Sage Journals: Discover world-class research

Abstract

Background:

With a 15% incidence, KRAS is one of the most common mutations in biliary tract cancer (BTC) and is a poor prognostic factor. Immune checkpoint inhibitors (ICIs) as salvage therapy have modest activity in BTC.

Objectives:

There are limited data on the efficacy of ICIs according to KRAS mutation in BTC. We evaluated the efficacy of ICIs in BTC patients with or without KRAS mutations.

Design:

Retrospective observational study.

Methods:

We conducted molecular profiling in BTC patients who received ICIs as salvage therapy. The expression of programmed death ligand 1 (PD-L1) on tumor cells was assessed using immunohistochemistry. The TruSight^TM Oncology 500 assay from Illumina was used as a cancer panel. We analyzed overall survival (OS) and progression-free survival (PFS) of ICI in BTC patients according to KRAS mutation and PD-L1 expression.

Results:

A total of 62 patients were included in this analysis. The median age was 68.0 years; 47 patients (75.8%) received pembrolizumab and 15 (24.2%) received nivolumab as salvage therapy. All patients received gemcitabine plus cisplatin as the frontline therapy, and 53.2% received fluoropyrimidine plus oxaliplatin (FOLFOX) before ICI. The median number of lines of prior chemotherapy was 2.5. The KRAS mutation was found in 13 patients (19.1%), and 28 patients (45.2%) showed 1% or more of tumor cells out of visible tumor cells positive for PD-L1. There was no statistical correlation between KRAS mutation and PD-L1 expression. The median OS and PFS with ICI were 5.6 [interquartile range (IQR): 3.3–8.0] and 3.8 (IQR: 3.0–4.5) months, respectively. There were no statistically significant differences in PFS with ICIs according to KRAS mutation (mutant type versus wild type) and PD-L1 expression (positive versus negative). In subgroup analysis, patients with both KRAS mutation and PD-L1 positivity had longer PFS compared with patients with KRAS mutation and PD-L1 negativity (10.1 versus 2.6 months, p = 0.047). This finding was not shown in patients with wild-type KRAS.

Conclusion:

Our analysis suggested that PD-L1 expression might be a useful biomarker for ICIs in BTC patients with KRAS mutation but not in those with wild-type KRAS.

Keywords

biliary tract cancer immune checkpoint inhibitor KRAS mutation PD-L1

Introduction

Kirsten rat sarcoma viral oncogene homolog (KRAS) is one of the most common oncogenes among all cancers. KRAS has been thought to be an undruggable target; however, recently, KRAS-targeting agents, which are designed to target the KRAS p.G12C mutation, received approval based on encouraging clinical results. With the start of the success of sotorasib in non-small-cell lung cancer (NSCLC),¹ sotorasib showed improved treatment outcomes in previously treated pancreatic cancer with 21% of objective response and 6.9 months of overall survival (OS).² Adagrasib, another agent targeting KRAS p.G12C mutation, also proved clinical efficacy in previously treated colorectal cancer with or without cetuximab.³

The KRAS protein can activate multiple signaling pathways, including the rapidly accelerated fibrosarcoma, mitogen-activated protein kinase (MEK), extracellular regulated protein kinases signaling pathway, and the phosphoinositide 3-kinase/protein kinase B (Akt)/mammalian target of rapamycin signaling pathway. KRAS pathway activation leads to cell proliferation, differentiation, and migration and inhibits apoptosis, all of which are hallmarks of cancers.⁴ During pathway activation, many inflammatory cytokines and chemokines are secreted, facilitating an inflammatory microenvironment and inducing oncogenesis, immune escape, and evasion.⁵

Biliary tract cancer (BTC) has a high incidence of KRAS mutation (12.7%⁴), and KRAS is a poor prognostic factor for BTC.⁶ Despite the only 10-month median survival of gemcitabine plus cisplatin (GP), the first-line standard therapy for BTC has not changed for more than 10 years.⁷ Recently, TOPAZ-1 trial (Durvalumab or Placebo in Combination With Gemcitabine/Cisplatin in Patients With 1st Line Advanced Biliary Tract Cancer) showed that adding durvalumab to GP increased the median OS to 12.8 months.⁸ However, there is few data for the efficacy of immune checkpoint inhibitors (ICIs) according to KRAS mutation in BTC. Herein, we analyzed the efficacy of ICIs according to KRAS mutation in BTC.

Method

Patient selection

This analysis retrospectively included patients who were diagnosed with BTC and received ICI as salvage therapy between March 2020 and August 2022 at our institute. Also, all analyzed patients had available data on programmed death ligand 1 (PD-L1) expression and next-generation sequencing. The following clinicopathologic characteristics were analyzed: age, sex, performance status, primary tumor site, disease status, disease classification, treatment history, and response to ICI.

Immunohistochemistry of PD-L1

The expression of PD-L1 on tumor cells was assessed using immunohistochemistry (IHC). Tumor samples obtained through endoscopic biopsy, percutaneous needle biopsy, or surgical resection at initial diagnosis or progression were used. Tissue sections were freshly cut into 4-μm sections, mounted on Fisherbrand Superfrost Plus Microscope Slides (Thermo Fisher Scientific, Waltham, MA, USA), and dried at 60°C for an hour. IHC staining was carried out on a Dako Autostainer Link 48 system (Agilent Technologies, Santa Clara, CA, USA) using a Dako PD-L1 IHC 22C3 PharmDx kit (Agilent Technologies, Santa Clara, CA, USA) with an EnVision FLEX visualization system. Then the samples were counterstained with hematoxylin according to the manufacturer’s instructions. PD-L1 expression was considered positive if it was observed in 1% or more of tumor cells out of visible tumor cells.⁹

TruSight^TM oncology 500 assay

Forty (40) ng of DNA was quantified with the Qubit dsDNA HS Assay (Thermo Fisher Scientific, Waltham, MA, USA) on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and then sheared using a Covaris E220 Focused-ultrasonicator (Woburn, MA, USA) and the 8 microTUBE–50 Strip AFA Fiber V2 following the manufacturer’s instructions. The treatment time was optimized for formalin-fixed paraffin-embedded material. The treatment settings were as follows: peak incident power (W): 75; duty factor: 15%; cycles per burst: 500; treatment time (s): 360; temperature (°C): 7; water level: 6. For DNA library preparation and enrichment, the TruSight^TM Oncology 500 Kit (Illumina Inc., San Diego, CA, USA) was used following the manufacturer’s instructions. Post-enriched libraries were quantified, pooled, and sequenced on a NextSeq 500 (Illumina Inc., San Diego, CA, USA). The quality of the NextSeq 500 (Illumina) sequencing runs was assessed with the Illumina Sequencing Analysis Viewer (Illumina Inc., San Diego, CA, USA). Sequencing data were analyzed with the TruSight Oncology 500 Local App Version 1.3.0.39 (Illumina Inc., San Diego, CA, USA). The TruSight^TM Oncology 500 is a comprehensive tumor profiling assay designed to identify known and emerging tumor biomarkers, including small variants, splice variants, and fusions. Importantly, the TruSight^TM Oncology 500 measures tumor mutational burden (TMB) and microsatellite instability (MSI), features that are potential key biomarkers for immunotherapy. TMB was reported as mutations per megabase (Mb) sequenced, and high TMB was defined as more than 10 mutations per Mb (⩾10 Mut/Mb).

Statistical analysis

The clinical features and treatment outcomes were analyzed, and categorical variables were evaluated using the chi-square test and Fisher’s exact test. OS was defined as the time from the first day of ICI to death from any cause, and living patients were censored at the time of analysis. Progression-free survival (PFS) was defined as the time from the first day of ICI to the date of confirmed progressive disease according to RECIST v1.1. We analyzed OS and PFS of ICI in BTC patients according to PD-L1 expression and KRAS mutation. Survival was estimated based on Kaplan–Meier curves and compared using a log-rank test. p Values from two-sided statistical tests were considered statistically significant at p < 0.05. Survival analyses were performed using IBM PASW version 24.0 software (SPSS Inc., Chicago, IL, USA).

We have followed TRIPOD guidelines. The study was conducted and reported according to the TRIPOD (Transparent Reporting of a multivariable prediction model for Individual Prognosis Or Diagnosis) statement.¹⁰

Results

Clinical features

A total of 62 BTC patients were included in this analysis. In all, 38 (61.3%) patients were male, and the median age was 68.0 [interquartile range (IQR): 59.3–73.7] years. The primary site was intrahepatic in 31 (50.0%) patients, extrahepatic in 21 (33.9%), and gallbladder in 10 (16.1%). Overall, 55 (88.7%) patients had metastatic disease, and 7 (11.3%) had locally advanced disease when they were considered in a palliative setting. Pembrolizumab was administered as a salvage therapy in 47 patients (75.8%), and nivolumab was given in 15 (24.2%). All patients received GP as the frontline therapy, and 53.2% received fluoropyrimidine plus oxaliplatin (FOLFOX) before ICIs. The median number of lines of prior chemotherapy was 2.5. TMB-high tumors were found in 9 (14.5%) patients and TMB-low tumors in 47 (75.8%) patients. Only one (1.6%) patient had an MSI-H tumor (Table 1).

Table 1.

Clinicopathologic characteristics of the patients.

Characteristics	Total number (%) n = 62	KRAS MT n = 13	KRAS WT n = 49
Male sex	38 (61.3)	11 (84.6)	27 (55.1)
Age, median (IQR)	68.0 (59.3–73.7)	68.7 (59.3–72.3)	67.8 (59.0–74.3)
ECOG
0, 1	59 (95.2)	13 (100)	46 (93.9)
⩾2	3 (4.8)	0 (0)	3 (6.1)
Primary tumor site
Intrahepatic cholangiocarcinoma	31 (50.0)	6 (46.2)	25 (51.0)
Extrahepatic cholangiocarcinoma	21 (33.9)	7 (53.8)	14 (28.6)
Gallbladder	10 (16.1)	0 (0)	10 (20.4)
Disease status
Initially unresectable	34 (54.8)	6 (46.2)	28 (57.1)
Recurrent	28 (45.2)	7 (53.8)	21 (42.9)
Disease classification
Locally advanced	7 (11.3)	0 (0)	7 (14.3)
Metastatic	55 (88.7)	13 (100)	42 (85.7)
Latest lines of chemotherapy
2L	31 (50.0)	6 (46.2)	25 (51.0)
3L	25 (40.3)	7 (53.8)	18 (36.7)
4, 5L	6 (9.7)	0 (0)	6 (12.2)
Prior chemotherapy
GP	62 (100.0)	13 (100)	49 (100.0)
Fluoropyrimidine	33 (53.2)	6 (46.2)	27 (55.1)
Other immunotherapy	6 (9.7)	1 (7.7)_	5 (10.2)
Types of immunotherapy
Pembrolizumab	47 (75.8)	11 (84.6)	36 (73.5)
Nivolumab	15 (24.2)	2 (15.4)	13 (26.5)
TMB
High	9 (14.5)	2 (15.4)	7 (14.3)
Low	47 (75.8)	10 (76.9)	37 (75.5)
Not assessed	6 (9.7)	1 (7.7)	5 (10.2)
MSI status
High	1 (1.6)	0 (0)	1 (2.0)
Stable	55 (88.7)	12 (92.3)	43 (87.8)
Not assessed	6 (9.7)	1 (7.7)	5 (10.2)

ECOG, Eastern Cooperative Oncology Group; GP, gemcitabine + cisplatin; IQR: interquartile range; MSI, microsatellite instability; MT, mutant type; TMB, tumor mutation burden; WT, wild type.

KRAS mutation and PD-L1 expression

The KRAS mutation was found in 13 patients (19.1%). The types of mutations were as follows: G12A in 2 (15.4%) patients, G12C in 1 (7.7%), G12D in 3 (23.1%), G12R in 1 (7.7%), G12V in 2 (15.4%), G13D in 1 (7.7%), Q61H in 1 (7.7%), Q61R in 1 (7.7%), and R68S in 1 (7.7%). We summarized the clinical features of patients with KRAS mutation in Figure 1. In these patients, PD-L1 positivity was detected in 28 (45.2%), showing no statistical correlation between KRAS mutation and PD-L1 expression (p = 0.589, Table 2).

Figure 1.

Summary of the clinicopathologic characteristics of patients with KRAS mutation.

Table 2.

PD-L1 status and KRAS status.

PD-L1 Status/KRAS status	KRAS MT	KRAS WT	Total
PD-L1 (+)	6	22	28 (45.2)
PD-L1 (−)	7	27	34 (54.8)
Total	13 (19.1)	49 (79.0)	62 (p = 0.589)

MT, mutant type; PD-L1, programmed death ligand 1; WT, wild type.

Treatment outcomes according to PD-1 expression, KRAS mutation, and TMB

The median OS and PFS with ICI were 5.6 (IQR: 3.3–8.0) and 3.8 (IQR: 3.0–4.5) months, respectively. Between mutant and wild-type KRAS, there were no statistically significant differences in OS and PFS with ICIs (OS; 5.0 versus 5.6 months, p = 0.775, PFS; 4.0 versus 3.8 months, p = 0.724, Figure 2(a)). The median OS and PFS between patients with PD-L1 positivity and those with PD-L1 negativity were not statistically different (OS; 6.1 versus 5.2 months, p = 0.812, PFS; 3.6 versus 4.1 months, p = 0.785, Figure 2(b)). However, in patients with KRAS mutation, those who were also PD-L1 positive had longer PFS than patients who were PD-L1 negative (10.1 versus 2.8 months, p = 0.047) (Figure 3(a)). This finding was not observed in patients with wild-type KRAS (Figure 3(b)). The tumor responses to ICI are presented in Tables 3 to 5.

Figure 2.

(a) OS and (b) PFS of patients with KRAS wild-type or mutation-type tumors. (c) OS and (d) PFS of patients with positive or negative PD-L1 expression.

Figure 3.

Subgroup analysis. (a) OS and (b) PFS of patients with KRAS mutation type according to PD-L1 expression. (c) OS and (d) PFS of patients with KRAS wild type according to PD-L1 expression.

Table 3.

Treatment response according to KRAS status.

Characteristics	Total number (%) n = 47	KRAS MT n = 9	KRAS WT n = 38
Best overall response
Complete response	1 (2.1)	0 (0)	1 (2.6)
Partial response	4 (8.5)	2 (25.0)	2 (5.1)
Stable response	28 (59.6)	4 (50.0)	24 (61.5)
Progressive disease	14 (29.8)	2 (25.0)	12 (30.8)
Overall response	5 (10.6)	2 (25.0)	3 (7.7)
Disease control rate	33 (70.2)	6 (75.0)	27 (71.1)

MT, mutant type; WT, wild type.

Table 4.

Treatment response according to PD-L1 status.

Characteristics	Total number (%) n = 47	PD-L1 (+) n = 21	PD-L1 (−) n = 27
Best overall response
Complete response	1 (2.1)	1 (4.8)	0 (0)
Partial response	4 (8.5)	3 (14.3)	1 (3.8)
Stable response	28 (59.6)	14 (66.7)	14 (53.8)
Progressive disease	14 (29.8)	3 (14.3)	11 (42.3)
Overall response	5 (10.6)	4 (19.0)	1 (3.8)
Disease control rate	33 (70.2)	18 (85.7)	15 (55.6)

PD-L1, programmed death ligand 1.

Table 5.

Treatment response according to KRAS and PD-L1 status.

Characteristics	Total number (%) n = 48	KRAS MT and PD-L1 (+) n = 3	KRAS MT and PD-L1 (−) n = 5	KRAS WT and PD-L1 (+) n = 18	KRAS WT and PD-L1 (−) n = 21
Best overall response
Complete response	1 (2.1)	0 (0)	0 (0)	1 (5.6)	0 (0)
Partial response	4 (8.5)	2 (66.6)	0 (0)	1 (5.6)	1 (4.8)
Stable response	28 (59.6)	1 (33.3)	3 (75.0)	13 (72.2)	11 (52.4)
Progressive disease	14 (29.8)	0 (0)	1 (25.0)	3 (16.76)	9 (42.9)
Overall response	5 (10.6)	2 (66.6)	0 (0)	2 (11.1)	1 (4.8)
Disease control rate	33 (70.2)	4 (100)	3 (75.0)	14 (77.8)	12 (57.1)

MT, mutant type; PD-L1, programmed death ligand 1; WT, wild type.

There were no statistically significant differences in OS and PFS between TMB-high and TMB-low tumors (OS; 10.3 versus 4.5 months, p = 0.235, PFS; 5.7 versus 4.5 months, p = 0.333).

Discussion

Among the 62 BTC patients herein, the KRAS mutation was found in 14 (22.6%), and PD-L1 positivity was detected in 28 (45.2%). There was no statistical correlation between KRAS mutation and PD-L1 expression. In patients with PD-L1 expression, those who also had KRAS mutation showed longer PFS with ICIs compared with patients with PD-L1 expression only. However, this difference was not observed in patients with wild-type KRAS. This finding suggests that PD-L1 expression might be a novel predictive marker for ICIs, especially in BTC patients with KRAS mutation.

PD-L1 expression has been used as a potential predictive biomarker for ICI. Nonetheless, negative PD-L1 expression does not mean that patients would not respond to ICI. We observed that PD-L1 status did not affect survival including OS or PFS of patients. Similarly, in a phase 2 study of nivolumab in refractory BTC patients, PD-L1 expression was not correlated with survival.⁹ The TOPAZ-1 trial showed similar efficacy between PD-L1-positive and PD-L1-negative patients.⁸

There are some data showing a correlation between KRAS mutation and PD-L1 expression. In NSCLC, several studies have reported significantly higher PD-L1 expression in NSCLC patients with KRAS mutation,^11–15 and the mechanisms are being researched.^16,17 It is not revealed much in BTC; however, some studies showed that KRAS mutation induces the expression of PD-L1.^18,19 Our data did not show any correlation between KRAS and PD-L1 in BTC patients; However, as this study only included small number of patients, further studies would be needed.

In this analysis, patients with both KRAS mutation and PD-L1 expression had better PFS than patients with PD-L1 expression and wild-type KRAS. Some studies have suggested KRAS mutation as a potential biomarker for ICI use. A meta-analysis reported that ICI as salvage therapy improved OS in NSCLC patients with KRAS mutation but not in NSCLC patients with wild-type KRAS.^20,21 Currently, combination treatments with KRAS inhibitors are being tried, such as those with agents targeting related singling pathways such as RTK, SHP2, SOS1, or MEK.⁴ As KRAS mutation fosters an immunosuppressed tumor microenvironment (TME), KRAS inhibitors change the TME from immunosuppressed to immunoreactive.^5,22 Therefore, a combination of KRAS inhibitors and ICI should be tried.

In conclusion, we observed that KRAS mutation and PD-L1 expression did not predict the efficacy of ICI in patients with BTC. However, PD-L1 expression might be a useful biomarker of ICI use in BTC patients with KRAS mutation but not in those with wild-type KRAS. Furthermore, we suggest that treatment with ICI plus anti-KRAS therapy might be valuable in BTC. However, as this study only included a small number of patients, further studies would be needed.

Supplemental Material

sj-docx-1-tag-10.1177_17562848231170484 – Supplemental material for The efficacy of immune checkpoint inhibitors in biliary tract cancer with KRAS mutation

Supplemental material, sj-docx-1-tag-10.1177_17562848231170484 for The efficacy of immune checkpoint inhibitors in biliary tract cancer with KRAS mutation by Sun Young Jeong, Jung Yong Hong, Joon Oh Park, Young Suk Park, Ho Yeong Lim, Jae Yeon Jang, Youngkyung Jeon and Seung Tae Kim in Therapeutic Advances in Gastroenterology

Footnotes

Acknowledgements

None.

Declarations

ORCID iD

Sun Young Jeong

Supplemental material

Supplemental material for this article is available online.

References

Skoulidis

, et al. Sotorasib for lung cancers with KRAS p.G12C mutation. N Engl J Med 2021; 384: 2371–2381.

Strickler

Satake

George

, et al. Sotorasib in KRAS p.G12C–mutated advanced pancreatic cancer. N Engl J Med 2022; 388: 33–43.

Yaeger

Weiss

Pelster

, et al. Adagrasib with or without cetuximab in colorectal cancer with mutated KRAS G12C. N Engl J Med 2022; 388: 44–54.

Huang

Guo

Wang

, et al. KRAS mutation: from undruggable to druggable in cancer. Signal Transduct Target Ther 2021; 6: 386.

Dias Carvalho

Guimarães

Cardoso

, et al. KRAS oncogenic signaling extends beyond cancer cells to orchestrate the microenvironment. Cancer Res 2018; 78: 7–14.

Javle

Bekaii-Saab

Jain

, et al. Biliary cancer: utility of next-generation sequencing for clinical management. Cancer 2016; 122: 3838–3847.

Valle

Wasan

Palmer

, et al. Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer. N Engl J Med 2010; 362: 1273–1281.

D-Y

Qin

, et al. Durvalumab plus gemcitabine and cisplatin in advanced biliary tract cancer. NEJM Evid 2022; 1. DOI: 10.1056/EVIDoa2200015.

Kim

Chung

Alese

, et al. A phase 2 multi-institutional study of nivolumab for patients with advanced refractory biliary tract cancer. JAMA Oncol 2020; 6: 888.

10.

Collins

Reitsma

Altman

, et al. Transparent reporting of a multivariable prediction model for individual prognosis or diagnosis (TRIPOD): the TRIPOD statement. Ann Intern Med 2015; 162: 55–63.

11.

Brody

Zhang

Ballas

, et al. PD-L1 expression in advanced NSCLC: insights into risk stratification and treatment selection from a systematic literature review. Lung Cancer 2017; 112: 200–215.

12.

Cronin-Fenton

Dalvi

Movva

, et al. PD-L1 expression, EGFR and KRAS mutations and survival among stage III unresected non-small cell lung cancer patients: a Danish cohort study. Sci Rep 2021; 11: 16892.

13.

Dong

Zhong

Zhang

, et al. Potential predictive value of TP53 and KRAS mutation status for response to PD-1 blockade immunotherapy in lung adenocarcinoma. Clin Cancer Res 2017; 23: 3012–3024.

14.

Schoenfeld

Rizvi

Bandlamudi

, et al. Clinical and molecular correlates of PD-L1 expression in patients with lung adenocarcinomas. Ann Oncol 2020; 31: 599–608.

15.

Holmes

Mahar

Lum

, et al. Real-world programmed death-ligand 1 prevalence rates in non-small cell lung cancer: correlation with clinicopathological features and tumour mutation status. J Clin Pathol 2021; 74: 123.

16.

Chen

Fang

Lin

, et al. KRAS mutation-induced upregulation of PD-L1 mediates immune escape in human lung adenocarcinoma. Cancer Immunol Immunother 2017; 66: 1175–1187.

17.

Coelho

de Carné Trécesson

Rana

, et al. Oncogenic RAS signaling promotes tumor immunoresistance by stabilizing PD-L1 mRNA. Immunity 2017; 47: 1083–1099.e6.

18.

Hashimoto

Furukawa

Hashimoto

, et al. ARF6 and AMAP1 are major targets of KRAS and TP53 mutations to promote invasion, PD-L1 dynamics, and immune evasion of pancreatic cancer. Proc Natl Acad Sci 2019; 116: 17450–17459.

19.

Gao

Chen

, et al. KRAS acting through ERK signaling stabilizes PD-L1 via inhibiting autophagy pathway in intrahepatic cholangiocarcinoma. Cancer Cell Int 2022; 22: 128.

20.

Kim

BJ.

Prognostic value of KRAS mutation in advanced non-small-cell lung cancer treated with immune checkpoint inhibitors: a meta-analysis and review. Oncotarget 2017; 8: 48248–48252.

21.

Liu

Zheng

Jin

, et al. The superior efficacy of anti-PD-1/PD-L1 immunotherapy in KRAS-mutant non-small cell lung cancer that correlates with an inflammatory phenotype and increased immunogenicity. Cancer Lett 2020; 470: 95–105.

22.

Cullis

Das

Bar-Sagi

Kras and tumor immunity: friend or foe?

Cold Spring Harb Perspect Med 2018; 8: a031849.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.09 MB

The efficacy of immune checkpoint inhibitors in biliary tract cancer with KRAS mutation