Genomic feature and potential therapeutic target for cholangiocarcinoma

doi:10.21203/rs.3.rs-2784205/v1

Background: The genomic feature of biliary tract carcinoma (BTC) has been characterized, but limited studies focus on the potential therapeutic target for cholangiocarcinoma patients.

Methods:

43 BTC patients were enrolled in this retrospective study. Genomic characteristics including genomic alterations and mutational signatures were detected and analyzed. Then, the correlation between the genomic characteristics and clinicopathologic features was investigated. Next, the prognostic significance of these altered genes was evaluated. Besides, personalized targeted therapies for patients harboring potentially actionable targets (PATs) were investigated.

Results:

Among 43 patients, the genomic mutation was detected in 38 patients. Among these mutations, KRAS (44.2%), TP53(37.2%), ARID1A (18.6%), SMAD4(18.6%), BRCA2, CDKN2A (11.6%), and VEGFA (11.6%) are the most frequently altered cancer-related genes. Besides, germline mutations mainly occurred in ERBB, PI3K/AKT, MAPK, and RAS signaling. Among detected mutations, we found that TP53, STK11, MYC, and ERBB3 are gene alternations with significant prognostic values. In terms of potentially actionable target (PAT) analysis, 19 genes were proposed to be PATs in BTCs. and we found out that 79.1% of patients have Tier II somatic mutation in our cohort.

Conclusions:

The molecular feature is closely related to clinical characteristics in cholangiocarcinoma patients. In this study, we identified several commonly altered genes in cholangiocarcinoma patients and determined potential prognostic biomarkers and therapeutic targets for cholangiocarcinoma patients.

Genomic

Cholangiocarcinoma

Target therapy

Clinical application

prognosis

Cholangiocarcinoma is defined as a kind of malignancy tumor that originates from the epithelial cells of the bile duct. According to the latest epidemiological data, the incidence (1.26 per 100000 people) and the mortality rate of cholangiocarcinoma are rising [1]. Currently, surgical resection remains to be the only potentially curative therapy for patients with cholangiocarcinoma[2]. However, due to the lack of significant symptoms and effective diagnostic biomarkers, the majority of patients were diagnosed at a late stage and could not receive surgery[3]. Recently, several new methods have been implicated to treat this disease, including offering neoadjuvant therapy before surgical resection, liver transplantation, and target therapy[4]. Despite the advancements in locally advanced or borderline resectable lesions, most patients were diagnosed at the advanced stage. For these patients, chemotherapy is the prior choice[5]. The first-line treatment in fit patients is the combination of gemcitabine and cisplatin. Besides, with the improvement in the understanding of the molecular feature of cholangiocarcinoma, target therapy has been widely used, including isocitrate dehydrogenase-1 (IDH1), fibroblast growth factor receptor (FGFR) and ERBB2 (HER2/neu)[6, 7]. Although such effort has been made to improve the treatment for this disease[8], cholangiocarcinoma remains to be a big challenge in medicine. To develop more treatment methods for cholangiocarcinoma patients, further effort should be taken to understand the molecular feature of this tumor. In addition, further exploration should be made to investigate the clinical and prognostic significance of the molecular feature in cholangiocarcinoma patients, thus providing a more therapeutic target for cholangiocarcinoma patients.

In this study, we conducted a comprehensive analysis of genome profiles of 43 cholangiocarcinoma patients, systematically analyzed the gene alternation in cholangiocarcinoma patients, and investigated their clinicopathological significance of these genomic features, thus providing clues to explore potential therapeutic targets for cholangiocarcinoma.

Patients and sample

In this retrospective study, a total of 43 cholangiocarcinoma patients were enrolled. The baseline clinicopathological characteristics are presented in Table 1. Tumor tissue samples were collected at the initial diagnosis (baseline) and disease progression by resection or needle biopsy. The samples are stored as formalin-fixed paraffin-embedded (FFPE) samples. Next-generation sequencing (NGS) testing was carried out by Burning Rock Biotech. This study was approved by the institutional review board of Zhongshan Hospital and carried out in accordance with the Declaration of Helsinki. Informed consent was obtained from each patient.

Table 1

Clinicopathological characteristics of the study population (n = 43)
	Overall (n = 43)	Ampullary (n = 5)	Extrahepatic (n = 32)	Intrahepatic	Pancreatic duct
Gender
Female	17(39.53%)	2(40.00%)	10	5	0
Male	26(60.47%)	3(60.00%)	22	0	1
Age
Mean (SD)	62.23(9.84)	66(8.08)	60.88	67.6	60
Median (IQR)	62.00[57.00,69.50]	66.00[62.00,74]	62.00	65	60
CA199_baseline
High	29(67.44%)	4[80.00%]	21	4	0
Low	14(32.56%)	1[20.00%]	11	1	1
Tumor site
Distal	26(60.47%)	5[100.0%]	20	0	1
Intrahepatic	5(11.63%)	0[0%]	0	5	0
Perihilar	12(27.91%)	0[0%]	12	0	0
Division degree
Low	3(6.98%)	0[0%]	2	0	1
Median	39(90.70%)	4[80.00%]	30	5	0
Missing	1(2.33%)	1[20.00%]	0	0	0
Organs_ invasion
-	8	1[20.00%]	7	0	0
+	28	4[80.00%]	22	1	1
Missing	7	0[0%]	3	4	0
Nerve invasion
-	1	0	1	0	0
+	35	4	26	4	1
Missing	7	1	5	1	0
Lymph metastasis
No	25	5	17	3	0
Yes	16	0	15	0	1
Missing	2	0	0	2	0
Stage
I	4	1	1	2	0
II	27	4	22	1	0
III	9	0	7	1	1
IV	2	0	2	0	0
Missing	1	0	0	1	0
Ki67
High	25	3	19	2	1(100%)
Low	17	2	12	3	0(0%)
Missing	1	0	1	0	0
CA19-9_P0
-	2(4.65%)	0(0.00%)	1(3.12%)	1(20.00%)	0
+	13(30.23%)	1(20.00%)	11(34.38%)	1(20.00%)	0
Missing	28(65.12%)	4(80.00%)	20(62.50%)	3(60.00%)	1(100%)

Tissue DNA isolation, capture-based targeted DNA sequencing, and sequence data analysis

This procedure was carried out as previously described[9]. Details were listed in the Additional files (see Additional file 1).

Determination of potentially actionable targets (PATs) in BTCs

To determine the potential actionabilities of genetic alterations in druggable targets, the two-leading evidence-based knowledge databases, OncoKB [15] and ESMO Scale for Clinical Actionability of molecular Targets (ESCAT) [16] were referenced. OncoKB[10] is a precision oncology knowledge base that comprehensively considers the guidelines and recommendations from the FDA, National Comprehensive Cancer Network (NCCN), and medical literature. The ESCAT[11] was launched by the European Society for Medical Oncology (ESMO) Translational Research and Precision Medicine Working Group to facilitate the implementation of precision medicine in the clinical management of cancer. In our study, we defined PATs as genomic alterations classified according to the scoring system of OncoKB (tier ≤ level-3A) or ESCAT (tiers ≤ II-B), and their matched targeted therapies have shown compelling clinical efficacy in treating BTC or other tumors. Consequently, a total of 19 genes could be classified as PATs in BTCs.

Statistical analysis

All statistical analyses were performed using R version 3.5.7. Normally distributed continuous variables are presented in the mean ± SD form; otherwise, they are presented in the form of the median with the interquartile range. A two-tailed unpaired t-test and fisher’s exact test were carried out as appropriate. Kaplan-Meier was used to select prognostic factors related to DFS and OS. The R package “ggplot2” was implicated to draw figures. A two-tailed P < 0.05 was considered statistically significant.

The baseline information and the genetic profile of cholangiocarcinoma patients

A total of 43 patients were selected for this study. Detailed clinical information were displayed in Table 1. Among 43 patients, 5 patients were diagnosed with intrahepatic cholangiocarcinoma, 32 patients were diagnosed with extrahepatic cholangiocarcinoma, 5 patients were diagnosed with ampullary carcinoma, and 1 patient was diagnosed with pancreatic duct carcinoma..

Besides, in the genetic analysis, a total of 8 kinds of genetic alterations were detected, including missense, copy number deletion, copy number amplification, indel, stop gained, frameshift, splice site, and gene fusion (Fig. 1A). Our data suggested that among 43 patients, gene alterations were observed in 38 patients, with a positive rate of 88.4%. The most commonly mutated genes included KRAS (44.2%), TP53 (37.2%), ARID1A (18.6%), SMAD4 (18.6%), CDKN2A (14.0%), BRAC2(11.6%), VEGFA (11.6%), KEAP1(9.3%), MYC (9.3%), and STK11(9.3%) (Fig. 1B). Only five patients harbor no somatic mutation. Among the top altered genes in our study, most high-frequency mutated genes are following previous sequencing results (Table 2). Of note, most mutations are enriched in PI3K/AKT, ERBB, MAPK, and RAS signaling ( see Additional file 2), indicating that these signaling might play essential roles in the pathological process of cholangiocarcinoma.

Table 2

Top mutated genes in cholangiocarcinoma in different cohorts.
	TCGA cohort (N = 36)	ICGC cohort (N = 489)	MSK cohort (N = 192)	Our study (N = 43)
Top mutated genes	PBRM1(19.4%)	TP53 (35.3%)	IDH1 (24.6%)	KRAS (44.2%)
	BAP1(19.4%)	ARID1A (19.2)	TP53 (23.6%)	TP53 (37.2%)
	IDH1(13.9%)	KRAS (18.2%)	ARID1A(20.5%)	ARID1A (18.6%)
	TTN(11.1%)	SMAD4 (14.4%)	BAP1(13.3%)	SMAD4(18.6%)
	EPHA2(11.1%)	SYNE1(11.0%)	KRAS(11.8%)	CDKN2A (14%)
	ARID1A(11.1%)	MUC16(10.6%)	PBRM1(10.3%)	BRCA2(11.6%)
	DNAH5(11.1%)	LRP1B(9.4%)	SMAD4(8.2%)	VEGFA (11.6%)
	KMT2C(11.1%)	BAP1(9.4%)	ATM(7.7%)	KEAP1(9.3%)
	TP53(11.1%)	FSIP2(8.9%)	PIK3CA (6.7%)	MYC (9.3%)
	ADGRV1(8.3%)	EPHA2(8.6%)	KMT2C (6.7%)	STK11(9.3)

In cholangiocarcinoma, the genetic mutation is closely associated with the clinicopathological feature.

After depicting the genomic profiling of cholangiocarcinoma, we investigated the associations between the genetic mutation and clinicopathological features (Figure 2A). At first, we investigated the correlations among the clinicopathological features, which turned out that patients with extrahepatic cholangiocarcinoma had a higher rate of lymph node metastasis, and all intrahepatic cholangiocarcinoma patients were female （Figure 2B）. Besides, distal cholangiocarcinoma is more common in male patients and male patients are diagnosed at earlier ages （Figure 2C）. In terms of the clinical significance of the Ki67 index, our data indicated that patients with a higher Ki67 index have a higher rate of lymph node metastasis. As for the correlation between the Ki67 index and tumor site, it was observed that distal cholangiocarcinoma had a higher Ki67 index than other subtypes of cholangiocarcinoma （P=0.002; Figure 2D）. Next, we analysis the correlation between the TNM stage and tumor site, which suggested that patients with distal cholangiocarcinoma were diagnostic with a relative earlier stage (Figure 2E). Moreover, we found that the CA19-9 level was positively related with the rate of organ invasion, suggesting that CA19-9 level was associated with the tumor invasion capacity in cholangiocarcinoma (Figure 2F).

As for the correlation between the gene mutation and the clinical information (Figure 3A), we found that the STK11 mutation was related to the tumor site, this mutation was mainly observed in the intrahepatic cholangiocarcinoma. Besides, we found out that TP53 mutation was associated with a low division degree (P=0.040)（Figure 3B）. Moreover, we observed that ERBB2 mutation was closely related to the organ invasion and TNM stage（Figure 3C）. These results suggested that these mutations might contribute to the pathological process of cholangiocarcinoma. Moreover, the correlations between the genetic mutations were presented in Figure 3D, reflecting the complex correlation between genetic mutations.

The prognostic significance of specific genetic mutation

We further investigated the prognostic value-specific genetic mutation, especially commonly altered genes, including ARID1A, BRCA2, CDKN2A, ERBB2, ERBB3, KEAP1, KRAS, MET, MSH6, MYC, NRAS, RET, RNF43, SMAD4, STK11, TP53 and VEGFA (Figure 4A). Kaplan-Meier analysis was applied to evaluate the prognostic significance of these genes, and it turned out that ERBB3, MYC, STK11, TP53, and KRAS were related to the prognosis of these patients. To be specific, patients with ERBB3 mutation have shorter RFS (P=0.007, Hazard ration (HR) [95% Confidence interval (CI)] =7.00 [1.34-36.55]) (Figure 4B). Besides, patients with MYC mutation have worse RFS (P=0.007, HR [95%CI] = 6.23 [1.37-28.39]) and OS (P=0.010, HR [95%CI] =7.57 [1.24-46.14]) (Figure 4C). Meanwhile, patients with STK11 mutation have worse RFS (P<0.001, HR [95%CI] =29.18 [2.94-289.77]) and OS (P<0.001, HR (95%CI) =18.79 [3.33-106.15]) (Figure 4D). The mutation types of TP53 and KRAS genes were presented in Figure 4E. Of note, patients with TP53 mutation have worse RFS and OS (Figure 4F). A similar result was observed for the patients with KRAS mutation (Figure 4G). Taken together, all these data indicated that the genetic feature could serve as a potential prognostic biomarker for cholangiocarcinoma patients.

PAT analysis

Massive efforts have been implicated to translate genomic alterations into actionable targets. In this study, we defined PATs as genomic alterations classified by the scoring system of OncoKB (tier ≤level-3A) or the ESCAT (tier≤II-B)， as their matched therapies have shown compelling clinical efficacy in treating BTC and other tumors. In this study, we propose 19 genes as PATs in BTC. In this context, 34 (71.4%) patients harbored at least one PAT（Figure 5A）. The most commonly detected PAT is the KRAS mutation (23.8%), followed by the TP53 mutation (20.2%), and the missense mutation was the most commonly detected mutation type (48.8%) (Figure 5B). These data indicated that this 168-gene panel could provide clues for the targeted therapy of cholangiocarcinoma patients.

In this study, a total of 43 cholangiocarcinoma patients were included and DNA-targeted sequencing of cancer-associated genes was performed. Among 43 patients, the genomic mutation was detected in 38 patients. Among these mutations, KRAS (44.2%), TP53(37.2%), ARID1A (18.6%), SMAD4(18.6%), BRCA2(11.6%), CDKN2A (11.6%), and VEGFA (11.6%) are the top frequently altered cancer-related genes. Besides, germline mutations occurred mostly in PI3K/AKT, ERBB, MAPK, and RAS signaling. Among detected mutations, we found that TP53, STK11, MYC, and ERBB3 are gene alternations with significant prognostic values. In terms of potentially actionable target (PAT) analysis, 19 genes were proposed to be PATs in BTCs. and we found out that 79.1% of patients have Tier II somatic mutation in our cohort.

Among the top altered genes in our study, most high-frequency mutated genes following previous studies, like KRAS, TP53, ARID1A, and SMAD4[12–16]. Despite these genes, the mutated frequency of several genes is higher in our cohort, including BRCA2, KEAP1, MYC, and STK11. These gene mutations have been reported as a potential target for cholangiocarcinoma. For mutated BRCA2, increasing evidence indicated that germline or somatic BRCA mutations are being detected in BTC, and these mutations have been shown to cause defective repair mechanisms through homologous recombination (HR) for double-strand DNA breaks[17, 18]. Herein, patients with mutated BRCA1/2 were associated with an increased sensibility to DNA-damaging therapies in other types of tumors [19]. Recent in vitro experimental data suggested that Poly (ADP-ribose) polymerase (PARP) inhibitors could selectively suppress the growth of tumor cells with mutated BRCA and PARP inhibitors were increasingly being used in BRCA-mutated malignancies like ovarian, breast, prostate, and pancreatic cancer [20, 21]. For advanced BTC patients, a case report reported the efficacy of PARP inhibitors in patients with BRCA-mutated advanced BTC with overall survival ranging from 11 to 65 months[22]. In terms of the STK11 mutation, it has been reported that suppressing the expression of STK11 could promote the progression and the expression level of PD-L1 in cholangiocarcinoma, indicating that STK11 mutation is a potential target for clinical diagnosis and treatment for cholangiocarcinoma[23]. Besides, the STK11 mutation has been reported as a therapeutic target for lung cancer[24, 25]. KEAP1 is an important tumor suppressor factor[26]. Recently, it is reported that in the mice model of cholangiocarcinoma, KEAP1 deletion could promote the formation of invasive cholangiocarcinoma, indicating that KEAP1 might be a potential therapeutic target for cholangiocarcinoma patients[27]. In addition, KEAP1 has been reported as a predictive factor for the response of immune checkpoint inhibitors in lung cancer[28]. Currently, the administration of immune checkpoint inhibitors (ICI) is a hotspot in cholangiocarcinoma, and the exploration of a potential biomarker to predict response to ICI is essential.

In terms of the prognostic significance of the mutated genes, we observed that TP53, STK11, MYC, KRAS, and ERBB3 were related to the prognosis of cholangiocarcinoma. Consistently to previous studies, patients with mutated TP53[29] or mutated KRAS[13] have shorter recurrence-free survival and overall survival.

In our study, we found that most mutated genes were enriched in the PI3K/AKT[30], ErbB [31, 32], MAPK[33], and RAS signaling indicating that these pathways might be involved in the pathological process of cholangiocarcinoma patients. Previously, it has been reported that these pathways were related to the progression of cholangiocarcinoma. Our data further validated the previous conclusion and indicated that these pathways were potential drug targets for cholangiocarcinoma patients.

However, there were several limitations in this study. First, the patient number included in this study is relatively small. Secondly, further investigation should be carried out to explore the clinical significance and biological function of specific gene mutations in cholangiocarcinoma.

In this study, we uncovered the genomic feature of cholangiocarcinoma patients and found potential drug targets for cholangiocarcinoma patients. In addition, nearly 90% of patients were identified with potentially actionable alterations, which supported that molecular profiling could be applied in clinical practice to change the management of cholangiocarcinoma in the future.

Ethics approval and consent to participate

This study was approved by the institutional review board of Zhongshan Hospital and carried out in accordance with the Declaration of Helsinki. Informed consent was obtained from each patient.

Consent for publication

Not applicable.

Availability of data and materials

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

Funding

This study was supported by grants from the National Natural Science Foundation of China (82272772,81872352, and 82072682), Natural Science Foundation of Shanghai Municipality (21ZR1459100, and 22ZR1457900), Science and Technology Commission of Shanghai Municipality(20DZ2254500), and Shanghai Anticancer Association EYAS PROJECT.

Acknowledgments

None

Author contributions

Tao Suo and Yuan Ji designed and supervised the study. B-H Zheng, Jing Han, and Sheng Shen performed data collection and analysis. Z-Z Jiang, Rui Peng, and J-R Cai collected and managed patient samples. H-B. Liu and B-H Zheng wrote and reviewed the manuscript. All authors read and approved the final manuscript.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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No competing interests reported.

Addtionalfile1.docx
Additional file 1: Supplementary materials Detail information of the sequence method.
Addtionalfile2.jpg
Additional file 2: Figure S1 Gene enrichment analysis for high-frequency mutated analysis.

Genomic feature and potential therapeutic target for cholangiocarcinoma

Status:

Version 1

Abstract

Figures

Introduction

Material And Method

Patients and sample

Tissue DNA isolation, capture-based targeted DNA sequencing, and sequence data analysis

Determination of potentially actionable targets (PATs) in BTCs

Statistical analysis

Results

The baseline information and the genetic profile of cholangiocarcinoma patients

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1