. Representative images of immunofluorescence double staining of TERT/Ki67 and TERT/p53. Cells positive for each protein were cancer cells in the mixed HCC-iCCA component (left panels). Representative images of three proteins in the noncancer region are shown on the right. B. Distributions of three types of cancer cell (TERT+, Ki67+, and TERT+/Ki67+) (TERT+, p53+, and TERT+/p53+) in two double-staining assays. The positive rate was determined in cancer cells. DP, double-positive cells (TERT+/Ki67+ and TERT+/p53+) shown in dark gray; C, cytoplasmic TERT-positive cells; single-positive cells for TERT+ shown in light gray and for Ki67+ or p53+ in white. C. Frequency of cytoplasmic TERT-positive cells among TERT+, TERT+/Ki67+, and TERT+/p53+ cancer cells. *, P<0.05 by the Mann-Whitney U test.

Discussion

We demonstrated herein that the TERT protein is expressed in all cancer components (HCC, iCCA and mixed HCC-iCCA) existing within a tumor nodule of cHCC-CCA carrying a promoter mutation, but not in noncancer regions without mutation. The similar mutation frequency of early HCC-specific driver genes in all cancer components suggests the possibility that the examined tumor type originates from transdifferentiation of partial HCC to iCCA, indicating the monoclonal origin of the tumor. This finding is supported by other current reports [6,7], including our previous study [8] on mutation profiles of cHCC-CCA. We showed, using clinical samples, that the TERT promoter mutation C228T is actually related to TERT protein expression, especially through multiple cancer components of cHCC-CCA. However, the rate of positivity was different in different cancer components, and not all cancer cells within a component were positive for the TERT protein (Figure 3B). For example, considering the frequency of TERT promoter mutation, 33.1%, in a tumor nodule of HCC+iCCA (Figure 2A), mutant cancer cells are estimated to compose 66.2% of total cells. Because mutant cancer cells are heterozygotes, the remaining cells are stromal cells without mutation. TERT protein positivity was detected in only 16.1±6.8% of cancer cells (Figure 3B) but not in all mutant cancer cells. This heterogeneity in TERT protein expression suggests that its expression is regulated by other factors; for example, TERT protein expression is more common in iCCA than in HCC (Figure 3B), suggesting a role for an iCCA-related factor. In general, overexpression of TERT in cancers is induced by a variety of mechanisms in addition to mutation of its promoter and may involve methylation, miRNA interference, and alternative splicing [9]. A more complicated mechanism is possibly involved in the observed heterogeneous expression of the TERT protein.

Cytoplasmic TERT expression was observed in the mixed HCC-iCCA component by immunofluorescence staining but not by IHC in this study, probably due to high reactivity of the secondary fluorescence antibody or high sensitivity of fluorescence acquisition. Although 16.1±6.8% of cancer cells were positive for TERT by IHC, 28.2±6.3% were positive for TERT (including 10.5±5.0% positive for cytoplasmic TERT) by immunofluorescence staining. Cytoplasmic TERT has also been observed in clinical samples of HCC, and this is the predominant site of TERT expression. Cytoplasmic TERT expression is associated with poor tumor differentiation and oxidative stress-related DNA damage [15]. In addition to the telomere-lengthening function (canonical function) of nuclear TERT, cytoplasmic TERT has many secondary telomere-independent roles (noncanonical functions), such as interaction with signaling pathways, stress protection, and binding to and protection of mitochondrial DNA [9]. Thus, the biologic function of cytoplasmic TERT remains to be elucidated; at least in our observation, Ki67-positive, actively proliferating cancer cells exhibited significantly less cytoplasmic TERT (Figure 4C). Ki67-positive cancer cells might prefer nuclear TERT with its canonical function; there seems to be a possible signaling linkage between cell proliferation and immortalization that involves telomere lengthening by nuclear translocation of TERT.

Second, we showed that the poor prognostic markers Ki67 and p53 were highly positive in iCCA; double-positive Ki67+/p53+ cells were also high-frequency compared to HCC (Figure 3D and 3E). However, among Ki67-positive and p53-positive cancer cells of the mixed HCC-iCCA component, TERT+/Ki67+ and TERT+/p53+ cells were similar in relative frequency (Figure 4B). These results suggest that the iCCA component and iCCA-containing mixed component have a more malignant phenotype than the HCC component but that at the individual cell level, these malignant phenotypes are independently expressed. There are several IHC studies on Ki67 and p53 in HCC and iCCA cases but few on cHCC-CCA cases (Table S1). The mini-review of these data is described in comparison with ours (Supplementary Materials, Mini-review of the literature). Since there are few IHC studies on the quantitative comparison of a target protein among cancer components within a cHCC-CCA tumor, further investigation by use of multiple cHCC-CCA cases is necessary to confirm the higher positivity rate of the Ki67 and p53 proteins in the iCCA component versus the HCC component, and the correlation of these marker proteins at the individual cell level.

Third, we found unexpected expression of differentiation markers in some cancer components (Figure 1B, lower). These results also indicate heterogeneous development of cancer cells within homogenous HCC and iCCA components that are morphologically determined. For example, HCC cells within an HCC component were negative for both HepPar-1 and CK7 (Figure 1B, lower). Such double-negative HCC cells were observed in the HCC component in 4 of 9 cHCC-CCA cases (data not shown). A portion of HCC cells positive for HepPar-1 probably become dedifferentiated. The mechanism for this dedifferentiation is interesting, and it is unknown whether such dedifferentiation of HCC is a step of transdifferentiation toward iCCA. We also found iCCA cells positive for both CK7 and HepPar-1 scattered within an iCCA component (Figure 1C, lower). Such iCCA cells were observed in 3 of 6 cHCC-CCA cases (data not shown) and in other reports [16,17]. Such simultaneously double-positive cells are typically observed in the ‘intermediate cell carcinoma’ of the 2019 WHO classification of cHCC-CCA [1,2]. However, double-positive iCCA cells in this study appear to be morphologically distinct from ‘intermediate cell carcinoma’ and are probably intermediate cells in transdifferentiation from HCC to iCCA. On the other hand, we found that the CA19-9-strongly positive cancer area consisted of three IHC-pattern regions: HepPar-1(+)/CK7(+), HepPar-1(+)/CK7(-), and HepPar-1(-)/CK7(+) regions (Figure 1C). These cancer regions morphologically corresponded to the mixture of HCC and iCCA, HCC and iCCA, respectively. Thus, CA19-9-positive, CK7-negative HCC cells were observed, but rarely, in cHCC-CCA (Figure 1C, high-magnification images). CA19-9 is aberrantly produced by gastrointestinal cancer cells, including biliary tract cancer cells. For HCC, dual-phenotype HCC (classical HCC expressing cholangiocyte markers) is strongly associated with the elevation of serum CA19-9 [18]. The CA19-9-positive HCC cells in our cHCC-CCA cases also represent an intermediate state in HCC to iCCA transdifferentiation.

To date, there have been few studies on the genetic and phenotypic characterization of individual cancer components within a given cHCC-CCA tumor, but such information is helpful for understanding the genesis and poor prognosis of this complex cancer. Recently, spatial omics and multiplexed imaging technologies have been developed to decipher cell-to-cell variation within and between individual tumors [19]. Trajectory inference approaches using single-cell transcriptome sequencing enable cells to be ordered within the lineage based on pseudotime [20]. These new technologies will drive future cancer biology research and aid in the development of diagnostic and therapeutic strategies for cHCC-CCA.

Disclosure of conflict of interest

None.