1. PARP inhibitor and immune checkpoint inhibitor in a case of gallbladder cancer with BRCA2 mutation.
A 56-year-old women was presented to our hospital in June, 2019 with the chief complaint of epigastric discomfort (case #1). Abdominal computed tomography (CT) scan showed a lesion in the gallbladder (Fig. 1A). Then positron emission tomography - computed tomography (PET-CT) was conducted and showed gallbladder cancer with liver portal and retroperitoneal lymph node metastasis, multiple intrahepatic metastasis, and anterior splenic metastasis. Later, the patient came to our hospital for treatment. After admission, physical examination, tumor biomarkers, routine blood counts, coagulation function, and liver and kidney function were all normal. Then, the patient underwent percutaneous liver biopsy (First biopsy) under B Ultrasound and pathological examination suggested that the liver lesion was moderately/poorly differentiated CCA. Immunohistochemical (IHC) staining showed CK19 (+), CK7 (+), CK20 (-), CDX-2 (-), Arg-1(-) and HER2(-) (Fig. 1E). The PD-L1 expression level showed a tumor proportion score (TPS) of 40% for PD-L1. A next generation sequencing (NGS) (by Geneplus Technology Limited.) in liver tissue was performed and showed gene mutation: BRCA2 c. 7008-2A > T (Figure S1A), and MSS, and all mutation types observed in the patient are displayed in Table 1. Clinical diagnosis of this patient were cTxN + M1 stage IV gallbladder cancer combined with liver, abdominal cavity and retroperitoneal lymph node metastases.
Table 1
Next generation sequencing analysis (NGS) of all the mutations detected in the patient.
Gene
|
Mutation type
|
Exon
|
cDNA change
|
Amin acid change
|
Abundance
|
BRCA2
|
Alternative splicing mutation
|
14
|
c.7008-2A > T
|
-
|
32.76%
|
FBXW7
|
Missense mutation
|
10
|
c.1513C > T
|
p. Arg505Cys
|
38.08%
|
TP53
|
Missense mutation
|
7
|
c.733G > A
|
p. Gly245Ser
|
20.66%
|
AMER1
|
Missense mutation
|
2
|
c.730G > A
|
p. Glu244Lys
|
16.33%
|
BRCA2
|
Missense mutation
|
13
|
c.6953G > A
|
p. Arg2318Gln
|
22.09%
|
EMSY
|
Missense mutation
|
21
|
c.3546A > G
|
p. Ile1182Met
|
13.14%
|
KLF4
|
Missense mutation
|
3
|
c.1262C > G
|
p. Ser421Trp
|
31.48%
|
NTRK1
|
Missense mutation
|
9
|
c.899C > G
|
p. Thr300Ser
|
10.34%
|
RAD51D
|
Missense mutation
|
3
|
c.154G > T
|
p. Ala52Ser
|
11.64%
|
SPEN
|
Missense mutation
|
11
|
c.2415G > T
|
p. Glu805Asp
|
10.46%
|
SPTA1
|
Missense mutation
|
44
|
c.6386G > T
|
p. Arg2129Met
|
24.63%
|
ZNF703
|
Missense mutation
|
2
|
c.959T > A
|
p. Val320Glu
|
5.55%
|
After admission, treatment with 4 cycles of gemcitabine and paclitaxel chemotherapy were initiated from June 19, to September 14 2019. After the initial 4 cycles of chemotherapy, a computed tomography (CT) scan suggested reduction of gallbladder wall thickness, which were suggestive of partial response (PR). Then, considering the 40% tumor proportion score (TPS), pembrolizumab was added in the next cycle of treatment. From September 14, to November 8 2019, the patient was administered 2 cycles GP chemotherapy plus Pembrolizumab. However, during the treatment, severe myelosuppression, agranulocytosis together with fever and femoral vein thrombosis occurred. Evaluating her intolerance of traditional chemotherapy, pembrolizumab monotherapy was applied to May 17 2020. CT scans suggested sustained stable disease (SD). In the meantime, multidisciplinary treatment was carried and surgeons suggested excision of lesions, but the patient refused. To better control the lesion, pembrolizumab combined with tegafur (S-1) were used for 3 cycles from May 20, to July 24 2020 according to MDT conclusion. The CT scan indicated progressive disease (PD) with gallbladder wall thickening after maintenance pembrolizumab with S-1 for only 2 months. Therefore, pembrolizumab plus lenvatinib were considered for 2 cycles. The evaluation after 2 cycles of pembrolizumab plus lenvatinib showed thickened gallbladder wall and liver metastasis were largely aggravated, which suggested PD (Fig. 1B). Furthermore, the CEA and CA199 level significantly increased to 61.56 ng/ml and 35.14kU/L (Figure S1B).
Considering the patient had BRCA2 gene mutation, she began to receive olaparib and durvalumab since September 29 2020. The measurements of the target lesions were evaluated by liver MRI and whole-body CT, which were basically examined every 2 months (Figure S1C). Surprisingly, the image examination suggested thinned gallbladder wall and diminished liver metastasis, which indicated PR (Fig. 1C). And CEA and CA199 level fell rapidly to normal after the first cycle. Up to now, the patient has received 57 cycles targeted therapy and immunotherapy and has an overall survival (OS) of over 40 months with no discomfort. However, in March 01 2023, the patient's condition exhibited progression, as evidenced by a CT scan on March 13 showing an increase in gallbladder wall thickness (Fig. 1D). The second percutaneous liver biopsy was performed and was considered to originate from the gallbladder by pathological examination (Fig. 1F).
Unfortunately, the disease further advanced in April 2023, leading to complications such as bowel obstruction, bone marrow suppression, anemia, liver impairment, and pulmonary infection. These adverse events resulted in the patient's inability to tolerate further chemotherapy and she underwent a third biopsy for relieving bowel obstruction by surgery and postoperative pathological findings were shown in Fig. 1G. Then the patient received postoperative maintenance treatment and remains alive to date. The timeline of patient treatment was shown in Fig. 1H.
2. Possible mechanisms of PBscore
Based on the available literature, it has been established that PARP inhibitors primarily target pathogenic variants in the BRCA1 and BRCA2 genes. Additionally, PARP1 serves as a crucial target for PARP inhibitors26,27. Consequently, our main emphasis was on evaluating the impact of PARP inhibitors by examining the activation level of the PARP1/BRCA1/BRCA2 pathway. Furthermore, since RNA sequencing data specifically for gallbladder cancer is lacking in the Cancer Genome Atlas (TCGA) dataset, we leveraged the similarities between cholangiocarcinoma and gallbladder cancer, as supported by previous studies28,29. Therefore, the TCGA dataset for cholangiocarcinoma (CHOL) was selected as the basis for our research23. The PARP1-BRCA1-BRCA2 pathway related risk score (PBscore) was developed to evaluate the risk associated with this pathway and its potential influence on the tumor immune response.
To investigate the association between PARP inhibitors and gallbladder cancer, we examined the differentially expressed genes (DEGs) between high- and low- PBscore groups (Fig. 2A and B). To gain deeper insights into the underlying molecular mechanisms, we conducted comprehensive Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional analyses. Notably, the KEGG analysis (Fig. 2C) demonstrated a significant enrichment in transmitter-gated ion channel activity involved in regulation of postsynaptic membrane potential, neurotransmitter receptoractivity involved in regulation of postsynaptic membrane potential, transmitter − gated ion channel activity and transmitter − gated channel activity. Gene Set Enrichment Analysis (GSEA) in Fig. 2D further demonstrated PBscore could significantly influence antigen processing and presentation (P = 0.00248). The GO analysis focusing on biological functions revealed that PBscore prominently contributes to alanine, aspartate and glutamate metabolism, crdiac muscle contraction, chemical carcinogenesis − receptor activation, cholinergic synapse, cytokine − cytokine receptor interaction, epithelial cell signaling in Helicobacter pylori infection, glycosphingolipid biosynthesis − lacto and neolacto series, natural killer cell mediated cytotoxicity, neuroactive ligand − receptor interaction, and viral protein interaction with cytokine and cytokine receptor (Fig. 2E). The potential impact of PBscore on GBC development and may serve as a valuable indicator for disease prognosis or therapeutic targeting.
Unfortunately, univariate Cox analysis was performed using the PARP1-BRCA1-BRCA2 pathway and PBscore (Figure S2A) to investigate the prognostic significance. The results of the univariate analysis revealed a negative association between OS in CHOL and the PARP1-BRCA1-BRCA2 pathway as well as PBscore. Then the analysis of the survival curve showed no significant difference in overall survival (OS) between the high and low PBscore groups (P = 0.43) (Figure S2B).
3. PARP inhibitor may activate tumor immune microenvironment of GBC via immune checkpoint
In our study, as shown in Fig. 3A, we observed a negative correlation between PBscore and the immune checkpoint molecule CEACAM1, while a positive correlation was observed with immune checkpoints such as C10orf54, CD200, and PDCD1. Additionally, we found that PBscore was associated with tumor-infiltrating immune cells (TIICs) (Fig. 3B). Specifically, the activated CD4 T cells were upregulated, whereas gamma delta T cells, Type 17 T helper cells, CD56 dim natural killer cells, macrophages, natural killer cells, and neutrophils were downregulated, as depicted in the figure. To further elucidate the relationship between PBscore and the immune microenvironment, we conducted a correlation analysis between PBscore and the cancer-immunity cycle. The Fig. 3C and D demonstrated that two steps of the cancer-immunity cycle, namely the release of cancer cell antigens (Step 1) and neutrophil recruitment (Step 4), were upregulated in association with PBscore. Conversely, the steps involving cancer antigen presentation (Step 2) and recognition of cancer cells by T cells (Step 6) were downregulated.
To investigate the disparities in the tumor microenvironment between patients with different BRCA2 status, we obtained paired samples from an additional patient (case #2) through biopsy. The patient was diagnosed with gallbladder cancer based on pathological and immunohistochemical examinations, and subsequent gene sequencing confirmed the patient's BRCA2 wild-type status. As is known to all, CD3 activation triggers T Cell and its various downstream signaling pathways, CD56 expression recruits NK cells and is associated with anti-tumor immune responses, and PD1 plays a critical role in regulating tumor immune microenvironment. Thus, CD3, CD56 and PD1 were selected to identify the relationship between BRCA2 wide-type and BRCA2 mutant patients. The results of multiplexed immunofluorescence imaging in Fig. 4A showed that compared with BRCA2 wide-type patient, BRCA2 mutant patient have a higher expression of CD3, CD56 and PD1.
Subsequently, with the patient's (case #1) consent, we obtained the progressive liver metastatic tumor tissue (second biopsy) by percutaneous liver biopsy after applying PARP inhibitor and immune checkpoint inhibitor combination therapy and surgically collected gastrointestinal metastatic tumor tissue (third biopsy) following resistance to durvalumab and GP. In order to further investigate the resistance mechanism of this combination therapy, we conducted an evaluation of CD8, CK and CEACAM1 expression. Cytokeratin (CK) is employed to distinguish between epithelial cells in gallbladder cancer and parenchymal cells. Building upon the earlier bioinformatic analysis, CEACAM1 was selected as an additional marker for multiplexed immunofluorescence imaging. The analysis demonstrated a significant increase in the infiltration of CD8 + T cells in gallbladder cancer tissue (Fig. 4B). And there was a notable increase in CEACAM1 + cells within the third biopsy resistant metastatic tumor tissue (Fig. 4C).