Direct molecular evidence for both multicentric and monoclonal carcinogenesis followed by transdifferentiation from hepatocellular carcinoma to cholangiocarcinoma in a case of metachronous liver cancer

Abstract

Frequent recurrence is a major issue in liver cancer and histological heterogeneity frequently occurs in this cancer type. However, it has remained elusive whether such cancers are multicentric or monoclonal. To elucidate the clonal evolution of hepatocellular carcinoma (HCC) recurrence and combined hepatocellular-cholangiocarcinoma (cHCC-CCA) development, the somatic mutation frequency and signatures in a patient with triple occurrence of liver cancer every three years were examined, with samples designated as #1HCC, #2HCC and #3cHCC-CCA, respectively. A total of four tumor regions, including HCC (#3HCC) and intrahepatic CCA (#3iCCA) components of #3cHCC-CCA, and three nontumor regions (#1N, #2N and #3N) were precisely dissected from formalin-fixed paraffin-embedded tissues of each surgical specimen. DNA was extracted and subjected to tumor-specific somatic mutation determination. Of note, five nonsynonymous single-nucleotide variants (SNVs), namely those of KMT2D, TP53, DNMT3A, PKHD1 and TLR4, were identified in #3cHCC-CCA. All five SNVs were detected in both #3HCC and #3iCCA and #2HCC but not in #1HCC. The telomerase reverse transcriptase (TERT) promoter mutation C228T, but not C250T, was observed in all tumors. Digital PCR of C228T also indicated the presence of the TERT promoter mutation C228T in nontumorous liver tissues (#1N, #2N and #3N) at a frequency of 0.11-0.83% compared with normal liver and blood samples. These results suggest the following phylogenetic evolution of three metachronous liver cancers: #1HCC was not related to #2HCC, #3HCC and #3iCCA; both #3HCC and #3iCCA arose from #2HCC. From the above, three novel findings were deduced: i) Both multicentric occurrence and intrahepatic metastasis may be involved in liver cancer in a three-year interval; ii) transdifferentiation from HCC to iCCA is a possible pathogenic mechanism of cHCC-CCA; and iii) a nontumorous, noncirrhotic liver may contain a preneoplastic region with a cancer driver mutation in the TERT promoter.

Keywords: cancer evolution; combined hepatocellular-cholangiocarcinoma; hepatocellular carcinoma; intrahepatic metastasis; laser capture microdissection; metachronous cancer; multicentric carcinogenesis; next-generation sequencing; transdifferentiation.

Figure 1.

Gross and histological features of three metachronous liver cancers curatively resected every three years (#1 to #3, respectively). #1, well-differentiated HCC, 2.6×1.7 cm in diameter; #2, moderately differentiated HCC, 1.8×1.4 cm in diameter; #3, cHCC-CCA, 4.5×4.5 cm in diameter. The outlined areas were macroscopically dissected for #1HCC, #2HCC and #3cHCC-CCA and laser capture microdissected for #3HCC and #3CCA after staining with toluidine blue (red scale bars, 1 cm). DNA was then extracted. Histological features of all cancerous regions are presented by H&E staining (scale bars, 50 µm). #3cHCC-CCA was subjected to immunohistochemical staining for CK7 and CA19-9 and each result for the #3HCC and #3iCCA regions is presented (scale bars, 50 µm). HCC, hepatocellular carcinoma; cHCC-CCA, combined hepatocellular-cholangiocarcinoma; iCCA, intrahepatic cholangiocarcinoma; CK7, cytokeratin 7; CA19-9, carbohydrate/cancer antigen 19-9; H&E, hematoxylin and eosin.

Figure 2.

TERT promoter mutation C228T. (A) Sanger sequencing of the TERT promoter region. Nested PCR was performed using seven DNA samples (#1N, #1HCC, #2N, #2HCC, #3N, #3HCC and #3iCCA), which were then subjected to Sanger sequencing. An arrow indicates the C228T single nucleotide variant. (B) dPCR of the C228T mutation. The numbers of Mu/WT/both allele amplifications in each nontumorous DNA sample (#1N, #2N and #3N) were as follows: 19/2429/3, 14/2233/0 and 10/8007/2, respectively. The levels in two negative control DNA samples, liver (nontumorous liver of a patient with liver metastasis of gallbladder cancer) and blood (healthy donor blood) were 0/9942/0 and 0/9318/0, respectively. TERT, telomerase reverse transcriptase; Wt, wild-type; Mu, mutant/mutation; N, nontumorous liver section; HCC, hepatocellular carcinoma; iCCA, intrahepatic cholangiocarcinoma; dPCR, digital PCR.

Figure 3.

Schematic of the molecular evolution of three metachronous liver cancers. Different allele frequencies of six SNVs, including the TERT promoter mutation, are presented in doughnut charts and used for the cluster analysis (Fig. S4). Mutant cancer cell populations were predicted as indicated in Table I and cancer cell evolution by accumulation of somatic mutations is presented from right to left in ellipses. Bar charts indicate the population of two types of PKDH1-mutant cells: Heterozygous with SNV (colored) and hemizygous with wild-type loss as the second hit (blank); the % population of the mutant hemizygote is also presented. There are two possibilities for #3iCCA generation: #3iCCA was directly generated from #2HCC or indirectly generated from #2HCC through #3HCC, as indicated by a narrow arrow in parentheses. SNV, single-nucleotide variant; HCC, hepatocellular carcinoma; cHCC-CCA, combined hepatocellular-cholangiocarcinoma; iCCA, intrahepatic cholangiocarcinoma; TERT, telomerase reverse transcriptase; KMT2D, histone-lysine N-methyltransferase 2D; TP53, tumor protein p53; DNMT3A, DNA (cytosine-5′)-methyltransferase 3α; PKHD1, polycystic kidney and hepatic disease 1; TLR4, toll like receptor 4.