Due to the fast progression in molecular technologies such as next-generation sequencing, knowledge of genetic alterations in gallbladder cancer (GBC) increases. This systematic review provides an overview of frequently occurring genetic alterations occurring in GBC and their possible therapeutic implications. A literature search was performed utilizing PubMed, EMBASE, Cochrane Library, and Web of Science. Only studies reporting genetic alterations in human GBC were included. In total, data were extracted from 62 articles, describing a total of 3893 GBC samples. Frequently detected genetic alterations (>5% in >5 samples across all studies) in GBC for which targeted therapies are available in other cancer types included mutations in ATM, ERBB2, and PIK3CA, and ERBB2 amplifications. High tumor mutational burden (TMB-H) and microsatellite instability (MSI-H) were infrequently observed in GBC (1.7% and 3.5%, respectively). For solid cancers with TMB-H or MSI-H pembrolizumab is FDA-approved and shows an objective response rates of 50% for TMB-H GBC and 41% for MSI-H biliary tract cancer. Only nine clinical trials evaluated targeted therapies in GBC directed at frequently altered genes (ERBB2, ARID1A, ATM, and KRAS). This underlines the challenges to perform such clinical trials in this rare, heterogeneous cancer type and emphasizes the need for multicenter clinical trials.

Gallbladder cancer (GBC) is a rare, highly aggressive malignancy with a 5-year survival rate of 5–10% in advanced cases, highlighting the need for more effective therapies. The aim of this study was to identify potentially actionable therapeutic targets for GBC. Specimens and clinicopathological data of 642 GBC patients, diagnosed between 2000 and 2019 were collected using the Dutch Pathology Registry (PALGA) and the Netherlands Cancer Registry. All cases were histologically reviewed and a subset was subjected to a comprehensive next generation sequencing panel. We assessed mutations and gene amplifications in a panel of 54 actionable genes, tumor-mutational burden (TMB), and microsatellite instability (MSI). Additionally, the entire cohort was screened for HER2, PD-L1, pan-TRK, and p53 expression with immunohistochemistry. Histopathological subtypes comprised biliary-type adenocarcinoma (AC, 69.6%), intestinal-type AC (20.1%) and other subtypes (10.3%). The median total TMB was 5.5 mutations/Mb (range: 0–161.1) and 17.7% of evaluable cases had a TMB of >10 mutations/Mb. MSI was observed in two cases. Apart from mutations in TP53 (64%), tumors were molecularly highly heterogeneous. Half of the tumors (50%) carried at least one molecular alteration that is targetable in other tumor types, including alterations in CDKN2A (6.0% biallelically inactivated), ERBB2 (9.3%) and PIK3CA (10%). Immunohistochemistry results correlated well with NGS results for HER2 and p53: Pearson r = 0.82 and 0.83, respectively. As half of GBC patients carry at least one potentially actionable molecular alteration, molecular testing may open the way to explore targeted therapy options for GBC patients.

Angiosarcomas (AS) are extremely rare and aggressive vascular malignancies subdivided in de novo primary AS (pAS) and secondary AS (sAS). We hypothesize that the combination of immunological and genomic profiles significantly differs between primary and secondary AS, with potential impact on treatment strategies and a role for immunotherapy. Tumor-infiltrating lymphocytes were analyzed using multiplex immunohistochemistry from 79 pAS and 178 sAS. Median cell density was significantly higher in sAS for CD3⁺ T-cells (p < 0.001), CD8⁺ cytotoxic T-cells (p = 0.033), CD4⁺ T-helper cells (p < 0.001) and FoxP3⁺ T-regulatory cells (p < 0.001). CD20⁺ B-cell density was comparable (p = 0.417). Comprehensive genomic profiling was performed in 25 pAS and 25 sAS. A (likely) pathogenic mutation was detected in 80% of pAS vs. 88% of sAS (p = 0.702). Amplifications were found in 15% of pAS vs. 84% of sAS (p < 0.001). DNA damage response (DDR) pathway mutations (p = 0.021) and MYC amplifications (p < 0.001) were predominantly seen in sAS. In conclusion we observed a clear and clinical relevant distinction in immune infiltration and genomic profiles between pAS and sAS. The T-cell infiltrated tumor microenvironment and frequent DDR gene mutations, especially in sAS, warrant clinical trials with immunotherapy.

PURPOSE: Neuroendocrine carcinomas and mixed neuroendocrine non-neuroendocrine neoplasms of the gallbladder (NE GBC) are rare and highly aggressive entities. The cell of origin of NE GBC has been a matter of controversy. Here, we performed a comparative histopathologic and molecular analysis of NE GBC cases and, if present, associated precancerous lesions. PATIENTS AND METHODS: We selected cases diagnosed between 2000 and 2019 in the Netherlands. Precursors and carcinomas were immunohistochemically compared and analyzed for mutations, gene amplifications, microsatellite instability, and tumor mutational burden using an next-generation sequencing panel containing 523 cancer-related genes. In addition, presence of fusion genes was analyzed using a panel of 55 genes. RESULTS: Sixty percent of neuroendocrine cases (6/10) presented with a precursor lesion, either intracholecystic papillary neoplasm (n = 3) or biliary intraepithelial neoplasia (n = 3). Immunohistochemically, neuroendocrine components were different from the epithelial precursor lesions. Molecular profiling, however, revealed TP53 mutations shared between different components in five of six cases, indicating a clonal relation. Furthermore, 40% of cases (4/10) harbored at least one potentially actionable alteration. This included (likely) pathogenic mutations in RAD54L, ATM, and BRCA2; amplifications of ERBB2 and MDM2; and a gene fusion involving FGFR3-TACC3. All cases were microsatellite-stable and had a tumor mutational burden of < 10 mutations/Mb. CONCLUSION: Our data provide insight into the development of NE GBC and suggest a common origin of precancerous epithelial lesions and invasive neuroendocrine components, favoring the hypothesis of lineage transformation. Moreover, nearly half of the NE GBCs carried at least one potentially actionable molecular alteration, highlighting the importance of molecular testing in this highly lethal cancer.

Background: Metastases to the gallbladder (GBm) are rare and pose a unique diagnostic challenge because they can mimic a second primary tumor. This study aimed to gain insight into the clinicopathological and epidemiological characteristics of GBm. Methods: A comprehensive literature review was performed (literature cohort) and compared with a nationwide cohort of GBm patients diagnosed between 1999 and 2015 in the Netherlands, collected via two linked registries (population cohort). Overall survival (OS) was estimated by Kaplan–Meier. Hazard ratios were determined by a Cox proportional hazard model. Results: The literature cohort and population cohort consisted of 225 and 291 patients, respectively. In the literature cohort, melanoma was the most frequent origin (33.8%), while colorectal cancer was the most frequent origin in the population cohort (23.7%). Prognosis was poor with median OS ranging from 6.0 to 22.5 months in the literature and population cohorts, respectively. Age, timing of GBm (synchronous/metachronous) and primary tumor origin were independent prognostic factors for OS. Discussion: Metastases to the gallbladder are rare and carry a poor prognosis. Differences between both cohorts can be attributable to the biased reporting of tumor types that are more easily recognized as GBm because of distinct histological features.

Aims: Outcomes of colorectal cancer (CRC) treatment and survival have steadily improved during the past decades, accompanied by an increased risk of developing second primary tumours and metastatic tumours at unusual sites. Metastatic CRC can show mucosal colonisation, thereby mimicking a second primary tumour. This potential confusion could lead to incorrect diagnosis and consequently inadequate treatment of the patient. The aim of this study was to differentiate between metastatic CRC and a second primary (gallbladder cancer, GBC) using a combination of standard histopathology and molecular techniques.Methods and results: Ten consecutive patients with both CRC and GBC were identified in our region using the Dutch National Pathology Archive (PALGA). Two patients served as negative controls. Histology of GBC was reviewed by nine pathologists. A combination of immunohistochemistry, microsatellite analysis, genomewide DNA copy number analysis and targeted somatic mutation analysis was used to aid in differential diagnosis. In two patients, CRC and GBC were clonally related, as confirmed by somatic mutation analysis. For one case, this was confirmed by genomewide DNA copy number analysis. However, in both cases, pathologists initially considered the GBC as a second primary tumour.Conclusions: Metastatic CRC displaying mucosal colonisation is often misinterpreted as a second primary tumour. A combination of traditional histopathology and molecular techniques improves this interpretation, and lowers the risk of inadequate treatment.