Efficacy and biomarker analysis of second-line nab-paclitaxel plus sintilimab in patients with advanced biliary tract cancer

Abstract

Biliary tract cancer (BTC) is a highly aggressive malignancy with limited second-line therapy. We conducted this phase 2 trial to evaluate the efficacy and safety of second-line nab-paclitaxel plus sintilimab in advanced BTC. Histologically confirmed advanced BTC patients with documented disease progression after first-line chemotherapy were enrolled. Subjects received nab-paclitaxel 125 mg/m² on days 1 and 8 plus sintilimab 200 mg on day 1, administered every 3 weeks. The primary end point was the objective response rate (ORR). The secondary end points were progression-free survival (PFS), overall survival (OS), and adverse reactions. Simultaneously, next-generation sequencing, programmed cell death ligand 1 immunohistochemistry and multiplex immunofluorescence of tumor-infiltrating lymphocytes were applied to explore potential biomarkers. Twenty-six subjects were consecutively enrolled. The ORR was 26.9% (7/26), including two complete responses and five partial responses, which met the primary end point. The disease control rate was 61.5% (16/26). The median PFS was 169 days (about 5.6 months, 95% confidence interval [CI] 60–278 days). The median OS was 442 days (about 14.7 months, 95% CI 298–586 days). Grade 3 treatment-related adverse events (TRAEs) were mainly anemia (27%), leukopenia (23%), neutropenia (19%), and peripheral sensory neuropathy (8%). No grade 4 or 5 TRAEs occurred. Biomarker analysis suggested that positive PD-L1 and high proportions of CD8⁺ T-cell infiltration were correlated with improved clinical outcome. Nab-paclitaxel plus sintilimab is a potentially effective and tolerable second-line regimen for advanced BTC that deserves to be studied in large-scale trials. PD-L1 status and CD8⁺ T cell infiltration might be promising biomarkers for efficacy prediction.

Abbreviations
• ASC
• active symptom control
• BTC
• biliary tract cancer
• CI
• confidence interval
• CPS
• combined positive score
• CR
• complete response
• CT
• computed tomography
• DCR
• disease control rate
• ECC
• extrahepatic cholangiocarcinoma
• GBC
• gallbladder cancer
• GemCis
• gemcitabine plus cisplatin
• HR
• hazard ratio
• ICC
• intrahepatic cholangiocarcinoma
• ICI
• immune checkpoint inhibitor
• IDH1
• isocitrate dehydrogenase 1
• MSI
• microsatellite instability status
• MSI-H
• microsatellite instability-high
• MSS
• microsatellite stable
• NA
• not available
• NGS
• next generation sequencing
• ORR
• objective response rate
• OS
• overall survival
• PD
• disease progression
• PFS
• progression-free survival
• PR
• partial response
• SD
• stable disease
• TILs
• tumor-infiltrating lymphocytes
• TPS
• tumor cell proportion score
• TRAEs
• treatment-related adverse events
• VEGFR
• vascular endothelial growth factor receptor

INTRODUCTION

Biliary tract cancer (BTC) arises from the epithelium of bile ducts and is the second most common primary hepatobiliary cancer.^1,2 Based on anatomic location, BTC is further classified into intrahepatic cholangiocarcinoma (ICC), extrahepatic cholangiocarcinoma (ECC), and gallbladder cancer (GBC). Due to the high aggressiveness of BTC, patients have a poor prognosis with a 5-year survival rate of less than 10%.³

Surgical resection is the only curative treatment option for BTC patients. Unfortunately, most BTC patients present with advanced disease at diagnosis and lose the opportunity for radical surgery. For advanced BTC, chemotherapy is the mainstay treatment approach, while immunotherapy and targeted drugs are emerging as promising therapeutic methods. The ABC-02 trial has demonstrated the regimen of gemcitabine plus cisplatin (GemCis) as a standard first-line treatment.⁴ In 2022, the phase 3 TOPAZ-1 trial reported a better effect of durvalumab plus GemCis chemotherapy compared with chemotherapy alone (median overall survival [OS] 12.8 vs. 11.5 months, hazard ratio [HR] = 0.80, 95% confidence interval [CI] 0.66–0.97, P = 0.021), which indicates the promising efficacy of immunotherapy in advanced BTC.⁵ Similar positive results have been obtained in the recently published phase 3 KEYNOTE-966 trial, which compared the efficacy of pembrolizumab plus GemCis with GemCis alone.⁶ Recently, a phase 2 trial (ChiCTR2000036652) has suggested that first-line sintilimab, an anti-PD-1 antibody approved by the State Food and Drug Administration of China for treating hepatocellular carcinoma and other malignancies, plus the GemCis regimen has promising efficacy and manageable safety in advanced BTC, with a median OS of 15.9 months.⁷

However, disease progression inevitably occurs after first-line therapy. Unfortunately, except for a small percentage of patients with certain targetable genetic alterations, such as fibroblast growth factor receptor 2 fusion^8,9 or isocitrate dehydrogenase 1 (IDH1) mutation,^10,11 second-line treatment options for most BTC cases are very limited and have disappointing efficacy. Most studies on second-line treatment for BTC patients without selective genetic mutations are phase 2 trials with small sample sizes, except for one phase 3 randomized trial (ABC-06 study) and one phase 2b randomized trial (NIFTY study).^12–14 The ABC-06 trial¹² showed that compared with active symptom control (ASC), second-line (fluorouracil, folinic acid, and oxaliplatin (FOLFOX) chemotherapy plus ASC in advanced BTC patients has very limited OS improvement (median OS 6.2 vs. 5.3 months, P = 0.031, HR = 0.69, 95% CI 0.50–0.97) but is accompanied by a higher incidence of grade 3–5 adverse events (69% vs 52%), which inhibits its broad clinical application. The NIFTY trial^13,14 suggested that second-line liposomal irinotecan plus fluorouracil and leucovorin could improve clinical outcome with median progression-free survival (PFS) of 4.2 months and median OS of 8.6 months. However, serious adverse events (such as neutropenia, abdominal pain, constipation, and diarrhea) occurred in 42% of patients receiving liposomal irinotecan and the dose of liposomal irinotecan is limited by UGT1A1 alleles. Thus, it is urgent to explore new second-line treatment regimens with better efficacy and tolerability.

Several studies have proven the effectiveness of nab-paclitaxel in advanced BTC.^15–20 A phase 2 trial has suggested that first-line treatment with nab-paclitaxel and gemcitabine plus cisplatin (GAP) is effective in advanced BTC, with a median PFS of 11.8 months and a median OS of 19.2 months.¹⁵ Although phase 3 SWOG 1815 trial has not proven statistically significant improvement in median OS with GAP versus GemCis regimen, but patients with locally advanced disease and GBC may benefit from GAP with a longer OS tendency (median OS in locally advanced disease 19.2 vs. 13.7 months, P = 0.09; in GBC 17.0 vs. 9.3 months, P = 0.33).¹⁹ A real-world study conducted by Cheon et al.¹⁶ showed that second-line nab-paclitaxel addition to GemCis chemotherapy after GemCis failure is a promising option with an objective response rate (ORR) of 16.0%, a median PFS of 4.4 months, and a median OS of 7.3 months. In addition, recent studies have indicated that nab-paclitaxel can enhance the antitumor activity of immune checkpoint inhibitors (ICIs), nab-paclitaxel benefits from its nanostructure.^21,22 Clinical trials have confirmed the efficacy superiority of nab-paclitaxel combined with ICIs in certain cancers, including breast cancer and urothelium carcinoma.^23,24

Given the effect of ICIs and nab-paclitaxel in advanced BTC, we conducted this phase 2 trial to investigate the therapeutic efficacy and tolerability of second-line nab-paclitaxel plus sintilimab in advanced BTC patients. As favorable predictive biomarkers are lacking for immunotherapy in BTC, we also tried to investigate potential biomarkers for efficacy prediction.

METHODS Study design and patient selection

The NapaSinti trial was an investigator-initiated, prospective, single-arm, phase 2 study. The study protocol was approved by the Ethics Committee on Biomedical Research, West China Hospital of Sichuan University. This study was conducted in accordance with the principles of the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice guidelines.

The main inclusion criteria were histologically confirmed advanced BTC, age 18–75 years, life expectancy of ≥3 months, Eastern Cooperative Oncology Group performance status score 0 or 1, documented disease progression after prior gemcitabine or fluorouracil-based systemic chemotherapy, not received Taxol drug treatment, including but not limited to paclitaxel, paclitaxel liposomes, nab-paclitaxel, and docetaxel, measurable disease according to the RECIST criteria version 1.1, and adequate hematologic, hepatic, and renal functions. The main exclusion criteria included malignancies of the ampulla, severe hepatic or renal insufficiency, other malignancy history with disease-free survival less than 5 years, ever received treatment of ICIs, history of autoimmune diseases, and immunosuppressant or corticosteroid use within 2 weeks before inclusion. Complete inclusion and exclusion criteria are listed in Table S1.

Treatment

Eligible patients received nab-paclitaxel 125 mg/m² intravenously guttae on day 1 and day 8 + sintilimab 200 mg intravenously guttae on day 1, every 3 weeks. Treatment continued until disease progression, intolerable toxicity, patient refusal, or investigator decision. Patients received maintenance treatment with sintilimab (200 mg, every 3 weeks) if the disease was stable or in remission after eight cycles of combination therapy. The treatment duration of sintilimab was at most 24 months.

Endpoints and assessments

The primary endpoint of this study was the ORR, which was defined as the percentage of patients who obtained complete response (CR) and partial response (PR). The secondary end points were PFS, OS, and adverse reactions. PFS was defined as the time from trial enrollment to first confirmed disease progression (PD) or death, whichever occurred first, and OS was defined as the time from trial enrollment to death from any cause. Disease evaluation was performed every 9 weeks by enhanced computed tomography (CT) based on RECIST version 1.1. Patients who received radiological PD could be permitted to continue treatment if investigators judged clinical benefit from the continued treatment according to physical status, clinical symptoms, and laboratory examination results. These patients should receive imaging examination again to determine whether they have confirmed PD after 4 weeks according to immune-related RECIST. All adverse events during the treatment period were assessed according to the Common Terminology Criteria for Adverse Events version 5.0.

DNA extraction and sequencing

Next-generation sequencing (NGS) of deoxyribonucleic acid (DNA) in formalin-fixed paraffin-embedded (FFPE) tissues was performed using the 769 cancer gene-targeted panel (Genecast Biotechnology Co., Ltd.). DNA was extracted from FFPE tissues using the black PREP DNA kit (Analytik Jena). Genomic DNA was fragmented into 150–200 bp fragments with a Covaris M220 Focused-Ultrasonicator. Fragmented DNA libraries were constructed by the KAPA highthroughput library preparation kit from the Illumina platform (KAPA Biosystems) following the manufacturers' instructions. DNA libraries were captured with a designed 2 Mega panel of the Integrated DNA Technologies library, which included major tumor-related genes. The captured samples were then subjected to NovaSeq 6000 processing for paired-end sequencing. The tumor mutational burden (TMB) was determined by analyzing non-silent somatic mutations, including single nucleotide variants, insertions, and deletions per megabase (Mb). TMB high was defined as ≥10 Muts/Mb.

PD-L1 immunohistochemistry

Programmed cell death ligand 1 (PD-L1) immunohistochemistry was performed using the Food and Drug Administration-cleared 22C3 assay (Dako PD-L1 IHC 22C3 pharmDx). PD-L1 expression was determined by a pathologist using the tumor-proportion score (TPS). A PD-L1 TPS ≥5% was considered PD-L1 positive. PD-L1 was defined as not available (NA) for patients whose collected tumor tissues cannot be evaluated for PD-L1 expression or whose tumor tissues cannot be collected.

Multiplex immunofluorescence

An Opal TM 7-color fluorescence immunohistochemistry kit was applied for multiplex immunofluorescence staining according to the manufacturer's instructions. The following primary antibodies were used for immunostaining: anti-CD4 (ZSGB-BIO, 1:100), anti-CD8 (ZSGB-BIO, 1:100), anti-FOXP3 (Abcam, 1:100), and anti-PD-1 (ZSGB-BIO, 1:50). We used 4′,6-diamidino-2-phenylindole (Sigma) to stain the nuclei. The detected markers included CD4, CD8, FOXP3, and PD1 in the tumor, stroma, and total regions. Analyzed tumor-infiltrating lymphocytes (TILs) included CD4⁺ T cells, CD8⁺ T cells, CD4⁺ FOXP3⁺ T regulatory cells, CD4⁺ FOXP3⁻ T cells, CD8⁺ PD-1⁺ T cells (exhausted CD8⁺ T cells), CD8⁺ PD-1⁻ T cells (nonexhausted CD8⁺ T cells), CD4⁺ PD-1⁺ T cells, and CD4⁺ PD-1⁻ T cells. The stained slides were scanned using Tissue FAXS software (TissueGnostics, version Tissue FAXS SL-7.1.120) and analyzed by StrataQuest software (version 7.1.129). The densities of TILs in the tumor, stroma, and total regions were calculated (cells/mm²), and their correlations with therapeutic efficacy and survival outcome were analyzed.

Statistical analysis

Simon's optimal two-stage design was used to determine the sample size. The previously reported ORR in a phase 3 trial of second-line treatment for advanced BTC was 5%.¹² In our study, the expected ORR was 15%. To achieve 80% power with a two-sided alpha level of 0.05, we needed to enroll 23 subjects with at least one subject achieving CR or PR in the first stage and another 33 subjects in the second stage. If at least five subjects had a response, the treatment regimen would be deemed a success and could be finished ahead of schedule. Considering a dropout rate of 10%, a total of 63 patients were needed. Statistical tests were two-sided, and P < 0.05 was considered statistically significant. All analyses were performed using SPSS Statistics 22.0 (IBM) and R 4.1.3.

RESULTS Patient characteristics

From November 2021 to November 2022, a total of 26 patients were consecutively enrolled. At the time of data cutoff (February 2, 2023), seven patients achieved a tumor response and the study met the primary endpoint of ORR, thus we stopped recruitment ahead of schedule based on the study design. The baseline characteristics of all participants are shown in Table 1. The median age was 57 years (range 31–71), 12 patients (46%) were male, and 10 patients (38%) had an ECOG score of 0. Most patients (50%) were diagnosed with ICC, 7 (27%) with GBC, and 6 (23%) with ECC. All patients were diagnosed with metastatic BTC at the time of screening. Most patients (92%) had extrahepatic metastases and received gemcitabine plus cisplatin as first-line chemotherapy (85%). The median number of cycles of nab-paclitaxel was 4 (range 2–8), and the median number of cycles of sintilimab was also 4 (range 2–20).

TABLE 1 Baseline clinical characteristics of all participants.

Characteristics	N = 26
Age, years
Median (range)	57 (31–71)
Sex, n (%)
Male	12 (46)
Female	14 (54)
ECOG score, n (%)
0	10 (38)
1	16 (62)
Tumor type based on primary tumor location, n (%)
ICC	13 (50)
ECC	6 (23)
GBC	7 (27)
HBV infection, n (%)
Yes	3 (12)
No	23 (88)
Cholelithiasis history, n (%)
Yes	10 (38)
No	16 (62)
Extrahepatic metastasis, n (%)
Yes	24 (92)
No	2 (8)
First-line treatment, n (%)
Gemcitabine + capecitabine	4 (15)
Gemcitabine + cisplatin	22 (85)
PD-L1 expression, n (%)
TPS <5%	17 (65)
TPS ≥5%	3 (12)a
NA	6 (23)
MSI status, n (%)
MSS	21 (81)
MSI-H	1 (4)
NA	4 (15)

Abbreviations: ECC, extrahepatic cholangiocarcinoma; ECOG, Eastern Cooperative Oncology Group; GBC, gallbladder cancer; HBV, Hepatitis B virus; ICC, intrahepatic cholangiocarcinoma; MSI, microsatellite instability status; MSI-H, microsatellite instability-high; MSS, microsatellite stable; NA, not available; PD-L1, programmed cell death ligand 1; TPS, tumor cell proportion score

^aTwo had ECC and one had GBC.

Fifteen patients (57.7%) accepted subsequent anticancer treatment after disease progression. Subsequent chemotherapy drugs included fluorouracil, oxaliplatin, cisplatin, and nab-paclitaxel. Targeted therapy included lenvatinib, regorafenib, disitamab vedotin, and bevacizumab. The details are shown in Table 2.

TABLE 2 Subsequent anticancer therapy.

	Third-line therapy	Fourth-line therapy	Fifth-line therapy
Chemotherapy	5	1	1
Chemotherapy + immune checkpoint inhibitor	7	1	0
Immune checkpoint inhibitor + targeted therapy	1	3	1
Targeted therapy	1	2	0
Chemotherapy + Immune checkpoint inhibitor + targeted therapy	1	0	0

Note: Subsequent chemotherapy drugs included fluorouracil, oxaliplatin, cisplatin, and nab-paclitaxel. Targeted therapy included lenvatinib, regorafenib, disitamab vedotin, and bevacizumab.

Efficacy

At the time of data cutoff (February 2, 2023), 19 patients (73.1%) had confirmed disease progression and 14 (53.8%) had died. The median follow-up time was 439 days (95% CI 338–540 days). For the best disease evaluation among the 26 patients, two patients achieved CR, five patients had PR, nine patients had stable disease (SD), and 10 patients experienced PD (Figure 1A), resulting in an ORR of 26.9% (7/26) and a DCR of 61.5% (16/26). Patients with GBC had a relatively higher ORR of 42.9%, while the ORR of ECC patients was 33.3% and the ORR of ICC patients was 15.4% (Figure 1B). Due to the small sample size, the ORRs of different tumor types had no statistically significant differences. The best change in the longest diameters of the target lesions from baseline is shown in Figure 1C.

FIGURE 1. Clinical response of patients based on RECIST 1.1. (A) Objective response rate (ORR) in all patients. (B) ORR in patients with different tumor types. (C) The best change in the longest diameters in target lesions from baseline. CR, complete response; ECC, extrahepatic cholangiocarcinoma; GBC, gallbladder cancer; ICC, intrahepatic cholangiocarcinoma; PD, disease progression; SD, stable disease.

In the survival analysis applying the Kaplan–Meier method, the median PFS was 169 days (approximately 5.6 months, 95% CI 60–278 days; Figure 2A) and the median OS was 442 days (approximately 14.7 months, 95% CI 298–586 days; Figure 2B). In addition, the median PFS of GBC patients was 314 days (95% CI 108–520 days), that of ECC patients was 48 days (95% CI 0–161 days), and that of ICC patients was 77 days (95% CI 18–320 days). The median OS of GBC patients was 378 days (95% CI NA), of ECC patients was 142 days (95% CI NA–474 days), and of ICC patients was 442 days (95% CI 271–613 days). Neither PFS nor OS had significant differences between the three tumor types, which might be due to the small sample size.

FIGURE 2. (A) Progression-free survival (PFS) curves in all patients. (B) Overall survival (OS) curves in all patients. (C) Representative computed tomography images of a patient who obtained a complete response after nab-paclitaxel plus sintilimab treatment.

Adverse events

Treatment-related adverse events (TRAEs) are presented in Table 3. Most patients experienced grade 1 or 2 TRAEs and tolerated the treatment regimen. The most common grade 3 TRAE was anemia (7/26, 27%), followed by leukopenia (6/26, 23%), neutropenia (5/26, 19%), peripheral sensory neuropathy (2/26, 8%), and sleep disorder (2/26, 8%). In this study, immune-related adverse events (irAEs) associated with sintilimab occurred infrequently. We only observed seven patients with immune-related hypothyroidism, one patient with myocarditis, one patient with hyperthyroidism, and one patient with colitis. Most irAEs were grade 1 or 2, except for one patient with grade 3 colitis. Treatment discontinuation occurred in three patients, including two patients with peripheral sensory neuropathy and one patient with colitis. No grade 4 or 5 TRAEs occurred. Overall, the nab-paclitaxel combined with sintilimab regimen had a favorable safety profile and was well tolerated.

TABLE 3 Treatment-related adverse events.

Adverse events	Any grade, n (%)	Grade 3, n (%)
Anemia	24 (92%)	7 (27%)
Sleep disorder	24 (92%)	2 (8%)
Peripheral sensory neuropathy	22 (85%)	2 (8%)
Alopecia	20 (77%)	0
Albumin decreased	18 (69%)	0
Fatigue	16 (62%)	0
Leukopenia	14 (54%)	6 (23%)
Neutropenia	14 (54%)	5 (19%)
Anorexia	12 (46%)	0
Itching	12 (46%)	0
Skin rash	10 (38%)	0
Myalgia	8 (31%)	0
Constipation	8 (31%)	0
Diarrhea	8 (31%)	0
AST elevation	7 (27%)	1 (4%)
Hypothyroidism	7 (27%)	0
Abdominal distension	7 (27%)	0
Thrombocytopenia	6 (23%)	1 (4%)
Fever	6 (23%)	0
Nausea	6 (23%)	0
Creatinine elevation	5 (19%)	1 (4%)
ALT elevation	4 (15%)	1 (4%)
Vomiting	4 (15%)	0
Total bilirubin elevation	3 (12%)	0
Weight decreased	2 (8%)	0
Arthralgia	2 (8%)	0
Myocarditis	1 (4%)	0
Hyperthyroidism	1 (4%)	0
Colitis	1 (4%)	1 (4%)

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase.

Biomarker detection

To explore potential biomarkers for efficacy prediction, enrolled patients received biomarker detection voluntarily before treatment, including gene sequencing, PD-L1 immunohistochemistry, and multiplex immunofluorescence analysis of TILs. Specimens with a tumor cell proportion score (TPS) <5% were identified as PD-L1 negative, while specimens with a TPS ≥5% were identified as PD-L1 positive.

NGS was applied to comprehensively explore genomic alterations in 22 patients with available tumor specimens. The most frequently mutated cancer-related genes were TP53 (32%), KRAS (26%), PBRM1 (16%), ATM (16%), PIK3CA (11%), IDH1 (11%), CHEK2 (11%), CDKN2A (11%), BRCA2 (11%), and APC (11%) (Figure 3). Compared to ICC, ECC and GBC had a higher mutational rate of the TP53 gene (Fisher's exact test, P = 0.04). However, there was no correlation between gene alterations and patients' clinical response. Additionally, TMB and microsatellite instability (MSI) status was detected in 22 patients. Median TMB was 6.08 Muts/Mb (range 1.0–68.16 Muts/Mb). The majority of patients had low TMB (TMB <10Muts/Mb) and microsatellite stable (MSS) status, while only one patient had high TMB (TMB = 68.16Muts/Mb) with MSI-high (MSI-H) status. The patient with TMB-H/MSI-H responded very well to the investigational regimen, obtained PR, and is still receiving maintenance treatment with sintilimab after eight cycles of nab-paclitaxel plus sintilimab.

FIGURE 3. Overview of the genomic mutation spectrum in the cohort. The most frequently mutated genes were TP53 (32%), KRAS (26%), PBRM1 (16%), ATM (16%), PIK3CA (11%), IDH1 (11%), CHEK2 (11%), CDKN2A (11%), BRCA2 (11%), and APC (11%). There was no statistically significant difference between genetic mutations and clinical response. CR, complete response; ECC, extrahepatic cholangiocarcinoma; ECOG, ; HBV, ; GBC, gallbladder cancer; GP, ; GX, ; ICC, intrahepatic cholangiocarcinoma; PD, disease progression; PR, ; SD, stable disease.

Among the 20 patients who had PD-L1 immunohistochemical tests, 17 were PD-L1 negative (TPS <5%), while three were PD-L1 positive (TPS ≥5%). Among the three patients with PD-L1 positive, two had ECC and one had GBC. Compared with the PD-L1-negative group, the ORR in the PD-L1-positive group was significantly higher (100% vs. 6%, P = 0.003, Fisher's exact test; Figure 4A). The median OS was longer in the PD-L1-positive group than in the PD-L1-negative group (median OS not reached vs. 442 days, P = 0.024, HR <0.01; Figure 4C). In addition, the median PFS showed a better trend in the PD-L1-positive group (median PFS 538 days vs. 110 days, p = 0.062, HR = 0.17; Figure 4B).

FIGURE 4. The association between programmed cell death ligand 1 (PD-L1) expression and clinical response. (A) PD-L1-positive patients presented a superior objective response rate (ORR) (100% vs. 6%, P = 0.003, Fisher's exact test). (B) The PD-L1-positive group showed a better trend in progression-free survival (PFS) than the PD-L1-negative group (median PFS 538 days vs. 110 days, P = 0.062, hazard ratio [HR] = 0.17). (C) The PD-L1-positive group had a significantly longer overall survival (OS) than the PD-L1-negative group (median OS not reached vs. 442 days, P = 0.024, HR [less than]0.01). CR, complete response; PD, disease progression; PR, partial response; SD, stable disease; TPS, tumor cell proportion score.

Furthermore, we used multiplex immunofluorescence to explore the association between TILs and therapeutic responses in 20 patients with available samples. Cell densities and spatial distribution of TILs were compared in different response groups.

In total, except for PD-1-positive T cells, other detected TILs were mainly distributed in the stroma region rather than the tumor region (Figure 5). In the tumor region, no significant differences were observed concerning the densities of all detected TILs in the responder and nonresponder groups (Wilcox test, P > 0.05). However, in the stroma region, CD8⁺ T cells (cytotoxic T cells) had a significantly higher density in responders than in nonresponders (Wilcox test, P = 0.024); the PD-1-negative subset of CD8⁺ T cells (CD8⁺ PD-1⁻ T cells, nonexhausted cytotoxic T cells) showed a higher density trend in responders (Wilcox test, P = 0.067) and the densities of other TILs in the stroma region presented no significant differences between responders and nonresponders. Similar results were observed in the total region: compared to nonresponders, responders had a significantly higher density of CD8⁺ T cells (Wilcox test, P = 0.019; Figure 6) and a higher density trend of CD8⁺ PD-1⁻ T cells (Wilcox test, P = 0.056; Figure 6).

FIGURE 5. TIL distribution pattern in the tumor and stromal regions. Except for PD-1-positive T cells, the densities of other detected TILs in the stroma region were significantly higher than those in the tumor region (paired Wilcox test, p [less than] 0.05). CR, complete response; PD, disease progression; PR, partial response; SD, stable disease.

FIGURE 6. The association between the density of tumor-infiltrating lymphocytes (TILs) and clinical response. (A) Overview of the TIL densities in the cohort. (B) Comparison of TIL densities in different response groups. CR, complete response; ECC, extrahepatic cholangiocarcinoma; ECOG, ; HBV, ; GBC, gallbladder cancer; GP, ; GX, ; ICC, intrahepatic cholangiocarcinoma; PD, disease progression; PR, ; SD, stable disease.

In addition, we analyzed the correlation between TILs and survival time. In the tumor region, no statistically significant association was observed between TILs and PFS. However, a higher density of CD8⁺ PD-1⁺ T cells (exhausted T cells) was associated with worse OS (log-rank test, cutoff density = 0.821 cells/mm², P = 0.012, HR = 7.127; Figure 7A). In the stroma region, no significant correlation was observed either between TILs and PFS or between TILs and OS. In the total region, higher densities of CD8⁺ and CD8⁺ PD-1⁻ T cells tended to be associated with better PFS (log-rank test, cutoff density of CD8⁺ T cells = 772.362 cells/mm², P = 0.069, HR = 0.262, Figure 7B; cutoff density of CD8⁺ PD-1⁻ T cells = 769.482 cells/mm², P = 0.069, HR = 0.262, Figure 7C) and OS (log-rank test, P = 0.08, HR = 0.181, Figure 7D; P = 0.08, HR = 0.181, Figure 7E). Detailed information is shown in Table S2.

FIGURE 7. The correlation between tumor-infiltrating lymphocytes and survival time. (A) A higher density of CD8+ PD-1+ T cells in the tumor region was associated with worse overall survival (OS) (cutoff density = 0.821 cells/mm2). (B) A higher density of CD8+ T cells in the total region presented a better progression-free survival (PFS) trend (cutoff density = 772.362 cells/mm2). (C) A higher density of CD8+ PD-1− T cells in the total region presented a better PFS trend (cutoff density = 769.482 cells/mm2). (D) A higher density of CD8+ T cells in the total region showed a better OS trend (cutoff density = 772.362 cells/mm2). (E) A higher density of CD8+ PD-1− T cells in the total region presented a better PFS trend (cutoff density = 769.482 cells/mm2). CR, complete response; HR, hazard ratio; PD, disease progression; PR, partial response; SD, stable disease.

DISCUSSION

Compared with published literature, this is the first study to investigate the efficacy and safety of nab-paclitaxel plus sintilimab as a second-line treatment for advanced BTC patients. The results of the NapaSinti trial indicated favorable antitumor activity with an ORR of 26.9%, a median PFS of approximately 5.6 months, and a median OS of approximately 14.7 months. Patients with positive PD-L1 and high proportions of CD8⁺ or CD8⁺ PD-1⁻ T cells showed potentially improved ORR and survival outcomes, which needs to be confirmed in large-scale trials.

As mentioned above, second-line therapeutic options for advanced BTC patients without certain targetable genetic mutations are very limited and have unsatisfactory effects. The phase 3 ABC-06 trial suggested that compared with ASC, second-line FOLFOX chemotherapy had unfavorable OS (median OS 6.2 vs. 5.3 months, P = 0.031) and a higher incidence of grade 3–5 adverse events.¹² In recent years, several phase 2 trials investigating monotherapy with ICIs (such as pembrolizumab²⁵ and nivolumab²⁶) or small molecule vascular endothelial growth factor receptor (VEGFR) inhibitors (such as lenvatinib,²⁷ regorafenib,²⁸ axitinib,²⁹ and surufatinib³⁰) have also resulted in disappointing effects. Recently, some single-arm trials with small sizes of 20–30 subjects have shown modest effectiveness of later-line ICIs combined with VEGFR inhibitors (such as pembrolizumab plus lenvatinib,³¹ camrelizumab plus apatinib,³² and sintilimab plus anlotinib³³) in advanced BTC. An exploratory study conducted by Lin et al.³¹ indicated that non-first-line treatment with pembrolizumab plus lenvatinib was a promising regimen for refractory BTC (ORR 25%, median PFS 4.9 months, median OS 11.0 months). Similarly, Jin et al.³³ showed encouraging antitumor activity with a tolerable safety profile of second-line sintilimab plus anlotinib in advanced BTC (ORR 30%, median PFS 6.5 months, median OS 12.3 months). The efficacy of our study was similar to that of combination therapy with ICIs plus VEGFR inhibitors. However, in terms of cost-effectiveness and for patients unfit for VEGFR inhibitors, our regimen had obvious advantages and provided a promising alternative option.

To date, the predictive biomarkers for combination immunotherapy are ambiguous. It is widely recognized that patients with MSI-H status are an effective population for ICIs, but unfortunately, these patients account for less than 5% of all advanced BTC patients, which limits the clinical application of MSI-H status in predicting ICI efficacy. Similarly, high TMB is a widely used immunotherapy predictive biomarker, but the majority of BTC patients show low TMB, with a median value of approximately 3.7 Muts/Mb.³⁴ In the present trial, there was only one patient with high TMB/MSI-H status who obtained PR and is still receiving maintenance sintilimab treatment. Recently, several studies have indicated that PD-L1-positive expression is associated with improved therapeutic outcomes, but the specific cutoff values of PD-L1-positive tumor cells (tumor cell proportion score, TPS) or tumor cells plus tumor-associated immune cells (combined positive score, CPS) are not clear.^5,31,33,35 In the study of pembrolizumab plus lenvatinib as a non-first-line treatment for advanced BTC, PD-L1 positivity was defined as TPS ≥5%, which was associated with improved clinical benefits.³¹ In the prospective study of sintilimab plus anlotinib, patients with CPS ≥10 resulted in prolonged survival time compared with CPS <10 patients.³³ In our study, PD-L1 positivity was defined as TPS ≥5%. Efficacy analysis suggested that PD-L1-positive patients showed improved clinical outcomes, but larger randomized trials are needed to confirm this finding.

TILs are the most important components of the tumor immune microenvironment. Many studies have proposed TILs as crucial prognostic and efficacy predictive indicators, including CD3⁺, CD4⁺, and CD8⁺ lymphocytes.^7,36–42 Previous studies have suggested that CD8⁺ T-cell infiltration is related to improved survival in patients receiving immunochemotherapy.^7,40,42 In the phase 2 study of sintilimab plus GemCis as a first-line treatment for patients with BTC, responders exhibited a higher proportion of CD8⁺ T cells than nonresponders (P = 0.0017). A higher proportion of CD8⁺ T cells was associated with a longer PFS (P = 0.012).⁷ In a systematic review analyzing 33 studies, investigators found that high CD8⁺ T cells were significantly associated with better ORR (OR 4.08, 95% CI 2.73–6.10, P < 0.001), OS (HR 0.52, 95% CI 0.41–0.67, p < 0.001), and PFS (HR 0.52, 95% CI 0.40–0.67, P < 0.001) in patients treated with ICIs.⁴⁰ Consistent with these studies, our results also demonstrated the potential protective role of CD8⁺ T cells and their subset CD8⁺ PD-1⁻ T cells.

Furthermore, emerging evidence has indicated that the spatial distribution of TILs could precisely reflect the interactions between immune and tumor cells, but the detailed distribution pattern for favorable response and prognosis is unclear.^43–47 In a retrospective study of pleural mesothelioma patients treated with nivolumab, total T-cell infiltration in the tumor nest was similar to that in the extratumoral area. Compared to the nonresponse group, CD8⁺ T cells in the response group were more clustered and located closer to tumor cells, whereas regulatory T cells were located further from tumor cells.⁴⁷ However, in a study of esophageal squamous cell carcinoma patients treated with first-line chemoradiotherapy plus the anti-PD-1 antibody camrelizumab, CD4⁺ and CD8⁺ T cells were mainly distributed in the stroma rather than in the tumor region before treatment. Higher PD-1-positive CD4⁺ and CD8⁺ T cells within a 100 μm distance from tumor cells predicted better OS.⁴⁶ In our study, the results suggested that TILs were mainly distributed in the stroma region, and high proportions of CD8⁺ T cells and CD8⁺ PD-1⁻ T cells in the stroma and total regions were associated with favorable clinical outcomes, whereas a higher density of CD8⁺ PD-1⁺ T cells in the tumor region was associated with worse OS. Further research is needed in this field.

There were some limitations in our study. First, this was a nonrandomized, open-label, investigator-initiated study, which might cause selection and participant bias. Second, as our study was designed and conducted in the era when ICIs were not standard therapy in first-line treatment, patients who ever received ICIs were excluded. It has not been demonstrated whether sintilimab plus nab-paclitaxel regimen was effective as second-line therapy after the failure of first-line GemCis with ICI, which is our future research topic. However, our study confirmed efficacy enhancement of nab-paclitaxel combined with ICI, which indicates that first-line nab-paclitaxel addition to standard GemCis chemotherapy with ICI might have efficacy superiority. Third, the sample size was small and all participants were Chinese, which might lead to bias and inaccuracy in the subgroup analysis. Moreover, not all patients in our study received biomarker analysis and there was a lack of whole-genome sequencing to identify other potential genetic biomarkers, therefore further large-scale, randomized, prospective clinical trials are warranted.

In conclusion, the present trial met its primary end point of an ORR increase. The clinical efficacy and manageable tolerability of sintilimab plus nab-paclitaxel provided a promising second-line treatment option for advanced BTC patients. Positive PD-L1 and CD8⁺ T-cell infiltration might be potential efficacy predictive biomarkers. Further large-scale trials and comprehensive biomarker analyses are necessary.

AUTHOR CONTRIBUTIONS

Xiaofen Li: Conceptualization; formal analysis; funding acquisition; investigation; writing – original draft; writing – review and editing. Nan Zhou: Data curation; investigation; writing – original draft; writing – review and editing. Yu Yang: Investigation; writing – review and editing. Zijian Lu: Investigation; writing – review and editing. Hongfeng Gou: Conceptualization; supervision; writing – review and editing.

ACKNOWLEDGMENTS

We appreciate the patients who volunteered to participate in this trial and the staff members in the hospital who cared for these patients.

FUNDING INFORMATION

The study was supported by the Health Commission of Sichuan Province Program (No. 21PJ007). The funding source was not involved in the study design, data analyses, or manuscript writing.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT

The data supporting the findings of this study will be available upon reasonable written request approved by the corresponding author.

ETHICS STATEMENT

Approval of the research protocol by an Institutional Review Board: This study was approved by the Ethics Committee on Biomedical Research, West China Hospital of Sichuan University.

Informed Consent: Written informed consent to participate was obtained from each subject.

Registry and the Registration No. of the study/trial: This trial was registered at Chinese Clinical Trial Registry (ChiCTR2100052118).

Animal Studies: N/A.

PREVIOUS PRESENTATION

The preliminary results of this trial were previously presented as a poster abstract at the 2023 American Society of Clinical Oncology Gastrointestinal Cancers Symposium (ASCO GI), January 19–21, 2023, titled “Nab-paclitaxel plus anti-PD-1 antibody as second-line treatment for advanced biliary tract cancer: an investigator-initiated phase 2 study (NapaSinti trial).”