Biliary tract cancer (BTC) accounts for approximately 3% and 10–15% of gastrointestinal and hepatobiliary malignancies, respectively (1). The biliary tract mainly comprises the biliary epithelium of small ducts in the liver periphery (intrahepatic), the common hepatic and common bile duct (extrahepatic), gallbladder, and ampulla of Vater. The epidemiological characteristics of sporadic BTCs include a higher and lower frequency of gallbladder cancer and BTC development, respectively, in females than in males (2). Compared with Caucasian or African Americans, Asian-Pacific Islander men and women have a higher rate of extrahepatic BTC development (2). Moreover, geographic regions, including North India, South Chili, and Poland, are risk factors for gallbladder cancer (3). Surgical resection with lymph node dissection is the main treatment (4-7). Although novel chemotherapies have been developed, BTCs remain highly malignant, with even complete surgical resection yielding a low 5-year survival rate (11–30%) (8,9).
Several risk factors for BTCs have been identified, including primary sclerosing cholangitis, biliary tract stones, pancreaticobiliary malfunction, infection, and toxic exposure (1,4). Additionally, several genetic factors, including Lynch syndrome (LS), BAP1 tumor predisposition syndrome, cystic fibrosis, and multiple biliary papillomatosis, are associated with an increased risk of BTC (10,11).
LS is among the most common inherited cancer syndromes related to pathogenic germline variants in mismatch repair (MMR) genes or epithelial cell adhesion molecule (EPCAM) (12). LS is associated with various tumor types, including colorectal, endometrial, ovarian, and biliary cancers (13,14). Compared with sporadic cancers, colorectal cancer (CRC) and endometrial cancer (EC) have longer survival (15,16). On the other hand, relatively rare LS-associated cancers, including brain tumors, pancreatic cancer, and BTC, have high mortality rates.
This study aimed to investigate the clinical features of BTC in individuals with LS, assess the utility of microsatellite instability (MSI) testing for LS identification in individuals with BTC, and discuss the surveillance indications for BTC in individuals with LS. We present the following article in accordance with the STROBE reporting checklist (available at https://jgo.amegroups.com/article/view/10.21037/jgo-22-165/rc).
Clinical features of BTC in LS
We conducted a retrospective review of LS individuals at the Iwakuni Clinical Center between January 2003 and April 2021. MSI testing on individuals who met Bethesda guidelines was performed (17). Probands underwent genetic testing if they presented with high-frequency of microsatellite instability (MSI-H) tumors or met a modified Amsterdam II criteria (18), which includes gastric cancer as among the LS-associated tumors since it is common in Asian individuals with LS. Data regarding age at diagnosis, variant type, tumor location, histological type, stage, treatment, follow-up, and surveillance were collected from pathological germline variant carriers and obligate carriers. The BTC location was classified according to WHO classification (5th edition). The spectrum and clinicopathological characteristics of BTCs in individuals with LS were explored.
Germline mutation analyses
Genomic DNA was extracted from peripheral blood samples using a standard phenol extraction and purification procedure. Germline variant analyses were performed by direct sequencing of the entire coding region of MLH1, MSH2, and MSH6 (19,20). In case there were no deleterious gene variants, multiplex ligation-dependent probe amplification was performed using a SALSA MLPA MLH1/MSH2 probemix assay. From 2014, genetic analysis was performed using a multi-gene panel that included MLH1, MSH2, MSH6, PMS2, and EPCAM (20), as previously described. Variants were assessed using the InSiGHT classification criteria (http://insight-group.org/variants/classifications/); moreover, individuals with Class 4 (likely pathogenic) or Class 5 (pathogenic) variants were considered to have LS. Before genetic testing, individuals underwent genetic counseling from clinical geneticists; further, they provided written informed consent. This study was approved by the institutional review board of the Iwakuni Clinical Center (No. 2774). The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).
MSI analysis for sporadic and LS cases
To compare the MSI status of BTC between sporadic and LS cases, we collected tissue samples from 21 consecutive individuals with primary BTC who underwent surgical resection between July 2008 and May 2014 at Iwakuni Clinical Center. Among 21 lesions of BTCs, 6 lesions have been obtained from 5 individuals with LS (Figure S1). MSI analysis was performed on surgically resected paraffin-embedded tumor specimens. Polymerase chain reaction analysis was performed targeting five markers: BAT25, BAT26, D2S123, D5S346, and D17S250. Tumors were classified as MSI-H (≥2 out of 5 markers), low-frequency of microsatellite instability (MSI-L, 1 out of 5), or microsatellite stable (MSS; 0 out of 5) (17).
Data are presented as medians (range) or percentages. The cumulative risk of developing BTC in individuals with LS was analyzed using a Kaplan-Meier plot. Statistical analyses were performed using JMP software (version 14, SAS Institute Inc.).
The spectrum of the initial cancer
A total of 92 individuals from 30 families were identified to have pathogenic MMR gene variants (Table 1). The male-to-female ratio of the 92 individuals was 42:50. Moreover, 75, 16, and 1 patient had MLH1, MSH2, and MSH6, respectively. Among the 92 individuals, 70 developed cancers. The initial cancers included colorectum (43/70, 61%), endometrium (14/38, 37% among females), gastric (5/70, 7%), ovary (4/38, 11% among females), and biliary tract (2/70, 3%) cancers. The median age at initial cancer diagnosis was 44 years (range, 17–78 years).
|MLH1 [obligation carrier]||75 |
|Age at the last follow-up, years||56 (23–90)|
|Individuals who developed any cancer||70 (76%)|
|Age at initial cancer, years||44 (17–78)|
|Origin of initial cancer|
|Individuals who developed BTC||6|
|Age at initial BTC, years||69 (34–78)|
Data are presented as number, median (range) or n (percentage). LS, Lynch syndrome; BTC, biliary tract cancer.
Clinical features of the BTC in individuals with LS
Among the 92 individuals, 6 individuals developed BTCs (Figure 1, Table 2). Six patients with BTC had MLH1 variants, with a male-to-female ratio of 2:1 (Tables 2,3). The median age at diagnosis of initial BTC was 69 years (range, 34–78 years). The BTC locations included 1 (1/9, 11%), 3 (3/9, 33%), 4 (4/9, 44%), 1 (1/9, 11%) in the intrahepatic biliary tract, peri-hilar biliary tract, distal biliary tract, and duodenal papilla of Vater (P. Vater), respectively. Histological subtypes of the nine specimens included four (4/9, 44%), four (4/9, 44%), and one (1/9, 11%) well, moderately, and poorly differentiated adenocarcinomas, respectively. Then, biliary intraepithelial neoplasia and intraductal papillary neoplasm of the bile duct were not detected in the nine lesions. Further, medullary growth pattern, which is the one of the unique features of LS associated CRC, was also not found in the lesions. Then, MSI analysis was tested in 7 lesions because other 2 lesions in case 3 was too small. All seven specimens showed MSI-H. The UICC (7th revision) stages included four (4/9, 44%), three (3/9, 33%), and two (2/9, 22%) at stages I, II, and IV, respectively. The incidence of synchronous and metachronous BTC was 17% (1/6) and 33% (2/6), respectively. Interestingly, case 3 had synchronous four lesions, in which two tumors showed MSI-H. The cumulative risks of individuals with an MLH1 pathogenic variant at 60, 70, and 75 years of age were 3.2%, 7.2%, and 13.4%, respectively (Figure 2).
|Case||Gender||Initial cancer||Age at initial cancer (years)||BTC|
|Age (years)||Location||Symptom||Family history of BTC||Histologic type||MSI status||Stage||Treatment||Outcome|
|1||Male||CRC||25||65||P. Vater||No||Yes||tub1||MSI-H||I||Partial resection||N.D|
|2||Male||CRC||54||76||Intrahepatic||No||Yes||tub2||MSI-H||II||Right posterior segmentectomy||Cancer death|
|3||Male||CRC||54||73||Perihilar||No||No||tub1||MSI-H||I||Left hepatectomy||Alive without recurrence|
|75||Distal||No||No||tub2||MSI-H||II||Pancreaticoduodenectomy||Alive without recurrence|
|75||Distal||No||No||tub1||N.A||I||Alive without recurrence|
|75||Distal||No||No||tub1||N.A||I||Alive without recurrence|
|4||Female||BTC||78||78||Perihilar||Yes||No||tub2||MSI-H||IVa||Extended left hepatectomy||Cancer death|
|5||Female||CRC||29||42||Perihilar||Yes||No||por||MSI-H||IVb||Best supportive care||Cancer death|
BTC, biliary tract cancer; LS, Lynch syndrome; MSI, microsatellite instability; CRC, colorectal cancer; P. Vater, duodenal papilla of Vater; tub1, well-differentiated tubular adenocarcinoma; MSI-H, high-frequency of microsatellite instability; N.D, no data; Distal, distal biliary tract; tub2, moderately differentiated tubular adenocarcinoma; Intrahepatic, intrahepatic biliary tract; Perihilar, perihilar biliary tract; N.A, not available; por, poorly differentiated adenocarcinoma.
|Case||Proband||Reason for genetic testing*||Gene variant||InSiGHT classification||Gene variant status|
|1||Yes||Colon cancer||MLH1 (c.381-415_453 + 733del)||Class 5||Carrier|
|2||No||Colon cancer||MLH1 (c.381-415_453 + 733del)||Class 5||Carrier|
|3||Yes||Colon cancer||MLH1 (c.70_72delAAG)||Class 5||Carrier|
|4||No||N.D||MLH1 (c.381-415_453 + 733del)||Class 5||Obligation|
|5||Yes||BTC||MLH1 (c.545+1G>C)||Class 5||Carrier|
|6||No||BTC||MLH1 (c.381-415_453 + 733del)||Class 5||Carrier|
*, cancer types that led to the implementation of genetic testing. MMR, mismatch repair; BTC, biliary tract cancer; N.D, no data.
MSI analysis for sporadic and LS cases
MSI analysis for BTC samples was performed to assess the MSI-H among individuals with BTC, including sporadic and LS cases. Among the 21 lesions, 6 and 15 lesions had MSI-H and MSI-L/MSS, respectively (Figure S1). All six MSI-H lesions were obtained from individuals with LS.
Our findings suggest that MSI analysis is an effective screening tool for identifying LS in individuals with BTC. Additionally, our findings indicate the need to conduct surveillance for BTC in MLH1 carriers living in countries with high risks for BTC.
Accurate LS diagnosis is crucial since various risk-reduction strategies, including surveillance, prophylactic surgery, and chemoprevention, might improve clinical outcomes. The efficacy of surveillance for LS has been demonstrated in CRC. Specifically, a 3-year colonoscopy surveillance was found to reduce CRC-caused mortality by 65% (21). Even an extended surgery can be an alternative treatment for CRC since there is a higher risk of metachronous CRC in MLH1 and MSH2 carriers (22).
Since MSI is a hallmark of LS tumors, many guidelines recommend universal screening for individuals with CRC and/or EC through MSI analysis. In LS, 90% of CRCs showed MSI-H. However, the frequencies of LS-associated tumors with MSI-H vary depending on the cancer origin. Specifically, colon, stomach, ovary, and ureter cancers show MSI-H >80%; bladder, endometrium, and kidney cancers show MSI-H ≤50%; and breast and brain tumors show MSI-H <35% (23). Unfortunately, there have been limited studies on the MSI status of BTC for both sporadic and LS cases. Additionally, MSI-H is not specific to LS. In most cases, MSI-H presence is indicative of epigenetic hypermethylation of the MLH1 promoter region. Recent studies have reported a 1–3% frequency of sporadic MSI-H tumors in BTCs (24,25). In this study, all BTC lesions (n=7) in 5 individuals with LS showed MSI-H. On the other hand, all BTC lesions (n=15) in 15 individuals with sporadic BTCs showed MSS or MSI-L. Therefore, MSI analysis might be an efficient screening tool for BTCs to identify LS. Given the fact that life time risk of BTC is lower than CRC, MSI analysis might be recommended when patients with BTC have family or personal history of LS.
Although the cumulative risk of BTC in LS is relatively low, BTC is highly malignant, with an average 5-year survival rate of 29% (26); further, it is among the main mortality causes in LS (27). Additionally, the MLH1 variant was predominant in BTC (28). A recent prospective study reported that the cumulative risk of BTC at 75 years of age was 3.7%, 1.7%, and 0% for MLH1, MSH2, and MSH6, respectively (26). In our study, the cumulative risk of BTC in Japanese MLH1 carriers was notably high, which was at 7.2% and 13.4% at 70 and 75 years of age, respectively. The BTC risk in Japanese individuals with the MLH1 variant is similar to that in individuals with primary sclerosing cholangitis, which is associated with an increased lifetime risk of BTC (5–20%) (29). Of the two patients who developed BTC as their initial cancer, one developed BTC at 78 years old. In individuals with LS, MLH1 variant is reported to be a risk factor to develop BTC (26). Besides, family history of BTC was observed in three of the six presented cases, suggesting that a family history of BTC may also be a risk factor. Aside from development of BTC as an initial cancer, it is important to detect asymptomatic BTCs by surveillance in order to improve the prognosis of individuals with LS. In our institution, we recommend that individuals with LS undergo surveillance with hematological examination and ultrasound (US), or computed tomography (CT) every 12 months beginning at age 40–50 years, 5 years prior to the earliest age of BTC diagnosis in the family according to some guidelines.
Despite the lack of evidence, several guidelines recommend surveillance for BTC in individuals with primary sclerosing cholangitis through imaging, including US, CT, magnetic resonance imaging, and/or serum carbohydrate antigen 19-9, at 6–12-month intervals (30). Since some BTC present with increased levels of the liver and bile duct enzymes, the blood biochemical tests are also applicable in surveillance for BTC (5). In this study, we have been able to diagnose 7 asymptomatic BTCs among 3 individuals by CT scan and blood biochemical tests performed during postoperative follow-up for CRCs or BTCs. Then, the three asymptomatic individuals with BTCs showed earlier clinical stages and better clinical outcomes than other three symptomatic individuals. Therefore, surveillance for BTC should be considered for MLH1 carriers in Japan with high risk for BTC.
This study has several limitations, including the small initial sample size (BTCs, n=10), the majority of MLH1 variant, and its retrospective single-center design. However, to our knowledge, this is the first report in Asia to demonstrate the importance of MSI analysis of BTCs in identifying LS, as well as to describe the clinical features of LS-associated BTC. Then, the unique features of LS-associated BTC should be investigate by prospective and/or multiple center design and further, it is very critical to build useful surveillance tool for BTC to improve overall survival.
Our findings suggested that LS-associated BTC has features similar to those of LS-associated CRC, including a high cumulative cancer risk, multiple occurrences, and high MSI-H incidence. MSI analysis could facilitate LS identification in patients with BTC when these patients are suspect to be LS by their family history and medical history. Therefore, BTC surveillance could be considered for individuals with MLH1 variants in countries with a high BTC incidence.
We would like to thank Editage (www.editage.com) for English language editing.
Funding: This research was supported in part by the Dial Study from the Japan Agency for Medical Research and Development, AMED. The research was also supported by Japan AMED under the grant reference JP18kk0205004 and JSPS KAKENHI grant reference JP18K07339.
Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-22-165/rc
Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-22-165/dss
Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-22-165/prf
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-22-165/coif). KA had lecture fee from Merck Sharp & Dohme (MSD). The other authors have no conflicts of interest to declare.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was approved by the institutional review board of the Iwakuni Clinical Center (No. 2774). The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). Before genetic testing, individuals received genetic counseling from clinical geneticists. Informed consent was obtained from all subjects involved in the study.
Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.
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