Clonal analysis of metachronous double biliary tract cancers

The molecular mechanisms underpinning the development of metachronous tumors in the remnant bile duct following surgical resection of primary biliary tract carcinomas (BTCs) are unknown. This study aimed to elucidate these mechanisms by evaluating the clinicopathologic features of BTCs, the alterations to 31 BTC‐related genes on targeted sequencing, and the aberrant expression of p53, p16, SMAD4, ARID1A and β‐catenin on immunohistochemistry. Twelve consecutive patients who underwent resection of metachronous BTCs following primary BTC resection with negative bile duct margins were enrolled. Among the 12 metachronous tumors, six exhibited anterograde growth in the lower portion and six exhibited retrograde growth in the upper portion of the biliary tree. Surgical resection of metachronous BTCs resulted in recurrence‐free survival in seven, local recurrence in five, and death in two patients. Nine achieved 5‐year overall survival after primary surgery. Molecular analyses revealed that recurrently altered genes were: TP53, SMAD4, CDKN2A, ELF3, ARID1A, GNAS, NF1, STK11, RNF43, KMT2D and ERBB3. Each of these was altered in at least three cases. A comparison of the molecular features between 12 paired primary and metachronous BTCs indicated that 10 (83%) metachronous tumors developed in clonal association with corresponding primary tumors either successionally or phylogenically. The remaining two (17%) developed distinctly. The successional tumors consisted of direct or evolved primary tumor clones that spread along the bile duct. The phylogenic tumors consisted of genetically unstable clones and conferred a poor prognosis. Metachronous tumors distinct from their primaries harbored fewer mutations than successional and phylogenic tumors. In conclusion, over 80% of metachronous BTCs that develop following primary BTC resection are probably molecularly associated with their primaries in either a successional or a phylogenetic manner. Comparison between the molecular features of a metachronous tumor and those of a preceding tumor may provide effective therapeutic clues for the treatment of metachronous BTC. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.


Introduction
Biliary tract carcinomas (BTCs) arise in the biliary mucosa; complete surgical resection is currently the sole treatment that prolongs the lifespan of patients [1][2][3].Although recent studies have indicated an increased incidence of BTCs, advances in perioperative management and surgical techniques have improved the survival of patients with these tumors.However, surgeons may unexpectedly encounter metachronous tumors in the remnant bile duct after pancreatoduodenectomy or hemihepatectomy with perihilar bile duct resection for primary BTC [4][5][6].In most cases, aggressive surgical resection may yield a better prognosis.
Multistep carcinogenesis has been widely accepted for the formation of BTCs.Biliary intraepithelial neoplasia (BilIN) and intraductal papillary neoplasm of the bile duct (IPNB) are precancerous lesions that exhibit low-grade to high-grade dysplasia, i.e. carcinoma in situ [7,8].These lesions eventually progress to invasive cholangiocarcinomas that subsequently metastasize to lymph nodes or invade adjacent organs.Multicentricity is one of the characteristics of BTCs.BilIN often surrounds cholangiocarcinoma; synchronous multicentric tumors arising in the biliary tree have been reported in 3-7% of IPNBs [9][10][11].Exposure to carcinogens induces precancerous changes in the biliary epithelium, i.e. field defect, and the additional accumulation of oncogenic alterations leads to cancers with multicentricity [12,13].
According to the concept of multistep and multicentric carcinogenesis of BTCs, metachronous tumors arising in the remnant bile duct after surgical resection are generally expected to be cancers arising independently from primary BTCs.However, some reports propose that metachronous BTCs are caused by multicentric carcinogenesis based on the observation that primary tumors present with extensive dysplastic epithelium in most cases [4][5][6]14].
In this study, we performed integrative clinicopathologic and molecular analyses to compare the signatures of metachronous multicentric tumors and to provide a deep insight into the biology of the metachronous development of BTCs.

Participants
This study retrospectively analyzed material from 12 consecutive patients who underwent resection of metachronous BTCs arising in the remnant bile duct following resection of a primary BTC between 2008 and 2020 at Tohoku University Hospital.This study was approved by the institutional review board of our institution, the Ethics Committee of Tohoku University Graduate School of Medicine (#2020-1-264).This study was carried out in accordance with the Declaration of Helsinki.

Surgical procedure
The surgical procedure was determined according to tumor location in the biliary system.Hemihepatectomy with or without extrahepatic bile duct resection was performed for perihilar BTC and intrahepatic cholangiocarcinoma, respectively.Pancreatoduodenectomy was performed for distal BTC and ampullary carcinoma.Cholecystectomy with or without extended liver-bed resection was performed for patients with gallbladder cancer.

Histological evaluation
The entire resected specimens were fixed in 10% formalin, sliced serially at 5-mm intervals, and embedded in paraffin.Formalin-fixed, paraffin-embedded tissues were sectioned at 4-μm thickness and stained with H&E.A pathological assessment was performed by two pathologists (YOm and TF) following the General Rules for Clinical and Pathological Studies on Cancer of the Biliary Tract of the Japanese Society of Hepato-Biliary-Pancreatic Surgery sixth edition and TNM staging of the Union for International Cancer Control eighth edition.
The extent of the invasive tumor and the superficial spreading lesion of carcinoma in situ/high-grade BilIN were determined by examination of the entire specimen.In addition, the status of the ductal margin during the primary operation, the status of the ductal stump during the second operation, and the resemblance of the tumor morphologies to one another were examined.Classification of subtypes, namely intestinal-type or pancreatobiliary-type, was informed by morphological and immunohistochemical evaluation [25].

Targeted amplicon sequencing
Targeted amplicon sequencing was performed on 12 primary carcinomas, their 12 paired metachronous carcinomas, and nine ductal margins, with normal tissues for reference.Neoplastic lesions were dissected macroscopically from formalin-fixed, paraffin-embedded tissues sectioned at 10-μm thickness.Genomic DNA was extracted using a GeneRead DNA FFPE Kit (Qiagen, Hilden, Germany) and quantified using the Qubit dsDNA HS Assay Kit on a Qubit4 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA).

Y Omori, S Aoki et al
Additional immunohistochemical analyses for HER2, MLH1, MSH2 and AFP were performed on selected cases depending on the sequencing results and histological findings.
Loss of expression of p16, overexpression or complete loss of expression of p53, loss or reduced expression of SMAD4 and ARID1A, and nuclear and cytoplasmic accumulation or reduced expression of β-catenin were designated aberrant and compared between paired primary and metachronous tumors.The expression of HER2 was evaluated according to the criterion of gastric cancer [26].

Statistical analyses
Categorical variables were compared using Fisher's exact test.Continuous variables were compared using the Mann-Whitney U-test and the Kruskal-Wallis test.Correlations were assessed using Spearman's rank correlation coefficient.Survival curves were plotted using the Kaplan-Meier method and compared using the logrank test.GraphPad Prism (San Diego, CA, USA) and R (version 3.6.3,R Foundation) were used to perform statistical analyses.Statistical significance was set at p < 0.05.

Clinicopathological characteristics of patients who underwent subsequent resection for metachronous BTC
Twelve patients underwent resections for metachronous BTC arising after the resection of a primary tumor (Table 1).The origins of the primary BTCs were four from the perihilar bile duct, three from the distal bile duct, three from the gallbladder, one from the ampulla, and one IPNB extended to the right anterior and posterior bile duct counted as intrahepatic bile duct origin; the locations of the metachronous BTCs were five perihilar, four distal, two ampulla, and one intrahepatic bile duct in the central part of the left lateral bile duct.Hepatectomies, pancreatoduodenectomies, extrahepatic bile duct resections, and cholecystectomies were performed for the primary tumors in five, four, one and two patients, and subsequently for metachronous tumors in five, seven, zero and zero patients, respectively.In six patients, metachronous tumors arose downstream of the primary tumor's location in the biliary tree, growing in an anterograde manner; in the other six, they exhibited retrograde growth from upstream of the primary BTC site.
The primary tumors tended to be more confined to the biliary wall compared with the metachronous tumors; UICC T stages were T1, T2 and T3 in seven, four, and one of the primary tumors, and two, six and four of the metachronous tumors, respectively ( p = 0.100, see supplementary material, Table S2).The histological downgrading along with recurrence was observed in five cases in conventional cholangiocarcinomas.
The metachronous tumors that arose following the resection of two IPNBs resembled primary neoplasms.Lymphatic invasion was more frequently observed in the metachronous than in the primary tumors.Mucosal carcinoma components were observed in all primary lesions and 11 metachronous lesions.Extended BilINs surrounding invasive carcinomas were observed in five primary lesions.R0 resections were successfully achieved in all patients during the initial operations; however, five patients were classified as R1 at the second operation due to positive vertical margins.No patients in this cohort exhibited anomalous pancreaticobiliary ductal union or Lynch syndrome.
Neoadjuvant therapy before initial surgery was administered only in one case (mBTC_005), using gemcitabine and radiation 1.8 Gy Â 25 fractions.Adjuvant chemotherapy, using gemcitabine or S1, was administered in six cases in the interval between initial surgery and the occurrence of the metachronous tumor.Adjuvant chemotherapy after the second surgery was administered in eight cases.

Comparison of pathological features between the primary and metachronous BTCs
The location and histology of the tumors are summarized in Figure 1 and supplementary material, Figure S1.In mBTC_009, a primary tumor was found in the cystic duct and that protruded into the common bile duct (Figure 1A).A metachronous tumor developed in the ampulla 36.8 months after cholecystectomy and extrahepatic bile duct resection with negative margins for the primary tumor.Pancreatoduodenectomy was subsequently performed for the metachronous lesion.Pathologically, both primary and metachronous tumors showed papillary-type growth of intestinal-type IPNBs with similar morphology.
In mBTC_011, a primary tumor was found in the ampulla, and a metachronous tumor developed in the perihilar bile duct 20.9 months after pancreatoduodenectomy for the primary tumor with a negative margin (Figure 1B).Subsequently, a right hemihepatectomy with extrahepatic bile duct resection was performed.Pathologically, the primary and metachronous tumors comprised CDX2-positive intestinal-type adenocarcinoma.The morphology of tumor cells was similar for both specimens and both exhibited nodular infiltrating-type growth.Their histological grades differed: moderately differentiated tubular adenocarcinoma was observed in the primary tumor and poorly differentiated adenocarcinoma in the metachronous tumor.
In mBTC_002, a papillary growth-type gallbladder carcinoma resected by cholecystectomy with a negative margin was diagnosed as papillary adenocarcinoma of the intestinal-type (Figure 1C).After 81.8 months, a metachronous tumor showing nodular infiltrating-type growth was detected in the distal bile duct and a pancreatoduodenectomy was performed.Pathologically, the metachronous tumor was a moderately to poorly    Noteworthy is the marked plug-like growth of tumor in the distal bile duct extending into the pancreatic duct through the ampulla in two cases of metachronous tumor

Clonal analysis of metachronous double biliary tract cancers 117
after resection of the IPNB in the intrahepatic bile duct in mBTC_007 (see supplementary material, Figure S1A) and for the moderately differentiated cholangiocarcinoma in the perihilar bile duct in mBTC_003 (see supplementary material, Figure S1G).
Molecular alterations in the 12 paired primary and metachronous BTCs In the 12 primary BTCs, 84 somatic alterations, including 38 mutations and 46 CNVs, were detected in 25 genes by targeted amplicon sequencing for 31 BTC-related genes (Figure 2 and supplementary material, Figure S2A-D, Table S3).On average, there were 3.2 mutations and 4.2 CNVs per tumor.No alterations were detected in nine ductal margin tissue samples, indicating negative surgical margins at the molecular level.In the 12 metachronous BTCs, 106 alterations, including 41 mutations and 65 CNVs, were detected in 28 genes.There were 3.4 mutations and 5.4 CNVs on average per tumor, and the number of altered genes was increased in six cases compared with their paired primary tumors.The genes recurrently altered in at least three cases were: TP53, SMAD4, CDKN2A, ELF3, ARID1A, GNAS, NF1, STK11, RNF43, KMT2D and ERBB3.ERBB2 amplification was detected in two paired tumors and one metachronous tumor.
The protein expression of tumor-associated molecules, namely p53, p16, SMAD4, ARID1A, β-catenin and HER2, was consistent with the genetic state detected by targeted amplicon sequencing (Figures 2, 3A-R).Thirtyeight and 41 aberrations in the protein expression were detected in the primary and metachronous tumors, respectively.

Comparison of molecular alterations between primary and metachronous BTCs
Comparisons of molecular features between 12 paired primary and metachronous BTCs indicated that 10 (83%) metachronous tumors developed in clonal association, either in a successional or a phylogenic manner, with their corresponding primaries (Figures 2, 3W).The remaining two tumors (17%) developed in a clonally distinct manner.In the successional pathway, a metachronous tumor is supposed to originate directly from its primary tumor clone: the metachronous tumor harbors all gene alterations detected in its primary with or without additional molecular alterations (4/12, 33%).In the phylogenic pathway, a metachronous tumor develops from an ancestor clone of its primary tumor: the metachronous tumor and its primary harbor identical as well as distinct gene alterations (5/12, 42%).One case (mBTC_010) showing the clonal association by genetic mutations was left undetermined whether it was successional or phylogenic because no CNV data were obtained for its primary tumor due to low tumor cellularity.In the distinct pathway, a metachronous tumor develops independently from its primary and harbors distinct gene alterations.
The number of altered genes in the primary tumors tended to be higher in the clonally related group, including successional and phylogenic pathways, than in the distinct pathway group (p = 0.078, Figure 4A).Moreover, the numbers of CNVs between the primary and metachronous tumors were higher in the phylogenic pathway than in the distinct pathway (p = 0.095 and p = 0.048, respectively, Figure 4A,B), and tended to increase on recurrence, as seen in four of five cases (80%).
The expression of the previously described six molecules in metachronous tumors was similar to that in the primary tumors of the clonally related group.The phenotype of AFP-producing adenocarcinoma in the perihilar bile duct was maintained in the recurring lesion in the ampulla of Vater by phylogenic pathway (mBTC_013, Figure 3S-V).In contrast, the expression patterns differed between the primary and metachronous tumors in the two cases of distinct pathways.

Clinicopathological assessment based on the clonality of BTCs
Clinicopathological factors were analyzed based on the clonality of double BTCs (see supplementary material, Tables S4-S7).All cases of cholangiocarcinoma, including a cystic duct carcinoma protruding into the common bile duct, developed clonally related metachronous carcinomas.Two IPNBs and their metachronous tumors were clonally related via successional or phylogenic pathways.On the other hand, the two cases of distinct pathways were early-stage gallbladder cancers that underwent primary cholecystectomy.
Among the 10 patients in the clonally related group, macroscopic growth patterns were nodular in six and papillary in three patients in paired BTCs, while the remaining patient showed nodular growth in the primary tumor and papillary growth in the metachronous tumor.In nine patients, histologic subtypes were identical between 118 Y Omori, S Aoki et al the primary and metachronous tumors: five pairs were intestinal-type and four were pancreatobiliary-type.The morphologies of carcinoma cells were similar between the primary and metachronous tumors in all 10 patients.However, the ductal differentiation according to the tumor grade decreased in one case of successional Clonal analysis of metachronous double biliary tract cancers 119  Clonal analysis of metachronous double biliary tract cancers 121 pathway (25%) and three cases of phylogenic pathway (60%, see supplementary material, Figure S3).In the successional pathway, three patients showed lymph node metastasis of the primary BTCs (p = 0.040, see supplementary material, Table S5) and venous invasion of the metachronous BTCs tended to be severe (p = 0.060, see supplementary material, Table S7).The only metachronous tumor without an obvious intramucosal carcinoma component pursued the successional pathway.
The primary BTC with neoadjuvant chemoradiation therapy before initial surgery showing the remarkable degeneration and necrosis of cancer cells harbored 13 pathogenic mutations (mBTC_005, see supplementary material, Figure S1F).All mutations succeeded in the paired metachronous BTC.The administration of chemotherapy in the interval between initial surgery and the occurrence of metachronous tumor was not associated with the molecular recurrence pattern (see supplementary material, Tables S6 and S7).The difference in the pattern, including the preference of selection or deselection along with the recurrence or number of altered genes was not significant between the clonally related cases with or without interval chemotherapy (see supplementary material, Figure S4).
Interestingly, five metachronous BTCs arose in the intrahepatic and perihilar bile duct.This clonally related group originated in a retrograde manner from the primary tumors located in the distal portion of the biliary tree; four from the distal bile duct and one from the ampulla.However, there was no difference between the clinicopathological features exhibited by the five downstream and five upstream recurrences (see supplementary material, Table S8).
The two cases of distinct pathway showed different tumor morphologies between the primary and metachronous BTCs; one case was a papillary adenocarcinoma of intestinal-type in the gallbladder and a pancreatobiliarytype moderately differentiated tubular adenocarcinoma infiltrated in the distal bile duct (mBTC_002, Figure 1C) and the other was a well-differentiated tubular adenocarcinoma arising in the pyloric gland adenoma of the gallbladder and a moderately differentiated tubular adenocarcinoma infiltrated in the perihilar bile duct (mBTC_001, see supplementary material, Figure S1I).

Survival analysis of patients who underwent surgical resection for double BTCs
The cohort collectively demonstrated favorable prognoses with a median disease-specific survival (mDSS) time of 141.2 months from the initial operation, regardless of the development pathway of metachronous BTCs (Figure 5A-C).The median disease-free survival (mDFS) after the initial operation was 41.1 months for the entire cohort; 41.1 months in the clonally related group and 50.0 months in the clonally independent group (Figure 5D-F).
Five patients developed local recurrences after the second operation; among them, two subsequently developed lung metastases.Two patients died due to disease progression.The mDSS and mDFS after the second operations of the clonally related group, especially of the phylogenic pathway, tended to be shorter than those of the clonally independent group (DSS; p = 0.184, Figure 5G-I, DFS; p = 0.094, Figure 5J-L).Two patients in the clonally independent group achieved 106.9 and 80.4 months of DFS after the second operation.The mDSS and mDFS of the clonally related group were both 54.5 months.The patients of the phylogenic pathway showed the shortest mDSS (53.3 months) and mDFS (25.4 months) among the three developmental pathways.However, this effect was statistically insignificant due to the small case number (Figure 5I,L).
The interval chemotherapy was not associated with the recurrence time from the initial surgery to the occurrence of metachronous tumor nor with the prognosis after the second surgery (see supplementary material, Figure S5A-C).Also, the adjuvant chemotherapy after the second surgery was not associated with the prognosis (see supplementary material, Figure S5D,E).

Discussion
The comparative genomic analysis of 12 paired BTCs revealed that metachronous BTCs were clonally related to their primary cancers in 10 cases (83%) in either a successional or a phylogenic manner.The absence of cancerous lesions and genetic alterations at the bile duct resection margins ruled out local recurrence due to a positive surgical margin (Figure 6A).Therefore, they were considered recurrences of the primary cancer clones due to intramural metastases via lymphovascular channels or seeding and implantation within the bile duct (Figure 6B,C).The two cases of IPNB were both clonally related recurrences.Moreover, the massive thrombus-like tumor growth in the pancreatobiliary system seemed to be a characteristic of the recurrence by intraductal seeding of clonally related BTCs.The clonally related tumors were not associated with cancer-predisposing mutations [27,28] or status, such as anomalous pancreaticobiliary ductal union or Lynch syndrome.On the contrary, in only two cases (17%) were the pairs clonally independent with distinct molecular alterations between the primary and metachronous tumors (Figure 6D).
To the best of our knowledge, this is the first study that identified the clonal relationship between metachronous double BTC series and their preceding primary tumors by using integrated genetic analysis.These clonal relationships can be classified as successional, phylogenic, or distinct (Figure 3W).We also clarified the long-term outcomes of repeated operations for metachronous tumors.The BTCs that pursued successional pathways showed higher potential for lymphovascular invasion and may have a predilection for intramural metastasis in the biliary tree.The primary cancer clone had already 122 Y Omori, S Aoki et al spread in the biliary tree through lymphovascular channels and its clones grew with time at a distant site.The BTCs of the phylogenic pathway harbored multiple CNVs and the number of CNVs tended to increase with recurrence.Mutations were generally common to the primary and metachronous BTCs and CNVs created intratumor heterogeneity simultaneously and metachronously.Their genomic instability may reflect the phylogenic progression of subclones and may contribute to poorer prognoses after the second operation.The double BTCs in the distinct pathway group presented as multicentric carcinogenesis.They tended Clonal analysis of metachronous double biliary tract cancers 123 to have fewer genetic alterations, indicating that a few driver genes with independent alterations may play a major role in their development.
The metachronous development of carcinoma in the same organ has generally been explained by different progression cascades, including local recurrences due to residual carcinoma at the resection margin, metastases via lymphovascular channels, de novo carcinogenesis of independent clones, and intraluminal spread as skip lesions in hollow organs, such as the urinary tract [29,30] and pancreatic duct [21][22][23]31].Several reports have described metastasis to the biliary epithelium from colon, lung, breast, testicular, pancreatic, or ovarian cancer [32,33] and most of these metastases occurred after surgical resection of the advanced primary cancers.
In contrast, several studies conducted by our group and others have demonstrated metachronous tumor development potentially via intraluminal seeding or dissemination in cases of pancreatic tumors [21,22,31].Pea et al [23] also proposed the mechanism of intraductal or intraparenchymal metastases from a primary intraductal papillary mucinous neoplasm of the pancreas based on integrated genetic analyses of metachronous neoplasm arising in the remnant pancreatic duct.Similar metastatic behavior can also be adopted by biliary cancer.Thus, even if the tumor resides within the biliary mucosa, a metastatic tumor might subsequently spread and develop in a distant area of the biliary epithelium as a skip lesion originating from the less-advanced primary tumor.Indeed, five primary BTCs showed less invasive characteristics with limited intramucosal growth and were less likely to have lymphovascular invasion and lymph node or distant metastasis.In contrast, the other seven BTCs invaded the submucosa or deeper layers so had the potential of metastasis via lymphovascular invasion.
Dissemination or implantation of cancer cells within the bile duct may occur by contact of contaminated biliary secretions with the biliary epithelium [34,35].Endoscopic retrograde cholangiopancreatography is the most common technique used to assess the spread and obtain histological evidence of BTCs, as well as to relieve biliary obstruction by the tumor.A multipoint sampling/mapping biopsy of the biliary tree is routinely carried out in Japan to accurately diagnose mucosal spread.Bile stasis or forced stenting due to biliary obstruction causes the non-physiological flow of cancer cell-contaminated biliary secretions.These procedures unique to BTC treatment practices may increase the risk of dissemination or implantation within the bile duct.In this cohort, preoperative endoscopic examinations were performed for multipoint biopsies or stenting for biliary obstruction in all 10 patients of the clonally related group.These examinations were not performed in the remaining two patients with primary gallbladder carcinomas and metachronous clonally independent BTCs.Damage to the biliary epithelium by the endoscopic procedure may cause subsequent cancer implantation or stealth dissemination [36]; however, direct evidence for this model is limited, and studies based on genomic tracing with a substantial case number will be required to prove the iatrogenic risk.
Favorable surgical outcomes have been reported in patients with synchronous multicentric BTCs.The spread of the mucosal neoplasia indicates early cancer characteristics of less lymphovascular invasiveness and postoperative distant metastases [4].However, the outcome of resection for metachronous BTC is not well documented.In this study, aggressive surgical procedures for metachronous BTCs conferred a favorable prognosis: mDSS from the initial operation was 141.2 months, mDFS from the second was 54.5 months, and only two patients died due to disease progression.Interestingly, all recurrences after the second surgery in five patients occurred at the local site, not in the distant organs.Reoperation of the remnant bile duct after hepatectomy or pancreatoduodenectomy demands highlevel surgical skills to secure sufficient surgical margins, and the difficulty increases the likelihood of postoperative local recurrences.With advances in surgical techniques [37], these risks have been decreasing.The two cases with early recurrence and short postoperative survival occurred more than 10 years ago.Even with repeated pancreatobiliary surgery, a favorable prognosis for patients with metachronous BTCs may be expected if curative resection with negative surgical margins can be achieved.
Postoperative recurrences after curative resection for BTCs are frequently observed and generally require systemic chemotherapy [38,39].Emerging metastatic tumors in distant organs are considered systemic diseases and surgical resection for these metastases cannot ensure sufficient postoperative survival [40,41].

Y Omori, S Aoki et al
However, as shown in this study, recurrence due to intraductal metastases of primary BTCs implies local spreading of cancer cells confined to the bile duct.Thus, the definitive surgical resection for the recurrence of cancer in the remnant bile duct may contribute to a better prognosis.Integrated genomic analysis using targeted sequencing revealed potentially actionable genetic alterations in the BTCs.Genome-wide analyses have highlighted heterogenetic molecular aberrations in BTCs [16,17], which facilitate the characterization of tumorigenesis and the development of individualized molecularly targeted therapy.Recurrently altered genes in our cohort were: TP53, SMAD4, CDKN2A, ELF3, ARID1A, GNAS, NF1, STK11, RNF43, KMT2D, ERBB3 and ERBB2.The number of gene alterations in the two cases that pursued the distinct pathway was relatively small; however, driver gene alterations in TP53, CTNNB1, SMAD4, PBRM1, KRAS, PIK3CA and ERBB2 characterizing each BTC were detected and we could discuss the clonal relationship.
According to diverse anatomical locations of the biliary tree system, BTCs can be classified into intrahepatic cholangiocarcinoma, extrahepatic cholangiocarcinoma, and gallbladder carcinoma.These anatomical locations are known to be associated with distinct mechanisms of carcinogenesis indicated by molecular characteristics: large duct-type intrahepatic cholangiocarcinoma shows a high frequency of TP53 and KRAS mutation and lower frequencies of ARID1A, BAP1, BRAF, CDKN2A, EGFR, ELF3, ERBB2, GNAS, MDM2, PIK3CA, PTEN and SMAD4 alterations, whereas small duct-type intrahepatic cholangiocarcinoma typically harbors mutation in IDH1/2 and FGFR2 fusions [42,43].Extrahepatic cholangiocarcinoma shares the heterogenous molecular pattern with large duct-type intrahepatic cholangiocarcinoma.Our genetic analysis using the extrahepatic cholangiocarcinoma-centered cohort revealed that the dominant molecular mechanism of the development of metachronous BTCs was clonally related.However, because the intrahepatic cholangiocarcinoma of large duct-or small duct-type was not included in our cohort, the molecular mechanisms of multiple hepatic lesions of these major types of intrahepatic cholangiocarcinoma are outside the scope of our study; studies elsewhere have discussed those issues [44].
Of note, the primary BTC that received neoadjuvant chemoradiation therapy showed remarkable degeneration of cancer cells and harbored 13 pathogenic mutations.The tumor was mismatch-repair proficient according to the retained expression of MLH1 and MSH2.That is, the numerous mutations may be induced by neoadjuvant therapy in this case.The chemoresistance molecular factor or the clinical influence of adjuvant treatment for metachronous double BTCs was not evident; however, this cohort was too small to conclude these issues.
The limitation of this study is that it consisted of retrospectively collected data concerning a small number of resected cases and sequencing data obtained for a limited number of genes.However, we collected 12 cases of metachronous double BTCs, which is the largest case series yet.If we applied a larger gene panel, ideally whole exome sequencing, we could have performed indepth analysis of the molecular recurrence mechanism, especially the clonal independence in the distinct pathway.Nonetheless, the BTC gene panel used in this study covered representative molecular alterations in 31 genes detected in previous comprehensive studies.Based on this analysis, the association of the molecular recurrence pathway and clinicopathological characteristics was revealed.
In conclusion, this study indicates that over 80% of metachronous BTCs developing after resection of primary BTC are probably molecularly associated with the primary in either a successional or a phylogenic manner.The successional tumors consist of directly evolved primary tumor clones that may have spread along the bile duct.The phylogenic tumors may consist of genetically unstable clones and cause poor prognosis.Metachronous tumors distinct from primary tumors may harbor fewer mutations than successional and phylogenic tumors.Aggressive surgical reoperation can achieve definitive local control and longer survival in patients with a metachronous BTC.Assessment of the molecular features of the metachronous tumor compared with those of its preceding tumor may provide effective therapeutic clues for these patients.
YOm, YOn and YM performed the genetic analyses and YOm and YOn interpreted the data.YOm and SA performed the statistical analysis.TF and MU supervised the project.All authors had access to the study data and reviewed and approved the final manuscript.
116Y Omori, S Aoki et al differentiated pancreatobiliary-type adenocarcinoma.The morphologies of the primary and metachronous tumors were different in this case.

Figure 1 .
Figure 1.Representative cases of the primary and metachronous BTCs.(A) Scale bars: 2.5 mm in the left panels, 50 μm in the right panels.(B) Scale bars: 2 mm in the left panels, 200 μm in the right panels.(C) Scale bars: 2 mm in the left panels, 100 μm in the right panels.AMP, amplification; DEL, deletion.

Figure 3 .
Figure3.Alterations of protein expressions in the primary and metachronous BTCs.The mBTC_011 primary (A) and metachronous (F) tumors, both harboring TP53 R282W, CDKN2A deletion, SMAD4 R135* and deletion, and ARID1A deletion, showed identical and consistent protein expressions, (B and G) overexpression of p53, (C and H) loss of p16, (D and I) loss of SMAD4, and (E and J) loss of ARID1A.In mBTC_003 primary (K) and metachronous (M) tumors, both harboring APC W157*, nuclear and cytoplasmic accumulation of β-catenin was observed (L and N).mBTC_004 primary (O) and metachronous (Q) tumors, both harboring ERBB2 amplification, showed strong membranous expression of HER2 (P and R).The mBTC_013 primary (S) and metachronous (U) tumors comprised clear cytoplasm and chromatin-aggregated, enlarged nuclei, and both showed positive immunostaining for AFP (T and V).(S) The mBTC_013 primary tumor showed marked perineural invasion.(W) The conceptual diagrams of molecular development pathways of the metachronous BTCs.prm, primary tumor; met, metachronous tumor.

Figure 4 .
Figure 4. Comparison of the number of gene alterations between (A) the primary tumors and (B) metachronous tumors.(C) The number of somatic mutations and (D) CNVs shared between the primary and metachronous tumors (green), only in the primary tumors (blue), and only in the metachronous tumors (magenta).Comparison of variant allele frequencies of detected mutations in (E) the primary tumors and (F) metachronous tumors.prm, primary tumor; met, metachronous tumor.

Figure 5 .
Figure 5. Survival analysis.Kaplan-Meier curves showing DSS and DFS after initial and second operations of total cohort (A, D, G, and J), groups according to clonality (B, E, H, and K), and molecular recurrence pattern (C, F, I, and L).MST, median survival time in months; R, clonally related; I, clonally independent; S, successional type; P, phylogenic type; D, distinct type; N.A., not available.

Figure 6 .
Figure 6.Four developmental mechanisms exhibited by metachronous tumors in the remnant bile duct.(A) Local recurrence by the residual microscopic carcinoma cells at the primary surgical margin.(B) Intraductal spread of neoplastic cells.(C) Intramural cancer cell dissemination via lymphovascular channels or perineural invasion of primary cancer clones at distant locations from the primary tumor with genetically related clones.(D) Multicentric carcinogenesis in the biliary tree with genetically distinct clones.

Table 1 .
Clinicopathological characters of 12 patients who underwent subsequent resection for metachronous BTCs.