Diagnostic Utility of Next-Generation Sequencing in Circulating Free DNA and a Comparison With Matched Tissue in Gallbladder Carcinoma.

Mutation detection for therapy monitoring in cell-free DNA (cfDNA) is used clinically for some malignancies. Gallbladder carcinoma (GBC) presents a diagnostic challenge and has limited late-stage treatment options. To our knowledge, this novel study examines, for the first time, genomic alterations in cfDNA from GBC to assess diagnostic accuracy and therapeutic options. The concordance of somatic genomic changes in cfDNA and DNA from paired tumor tissue was analyzed. Paired serum and tissue samples from 40 histologically proven GBC, 20 cholecystitis, and 4 normal (noninflamed gallbladder) controls were included. Targeted next-generation sequencing with a 22-gene panel (Colon and Lung Cancer Research Panel v2, Thermo Scientific) in cfDNA and tumor tissue with high depth and uniform coverage on ION Personal Genome Machine (ION, PGM) was performed. A spectrum of 223 mutations in cfDNA and 225 mutations in formalin-fixed paraffin-embedded tissue DNA were identified in 22 genes. Mutations ranged from 1 to 17 per case. In cfDNA frequent alterations were in TP53 (85.0%), EGFR (52.5%), MET (35%) CTNNB1, SMAD4, BRAF (32.5%), PTEN (30%), FGFR3 and PIK3CA (27.5%), NOTCH1 (25.0%), and FBXW7 and ERBB4 (22.5%). At least one clinically actionable mutation was identified in all cfDNA samples. Paired samples shared 149 of 225 genetic abnormalities (66.2%). Individual gene mutation concordance ranged from 44.44% to 82.0% and was highest for EGFR (82.0%), BRAF and NOTCH1 (80.0%), TP53 (73.08%), MET (72.22%), and ERBB4 (71.42%) with a significant level of correlation (Spearman r = 0.91, P ≤ .0001). The sensitivity and specificity of the TP53 gene at the gene level was the highest (94.44% and 100.0%, respectively). Overall survival was higher for ERBB4 and ERBB2 mutant tumors. The adenocarcinoma subtype revealed specific genetic changes in ERBB4, SMAD4, ERBB2, PTEN, KRAS, and NRAS. NGS-based cfDNA mutation profiling can be used to diagnose GBC before surgery to guide treatment decisions. Targeted therapy identified in GBC included SMAD4, ERBB2, ERBB4, EGFR, KRAS, BRAF, PIK3CA, MET, and NRAS.

Prediction of neoplastic gallbladder polyps in patients with different age level based on preoperative ultrasound: a multi-center retrospective real-world study.

BACKGROUND: The prevalence of neoplastic polyps in gallbladder polyps (GPs) increases sharply with age and is associated with gallbladder carcinoma (GBC). This study aims to predict neoplastic polyps and provide appropriate treatment strategies based on preoperative ultrasound features in patients with different age level. METHODS: According to the age classification of WHO, 1523 patients with GPs who underwent cholecystectomy from January 2015 to December 2019 at 11 tertiary hospitals in China were divided into young adults group (n=622), middle-aged group (n=665) and elderly group (n=236). Linear scoring models were established based on independent risk variables screened by the Logistic regression model in different age groups. The area under ROC (AUC) to evaluate the predictive ability of linear scoring models, long- and short- diameter of GPs. RESULTS: Independent risk factors for neoplastic polyps included the number of polyps, polyp size (long diameter), and fundus in the young adults and elderly groups, while the number of polyps, polyp size (long diameter), and polyp size (short diameter) in the middle-aged groups. In different age groups, the AUCs of its linear scoring model were higher than the AUCs of the long- and short- diameter of GPs for differentiating neoplastic and non-neoplastic polyps (all P<0.05), and Hosmer-Lemeshow goodness of fit test showed that the prediction accuracy of the linear scoring models was higher than the long- and short- diameter of GPs (all P>0.05). CONCLUSION: The linear scoring models of the young adults, middle-aged and elderly groups can effectively distinguish neoplastic polyps from non-neoplastic polyps based on preoperative ultrasound features.

Prognostic value of combined preoperative inflammatory marker neutrophil-lymphocyte ratio and platelet distribution width in patients with gallbladder carcinoma.

BACKGROUND: The neutrophil-lymphocyte ratio (NLR) and platelet distribution width (PDW) are associated with poor prognosis in various cancers. We aimed to analyze the prognostic value of the combination of preoperative NLR and PDW in patients with gallbladder carcinoma (GBC). METHODS: A total of 287 GBC patients who underwent curative-intent surgery in our institution was included. The relationship between NLR and PDW and clinicopathological features were analyzed. The receiver operating characteristic (ROC) curves were used to determine the optimal cutoff value for NLR and PDW. Overall survival (OS) was estimated using the Kaplan-Meier method. Meanwhile, the univariate and multivariate Cox regression models were used to assess the risk factors for OS. RESULTS: The optimal cutoff value of NLR and PDW was 3.00 and 14.76, respectively. In addition, survival analysis demonstrated that patients with NLR > 3.00 and PDW > 14.76 had a worse prognosis than patients with NLR ≤ 3.00 and PDW ≤ 14.76, respectively. The multivariate analysis showed that NLR and PDW were independent prognostic factors in the patients with GBC. When we combined NLR and PDW, the area under the ROC curve increased from 0.665 (NLR) and 0.632 (PDW) to 0.676. Moreover, the 1-, 3-, and 5-year OS of group A (patients with NLR ≤ 3.00 and PDW ≤ 14.76), group B (patients with either of NLR > 3.00 or PDW > 14.76) and group C (patients with NLR > 3.00 and PDW > 14.76) were 88.7%, 62.6%, 28.1%, 65.1%, 26.9%, 13.1%, and 34.8%, 8.3%, 0%, respectively. CONCLUSION: The combination of NLR and PDW may serve as a significant prognostic biomarker for GBC patients superior to either NLR or PDW alone.

A deep unenhanced area on contrast-enhanced ultrasound suggests T2 gallbladder carcinoma.

CE-US (Advanced Dynamic Flow, mechanical index: 1.6) in the perfusion phase (48 s after injection of Sonazoid®, marked with a red underline): A heterogeneously enhanced, sessile elevated lesion with a clearly visible deep unenhanced area (arrowhead), 3 mm in diameter, at the fundus.

GBCdb: RNA expression landscapes and ncRNA-mRNA interactions in gallbladder carcinoma.

Gallbladder carcinoma (GBC), an aggressive malignant tumor of the biliary system, is characterized by high cellular heterogeneity and poor prognosis. Fewer data have been reported in GBC than other common cancer types. Multi-omics data will contribute to the understanding of the molecular mechanisms of cancer, cancer diagnosis and prognosis. Herein, to provide better understanding of the molecular events in GBC pathogenesis, we developed GBCdb ( http://tmliang.cn/gbc/ ), a user-friendly interface for the query and browsing of GBC-associated genes and RNA interaction networks using published multi-omics data, which also included experimentally supported data from different molecular levels. GBCdb will help to elucidate the potential biological roles of different RNAs and allow for the exploration of RNA interactions in GBC. These resources will provide an opportunity for unraveling the potential molecular features of Gallbladder carcinoma.

Microcystin-leucine-arginine promotes the development of gallbladder carcinoma via regulating ELAC2.

Gallbladder carcinoma (GBC) is the most prevalent cancer of the bile tract, with unexpected GBC accounting for almost half of all GBC cases in some tertiary medical centers. Although the involvement of microcystin-leucine-arginine (MC-LR) in the development of intrahepatic cholangiocarcinoma has been established, there is a paucity of data regarding its association with GBC. The present study aims to investigate whether MC-LR level in the gallbladder of patients is associated with GBC development and, if so, to characterize the underlying mechanism in GBC cells. Our clinical data revealed that MC-LR level was significantly increased in GBC patients compared to patients with gallbladder stones only (P = 0.009). Moreover, our findings demonstrated that MC-LR could promote the proliferation and metastasis of human GBC cell lines. Furthermore, ELAC2 was identified as a critical mRNA involved in GBC progression through RNA sequencing. Collectively, our study suggests that MC-LR might be involved in the development of GBC by modulating the expression of ELAC2.

Tissue proteome analysis for profiling proteins associated with lymph node metastasis in gallbladder cancer.

Lymph node (LN) metastasis is the earliest sign of metastatic spread and an established predictor of poor outcome in gallbladder cancer (GBC). Patients with LN positive GBC have a significantly worse survival (median survival- 7 months) than patients with LN negative disease (median survival- ~ 23 months) in spite of standard treatment which includes extended surgery followed by chemotherapy, radiotherapy and targeted therapy. This study aims at understanding the underlying molecular processes associated with LN metastasis in GBC. Here, we used iTRAQ-based quantitative proteomic analysis using tissue cohort comprising of primary tumor of LN negative GBC (n = 3), LN positive GBC (n = 4) and non-tumor controls (Gallstone disease, n = 4), to identify proteins associated with LN metastasis. A total of 58 differentially expressed proteins (DEPs) were found to be specifically associated with LN positive GBC based on the criteria of p value ≤ 0.05, fold change ≥ 2 and unique peptides ≥ 2. These include the cytoskeleton and associated proteins such as keratin, type II cytoskeletal 7 (KRT7), keratin type I cytoskeletal 19 (KRT19), vimentin (VIM), sorcin (SRI) and nuclear proteins such as nucleophosmin Isoform 1 (NPM1), heterogeneous nuclear ribonucleoproteins A2/B1 isoform X1 (HNRNPA2B1). Some of them are reported to be involved in promoting cell invasion and metastasis. Bioinformatic analysis of the deregulated proteins in LN positive GBC using STRING database identified 'neutrophil degranulation' and 'HIF1 activation' to be among the top deregulated pathways. Western blot and IHC analysis showed a significant overexpression of KRT7 and SRI in LN positive GBC in comparison to LN negative GBC. KRT7, SRI and other proteins may be further explored for their diagnostics and therapeutic applications in LN positive GBC.

A Bayesian network prediction model for gallbladder polyps with malignant potential based on preoperative ultrasound.

BACKGROUND: It is important to identify gallbladder polyps (GPs) with malignant potential and avoid unnecessary cholecystectomy by constructing prediction model. The aim of the study is to develop a Bayesian network (BN) prediction model for GPs with malignant potential in a long diameter of 8-15 mm based on preoperative ultrasound. METHODS: The independent risk factors for GPs with malignant potential were screened by χ2 test and Logistic regression model. Prediction model was established and validated using data from 1296 patients with GPs who underwent cholecystectomy from January 2015 to December 2019 at 11 tertiary hospitals in China. A BN model was established based on the independent risk variables. RESULTS: Independent risk factors for GPs with malignant potential included age, number of polyps, polyp size (long diameter), polyp size (short diameter), and fundus. The BN prediction model identified relationships between polyp size (long diameter) and three other variables [polyp size (short diameter), fundus and number of polyps]. Each variable was assigned scores under different status and the probabilities of GPs with malignant potential were classified as [0-0.2), [0.2-0.5), [0.5-0.8) and [0.8-1] according to the total points of [- 337, - 234], [- 197, - 145], [- 123, - 108], and [- 62,500], respectively. The AUC was 77.38% and 75.13%, and the model accuracy was 75.58% and 80.47% for the BN model in the training set and testing set, respectively. CONCLUSION: A BN prediction model was accurate and practical for predicting GPs with malignant potential patients in a long diameter of 8-15 mm undergoing cholecystectomy based on preoperative ultrasound.

A Bayesian network model to predict neoplastic risk for patients with gallbladder polyps larger than 10 mm based on preoperative ultrasound features.

BACKGROUND: Polyp size of 10 mm is insufficient to discriminate neoplastic and non-neoplastic risk in patients with gallbladder polyps (GPs). The aim of the study is to develop a Bayesian network (BN) prediction model to identify neoplastic polyps and create more precise criteria for surgical indications in patients with GPs lager than 10 mm based on preoperative ultrasound features. METHODS: A BN prediction model was established and validated based on the independent risk variables using data from 759 patients with GPs who underwent cholecystectomy from January 2015 to August 2022 at 11 tertiary hospitals in China. The area under receiver operating characteristic curves (AUCs) were used to evaluate the predictive ability of the BN model and current guidelines, and Delong test was used to compare the AUCs. RESULTS: The mean values of polyp cross-sectional area (CSA), long, and short diameter of neoplastic polyps were higher than those of non-neoplastic polyps (P < 0.0001). Independent neoplastic risk factors for GPs included single polyp, polyp CSA ≥ 85 mm 2, fundus with broad base, and medium echogenicity. The accuracy of the BN model established based on the above independent variables was 81.88% and 82.35% in the training and testing sets, respectively. Delong test also showed that the AUCs of the BN model was better than that of JSHBPS, ESGAR, US-reported, and CCBS in training and testing sets, respectively (P < 0.05). CONCLUSION: A Bayesian network model was accurate and practical for predicting neoplastic risk in patients with gallbladder polyps larger than 10 mm based on preoperative ultrasound features.

Comparative analyses between radically re-resected incidental gallbladder carcinoma and primary radically resected gallbladder carcinoma: a single-center experience in China.

PURPOSES: The current study was performed to comparatively evaluate the similarities and differences between cases with radically re-resected incidental gallbladder carcinoma (RRIGBC) and those with primary radically resected gallbladder carcinoma (PRGBC). METHODS: Comparative analysis between patients with RRIGBC and those with PRGBC were performed in terms of clinic-pathological features and long-terms survival. RESULTS: A total of 330 surgically treated GBC patients with 110 patients with IGBC were identified. PRGBCs were generally in a more advanced tumor stage, sharing more aggressive tumor biological features and worse prognosis than those with RRIGBC. Subgroup analyses indicated a comparable prognosis among T1-2 patients between RRIGBC and PRGBC groups. However, among T3-4 patients, patients in the PRGBC group shared a much worse prognosis. Moreover, IGBC itself can be regarded as a prognostic factor but cannot be regarded as an independent prognostic factor. It is the tumor stage which really determined the overall prognosis. CONCLUSION: Patients with RRIGBC were generally in a much earlier tumor stage and shared a much better prognosis than those with PRGBC. IGBC itself can be regarded as a prognostic factor but cannot be regarded as the independent prognostic factors. It is the tumor stage which really determine the overall prognosis.

Metastatic lymph node ratio as an important prognostic factor in advanced gallbladder carcinoma with at least 6 lymph nodes retrieved.

BACKGROUND: The metastatic lymph node (LN) ratio (LNR) has shown to be an important prognostic factor in various gastrointestinal malignancies. Nevertheless, the prognostic significance of LNR in gallbladder carcinoma (GBC) remains to be determined. METHODS: From January 2007 to January 2018, 144 advanced GBC patients (T2-4 stages) who underwent curative surgery with at least 6 LNs retrieved were enrolled. Receiver operating characteristic (ROC) curve was performed to identify the optimal cut-off value for LNR. The clinicopathological features stratified by LNR level were analyzed. Meanwhile, univariate and multivariate Cox regression proportional hazard models were performed to identify risk factors for overall survival (OS). RESULTS: The optimal cut-off point for LNR was 0.28 according to the ROC curve. LNR>0.28 was associated with higher rate of D2 LN dissection (P=0.004) and higher tumor stages (P<0.001). Extent of liver resection, extrahepatic bile duct resection, tumor stage, LNR, margin status, tumor differentiation, and perineural invasion were associated with OS in univariate analysis (all P<0.05). GBC patients with LNR≤0.28 had a significantly longer median OS compared to those with LNR>0.28 (27.5 vs 18 months, P=0.004). Multivariate analysis indicated that tumor stage (T2 vs T3/T4; hazard ratio (HR) 1.596; 95% confidence interval (CI) 1.195-2.132), LNR (≤0.28 vs >0.28; HR 0.666; 95% CI 0.463-0.958), margin status (R0 vs R1; HR 1.828; 95% CI 1.148-2.910), and tumor differentiation (poorly vs well/moderately; HR 0.670; 95% CI 0.589-0.892) were independent prognostic factors for GBC (all P<0.05). CONCLUSIONS: LNR is correlated to advanced GBC prognosis and is a potential prognostic factor for advanced GBC with at least 6 LNs retrieved.

The diagnostic value of staging laparoscopy in gallbladder cancer: a nationwide cohort study.

BACKGROUND: Disseminated disease (DD) is often found at (re-)exploration in gallbladder cancer (GBC) patients. We aimed to assess the yield of staging laparoscopy (SL) and identify predictors for DD. METHODS: This retrospective study included patients from all Dutch academic centres with primary GBC (pGBC) and incidentally diagnosed GBC (iGBC) planned for (re-)resection. The yield of SL was determined. In iGBC, predictive factors for DD were assessed. RESULTS: In total, 290 patients were included. Of 183 included pGBC patients, 143 underwent laparotomy without SL, and 42 (29%) showed DD perioperatively. SL, conducted in 40 patients, identified DD in eight. DD was found in nine of 32 patients who underwent laparotomy after SL. Of 107 included iGBC patients, 100 underwent laparotomy without SL, and 19 showed DD perioperatively. SL, conducted in seven patients, identified DD in one. Cholecystitis (OR = 4.25; 95% CI 1.51-11.91) and primary R1/R2 resection (OR = 3.94; 95% CI 1.39-11.19) were independent predictive factors for DD. CONCLUSIONS: In pGBC patients, SL may identify DD in up to 20% of patients and should be part of standard management. In iGBC patients, SL is indicated after primary resection for cholecystitis and after initial R1/R2 resection due to the association of these factors with DD.

A deep learning model based on contrast-enhanced computed tomography for differential diagnosis of gallbladder carcinoma.

BACKGROUND: Gallbladder carcinoma (GBC) is highly malignant, and its early diagnosis remains difficult. This study aimed to develop a deep learning model based on contrast-enhanced computed tomography (CT) images to assist radiologists in identifying GBC. METHODS: We retrospectively enrolled 278 patients with gallbladder lesions (> 10 mm) who underwent contrast-enhanced CT and cholecystectomy and divided them into the training (n = 194) and validation (n = 84) datasets. The deep learning model was developed based on ResNet50 network. Radiomics and clinical models were built based on support vector machine (SVM) method. We comprehensively compared the performance of deep learning, radiomics, clinical models, and three radiologists. RESULTS: Three radiomics features including LoG_3.0 gray-level size zone matrix zone variance, HHL first-order kurtosis, and LHL gray-level co-occurrence matrix dependence variance were significantly different between benign gallbladder lesions and GBC, and were selected for developing radiomics model. Multivariate regression analysis revealed that age ≥ 65 years [odds ratios (OR) = 4.4, 95% confidence interval (CI): 2.1-9.1, P < 0.001], lesion size (OR = 2.6, 95% CI: 1.6-4.1, P < 0.001), and CA-19-9 > 37 U/mL (OR = 4.0, 95% CI: 1.6-10.0, P = 0.003) were significant clinical risk factors of GBC. The deep learning model achieved the area under the receiver operating characteristic curve (AUC) values of 0.864 (95% CI: 0.814-0.915) and 0.857 (95% CI: 0.773-0.942) in the training and validation datasets, which were comparable with radiomics, clinical models and three radiologists. The sensitivity of deep learning model was the highest both in the training [90% (95% CI: 82%-96%)] and validation [85% (95% CI: 68%-95%)] datasets. CONCLUSIONS: The deep learning model may be a useful tool for radiologists to distinguish between GBC and benign gallbladder lesions.

Contrast-enhanced CT radiomics for prediction of recurrence-free survival in gallbladder carcinoma after surgical resection.

OBJECTIVES: Gallbladder carcinoma (GBC) is the most common and aggressive biliary tract malignancy with high postoperative recurrence rates. This single-center study aimed to develop and validate a radiomics signature to estimate GBC recurrence-free survival (RFS). METHODS: This study retrospectively included 204 consecutive patients with pathologically diagnosed GBC and were randomly divided into development (n = 142) and validation (n = 62) cohorts (7:3). The radiomics features of tumor were extracted from preoperative contrast-enhanced CT imaging for each patient. In the development cohort, the least absolute shrinkage and selection operator (LASSO) Cox regression was employed to develop a radiomics signature for RFS prediction. The patients were stratified into high-score or low-score groups according to their median value of radiomics score. A nomogram was established using multivariable Cox regression by incorporating significant pathological predictors and radiomics signatures. RESULTS: The radiomics signature based on 12 features could discriminate high-risk patients with poor RFS. Multivariate Cox analysis revealed that pT3/4 stage (hazard ratio, [HR] = 2.691), pN2 stage (HR = 3.60), poor differentiation grade (HR = 2.651), and high radiomics score (HR = 1.482) were independent risk variables associated with worse RFS and were incorporated to construct a nomogram. The nomogram displayed good prediction performance in estimating RFS with AUC values of 0.895, 0.935, and 0.907 at 1, 3, and 5 years, respectively. CONCLUSIONS: The radiomics signature and combined nomogram may assist in predicting RFS in GBC patients. KEY POINTS: • A radiomics signature extracted from preoperative contrast-enhanced CT can be a useful tool to preoperatively predict RFS of GBC. • T3/T4 stage, N2, poor tumor differentiation, and high radiomics score were positively associated with postoperative recurrence.

Radical surgery for de novo gallbladder carcinoma-Single-center analysis of prognostic factors and survival outcomes from an endemic region.

BACKGROUND AND OBJECTIVE: Gallbladder cancer (GBC) is an aggressive malignancy where curative resection is possible in few and survival is poor. There are limited data on outcomes in patients with de novo GBC from endemic regions undergoing surgery for curative intent. We report survival outcomes in this group of patients from a region with high incidence of disease. METHODS: We reviewed the records of all GBC patients (2014-2018) and included those who underwent radical cholecystectomy (RC) for de novo GBC. Univariable and multivariable analyses were performed to identify factors influencing recurrence and survival. RESULTS: A total of 649 patients with GBC were evaluated for surgery and curative intent surgery was attempted in 246 (38%) patients. Of these 246 patients, RC was performed in 115 patients, with histologically confirmed de novo GBC. Locally advanced disease (≥stage IIIB) was present in 52 (45.2%) patients. Median time to recurrence and overall survival (OS) were 31 and 36 months, respectively. Lymph node positivity (p = 0.005) and grade significantly influenced OS on multivariable analysis. CONCLUSION: Satisfactory survival outcomes are possible after RC for de novo GBC. Extended resections performed in high volume centers combined with appropriate adjuvant treatment can offer significant survival benefits, with acceptable morbidity and mortality rates.

Neutrophilic inflammation in gallbladder carcinoma correlates with patient survival: A case-control study.

Gallbladder carcinoma is an uncommon malignancy with an overall 5-year survival of less than 5%. Gallbladder carcinoma has been strongly linked with cholelithiasis and chronic inflammation. Case reports and series have described cholecystitis with acute (neutrophilic) inflammation in association with gallbladder carcinoma, although a clear relationship to patient outcome has not been established. Our series included 8 cases of gallbladder carcinoma with high tumor-associated neutrophils (>25 per high power field) that were associated with shorter patient survival (Cox regression coefficient 6.2, p = 0.004) than age- and stage-matched controls. High tumor-associated neutrophils were not associated with gallbladder rupture/perforation or increased bacterial load measured by 16S PCR. Neutrophilic inflammation with gallbladder carcinoma correlates to shorter survival, independent of patient age and stage of carcinoma. The findings suggest that the degree of neutrophilic inflammation may have prognostic significance in specimens from patients with gallbladder carcinoma after cholecystectomy. Further studies with larger case numbers are needed to confirm and generalize these findings.

Single-cell RNA-sequencing atlas reveals an MDK-dependent immunosuppressive environment in ErbB pathway-mutated gallbladder cancer.

BACKGROUND & AIMS: Our previous genomic whole-exome sequencing (WES) data identified the key ErbB pathway mutations that play an essential role in regulating the malignancy of gallbladder cancer (GBC). Herein, we tested the hypothesis that individual cellular components of the tumor microenvironment (TME) in GBC function differentially to participate in ErbB pathway mutation-dependent tumor progression. METHODS: We engaged single-cell RNA-sequencing to reveal transcriptomic heterogeneity and intercellular crosstalk from 13 human GBCs and adjacent normal tissues. In addition, we performed WES analysis to reveal the genomic variations related to tumor malignancy. A variety of bulk RNA-sequencing, immunohistochemical staining, immunofluorescence staining and functional experiments were employed to study the difference between tissues with or without ErbB pathway mutations. RESULTS: We identified 16 cell types from a total of 114,927 cells, in which epithelial cells, M2 macrophages, and regulatory T cells were predominant in tumors with ErbB pathway mutations. Furthermore, epithelial cell subtype 1, 2 and 3 were mainly found in adenocarcinoma and subtype 4 was present in adenosquamous carcinoma. The tumors with ErbB pathway mutations harbored larger populations of epithelial cell subtype 1 and 2, and expressed higher levels of secreted midkine (MDK) than tumors without ErbB pathway mutations. Increased MDK resulted in an interaction with its receptor LRP1, which is expressed by tumor-infiltrating macrophages, and promoted immunosuppressive macrophage differentiation. Moreover, the crosstalk between macrophage-secreted CXCL10 and its receptor CXCR3 on regulatory T cells was induced in GBC with ErbB pathway mutations. Elevated MDK was correlated with poor overall survival in patients with GBC. CONCLUSIONS: This study has provided valuable insights into transcriptomic heterogeneity and the global cellular network in the TME, which coordinately functions to promote the progression of GBC with ErbB pathway mutations; thus, unveiling novel cellular and molecular targets for cancer therapy. LAY SUMMARY: We employed single-cell RNA-sequencing and functional assays to uncover the transcriptomic heterogeneity and intercellular crosstalk present in gallbladder cancer. We found that ErbB pathway mutations reduced anti-cancer immunity and led to cancer development. ErbB pathway mutations resulted in immunosuppressive macrophage differentiation and regulatory T cell activation, explaining the reduced anti-cancer immunity and worse overall survival observed in patients with these mutations.

Prolonged Response to HER2-Directed Therapy in Three Patients with HER2-Amplified Metastatic Carcinoma of the Biliary System: Case Study and Review of the Literature.

HER2 amplification, which results in overexpression of the receptor tyrosine kinase HER2, has been described in a wide variety of malignancies. HER2-targeting agents have been incorporated into the treatment paradigms for HER2-overexpressing breast and gastric cancer. More recently, these agents have shown promise in other gastrointestinal malignancies, such as colon cancer and biliary tract tumors. This study discusses two patients with gallbladder carcinoma and a third with ampullary carcinoma who were able to achieve marked responses to HER2-directed therapy. These cases underscore the importance of molecular analysis for HER2 amplification/HER2 overexpression, irrespective of tumor histology, and highlight a need for further investigation of HER2-directed therapy beyond breast and gastroesophageal cancers. KEY POINTS: Current guidelines recommend molecular assessment for HER2 overexpression exclusively in breast and gastric adenocarcinoma. The focus of this report is on three cases (two biliary tract and one ampullary carcinoma) in which amplification of HER2 or overexpression of HER2 was detected and treatment with HER2-directed therapy resulted in robust responses. These cases exemplify responsiveness of non-breast/gastric histologies to HER2-directed therapies, highlighting several promising new settings for these agents. Testing for amplification of HER2 or overexpression of HER2 should be considered especially in rare diseases with limited treatment options.

Dysplasia and carcinoma of the gallbladder: pathological evaluation, sampling, differential diagnosis and clinical implications.

Pathological evaluation of gallbladder neoplasia remains a challenge. A significant proportion of cases presents as clinically and grossly inapparent lesions, and grossing protocols are not well established. Among epithelial alterations, pseudo-pyloric gland metaplasia is ubiquitous and of no apparent consequence, whereas goblet cell metaplasia and a foveolar change in surface cells require closer attention. Low-grade dysplasia is difficult to objectively define and appears to be clinically inconsequential by itself; however, extra sampling is required to exclude the possibility of accompanying more significant lesions. For high-grade dysplasia ('high-grade BilIN', also known as 'carcinoma in situ'), a complete sampling is necessary to rule out invasion. Designating in-situ or minimally invasive carcinomas limited to muscularis or above as early gallbladder carcinoma (EGBC) helps to alleviate the major geographical differences (West/East) in the criteria for 'invasiveness' to assign a case to pTis or pT1. Total sampling is crucial in proper diagnosis of such cases. A subset of invasive GBCs (5-10%) arise from the intracholecystic neoplasm (ICN, 'adenoma-carcinoma sequence') category. Approximately two-thirds of ICNs have invasive carcinoma. However, this propensity differs by subtype. True 'pyloric gland adenomas' (> 1 cm) are uncommon and scarcely associated with invasive carcinoma. A distinct subtype of ICN composed of tubular, non-mucinous MUC6+ glands [intracholecystic tubular non-mucinous neoplasm (ICTN)] forms a localised pedunculated polyp. Although it is morphologically complex and high-grade, it appears to be invasion-resistant. Some of the invasive carcinoma types in the gallbladder have been better characterised recently with adenosquamous, neuroendocrine, poorly cohesive and mucinous carcinomas often being more advanced and aggressive.

Age, operation time and surgical approach can be used to detect incidental gallbladder carcinoma in cholecystectomy specimens from low-incidence settings.

AIMS: Gallbladders resected for non-neoplastic diseases are systemically examined microscopically to rule out incidental dysplasia and carcinoma. The main aim of this study was to test whether a pre-grossing algorithm can detect incidental gallbladder carcinoma. The secondary aim was to test whether the algorithm can detect high-grade dysplasia. METHODS AND RESULTS: A retrospective study of clinical, pathological and radiological findings in cholecystectomy recipients was performed on a test set to develop a classification and regression tree algorithm. Cholecystectomy cases were included; exclusion criteria were age <18 years, missing pathology reports, preoperative suspicion of neoplastic disease, and cholecystectomy for non-gallbladder oncological disease. Five thousand nine hundred and eighty-two cholecystectomies from 2006 to 2018 were included in the study, with 18 cases of incidental gallbladder carcinoma and 11 cases of high-grade dysplasia. Three hundred and ninety controls were randomly selected for the testing set. Patient age, surgical approach, operation duration, dilatation of the biliary tract and gallbladder gross anomalies were statistically significant distinguishing factors in multivariate analysis (P < 0.00-0.026). Unsupervised testing with a conditional inference tree suggested that age, procedure type and operation duration can be used to identify incidental gallbladder carcinoma from controls, whereas high-grade dysplasia also requires grossing parameters to identify half of the cases (5/11). CONCLUSION: Readily available clinical parameters and postoperative data can be used to detect incidental gallbladder carcinoma. High-grade dysplasia mostly requires grossing and microscopic examination.