Gallbladder cancer (GBC) is the most common type of biliary tract malignant tumor and resists most chemotherapeutic drugs (1). The incidence of gallbladder cancer in different regions varies, as it depends on multiple cancer risk factors, including geographical region, gender, age, and food intake (2). According to the database of the International Agency for Research on Cancer (IARC) Globocan in 2020, there were an estimated 115,949 new cases of GBC worldwide (contributing 0.6% of new cases, ranking 25th out of 36 cancers in 185 countries), and gallbladder cancer contributes to 0.9% of all cancer deaths annually.

Although divergent therapeutic approaches are currently available to treat GBC patients, surgery is still the most acceptable and effective means. However, a fairly large proportion of patients missed the opportunity of surgical resection due to the lack of early typical symptoms, thus accepting alternative non-surgical treatment including chemo- and/or radiotherapy. In order to improve early diagnosis, considerable efforts have been made to develop detecting technologies with minimally invasive approaches. For example, liquid biopsies containing circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) from the blood have received significant attention in the assistance of clinical diagnosis. The high sensitivity of these techniques allows us to reveal tumor genetic features with aberrant gene expressions and gene mutations, thus providing great value for early diagnosis and therapy.

A plethora of research evidence has established the paradigm that altered expressions of tumor oncogenes and/or suppressor genes drive tumor cell proliferation and transformation in tumor development. Elevated or mutated genes including ERBBs, TP53, EGF, CYP7A1, and CCR5 emerge to regulate a variety of human cancer development (1). For instance, mutation of BRCA1, a tumor suppressor gene, has been shown to be intimately associated with the formation of familial breast and ovarian cancer (3). Olaparib, a Food and Drug Administration (FDA)-approved drug targeting BRCA1 and BRCA2 (BRCA1/2) mutations, functions to inhibit poly ADP-ribose polymerase inhibitor (PARPi). In clinical trials, PARPi has improved outcomes of BRCA1/2 mutated cancer patients including breast, ovarian, pancreatic, and prostate cancers (4). While ERBB2/3 mutation was previously discovered to mediate the malignancy of GBC, we still lack sufficient knowledge on other gene mutations and the role of the co-existence of ERBB2/3 mutation with BRAC1/2 mutation in the malignant transformation of patients with GBC. Here, we reported that a gallbladder cancer patient with BRCA1 p.Arg1325Lys mutation responded well to olaparib, but 6 months later, the patient exhibited cancer relapse and developed ERBB3 mutation. Tumor cell lines carrying BRCA1 and ERRB3 mutations were responsive to olaparib and afatinib, suggesting that a combined regimen targeting BRCA1/2 and ERBB2/3 mutations may be an optimal strategy to treat GBC patients who carried both gene mutations.

This study is a case report and agreed with the principles of the CARE guidelines (5).

While BRCA1/2 mutation has frequently been identified in a wide spectrum of cancers, including breast (10), ovarian (11), pancreatic (12), and prostate (13) cancers, BRCA1 p.Arg1325Lys has not been reported in GBC. This is the first time to demonstrate that BRCA1 p.Arg1325Lys exists in GBC.

In concert with the sensitivities of mutated BRCA1/2 cancer cells to PARPi, the patient responded well to olaparib, the first line of targeted therapy approved by the FDA for BRCA1/2 mutated cancers (14). After treatment with olaparib, CT and PET/CT showed dramatic restriction of the tumor. Histopathological examination also showed only a few remaining tumor cells with no lymph node metastasis. The blood levels of CA19-9 declined from 328 U/ml to the normal range. All the results demonstrated favorable responses to BRCA1 mutation-targeted therapy. Although it is one case reported with BRCA1/2 mutation in GBC, the favorable clinical outcome has encouraged us to extend our research in clinical trials with GBCs expressing BRCA1/2 mutation in the near future.

Accumulating evidence indicated that cancer cells harboring BRCA1/2 mutation are defective in double-strand DNA break (DSB) repair, and these cells are sensitive to PARP inhibitors (15–18). However, prolonged treatment with PARPi can eventually lead to the occurrence of drug resistance. At present, at least four distinct molecular mechanisms have been documented to explain PARPi resistance (19): i) increased ability to export drugs, which could be caused by overexpression of drug-efflux transporter genes (Abcb1a and Abcb1b encoding for MDR1/P-gp and Abcg2) in cancer cells (7), ii) increased activity and level of PAR chains through loss of PARG (20) or PARP1 mutations that diminish trapping of protein on DNA (21), iii) increased homologous recombination (HR) (22), and iv) replication fork protection. For instance, there are defects of SMARCAL1, ZRANB3, and HLTF that mediate double DNA chain fork, MRE11-dependent degradation of nascent DNA in BRCA1/2-deficient human cell lines (23). In addition, other mechanisms that downregulate DNA repair pathways such as overexpression of histone methyltransferases EHMT1/2 have also been reported to mediate PARPi resistance (24, 25). It is worthwhile to further decipher the molecular mechanisms underpinning the acquired resistance to olaparib in our case.

The safety of PARPi has received significant attention, though no obvious adverse and unanticipated events were observed in this patient. Adverse events (AEs) may result in the termination of PARPi treatment when AE has threatened the patients’ life. PARPi has a high risk of serious AE (SAE)- and AE-related discontinuation of treatment compared with placebo (26). This suggests that in the clinical application of PARPi, drug safety and AE should be particularly taken into account.

The combined therapy of PARPi with a DNA damage repair blocker such as topoisomerase I poison inhibitor was reported to sensitize tumor cell death and improve patient prognosis (27). In addition, a conjunction treatment of PARPi with anti-angiogenesis and anti-immune checkpoint drugs was also practiced in the clinical trials. However, the underlying mechanisms of these treatments remain to be clarified. Here, we found that the patient developed a new gene mutation with ERBB3 p.Gly337Arg when the disease did not respond to olaparib. It remains to be investigated if ERBB3 mutation is mechanistically associated with BRCA1 mutation. Nevertheless, our results with GBC cultured cell lines, which ectopically expressed both BRCA1 p.Arg1325Lys and ERBB3 p.Gly337Arg, developed resistance to olaparib. In contrast, single BRCA1 p.Arg1325Lys-mutated cells failed to grow in the presence of olaparib. Thus, dual inhibitors with olaparib and afatinib recapitulated the sensitivity of these cells to decreased cell growth, offering effective means to treat both mutations in GBC.

Compared with other previous cases of olaparib in the treatment of BRCA1 mutant gallbladder cancer (28, 29), the progression-free survival of the patient in our case was similar, but the mutation sites were different, which further demonstrated the feasibility of olaparib in the treatment of BRCA1 mutant gallbladder cancer. Different from other cases, the patient in this case was first treated with olaparib to shrink the tumor and successfully achieve R0 resection, thus achieving conversion therapy, which also provides a new idea for the surgical treatment of gallbladder cancer. In addition, this patient developed a new gene mutation with ERBB3 p.Gly337Arg, which was also one of the possible reasons for the progression of the disease. We also verified the effectiveness of dual-targeted drug therapy to a certain extent through an in vitro experiment, providing some ideas and references for similar situations that may occur in the future.

In summary, we reported that a patient with GBC harboring BRCA1 mutation initially responded well to olaparib. After a 6-month course of treatment, the patient developed an additional ERBB3 mutation, which led to the failure of the PARPi therapy. A dual BRCA1/2 and ERBB2/3 mutation-targeted therapy may suggest a benefit to GBC patients who carry both mutated genes.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the [individual(s) AND/OR minor(s)’ legal guardian/next of kin] for the publication of any potentially identifiable images or data included in this article.

(I) Conception and design: YL, M-LL and Y-BL. (II) Administrative support: M-LL, Y-BL, S-YP and WL. (III) Provision of study materials or patients: Y-DZ, W-GW, X-AW, H-FL, FZ and R-FB. (IV) Collection and assembly of data: Z-YJ, J-XY, X-CL and LZ. (V) Data analysis and interpretation: F-TL, Z-YJ, J-XY, X-CL and LZ. (VI) Manuscript writing: all authors. All authors contributed to the article and approved the submitted version.