Synergistic combination of cytotoxic chemotherapy and cyclin-dependent kinase 4/6 inhibitors in biliary tract cancers

Background and aims: Biliary tract cancers (BTCs) are uncommon, but highly lethal, gastrointestinal malignancies. Gemcitabine/cisplatin is a standard-of-care systemic therapy, but has a modest impact on survival and harbors toxicities, including myelosuppression, nephropathy, neuropathy, and ototoxicity. Whereas BTCs are characterized by aberrations activating the cyclinD1/cyclin-dependent kinase (CDK)4/6/CDK inhibitor 2a/retinoblastoma pathway, clinical use of CDK4/6 inhibitors as monotherapy is limited by lack of validated biomarkers, diffident preclinical efficacy, and development of acquired drug resistance. Emerging studies have explored therapeutic strategies to enhance the antitumor efficacy of CDK4/6 inhibitors by the combination with chemotherapy regimens, but their mechanism of action remains elusive.Approach and results: Here, we report in vitro and in vivo synergy in BTC models, showing enhanced efficacy, reduced toxicity, and better survival with a combination comprising gemcitabine/cisplatin and CDK4/6 inhibitors. Furthermore, we demonstrated that abemaciclib monotherapy had only modest efficacy attributable to autophagy-induced resistance. Notably, triplet therapy was able to potentiate efficacy through elimination of the autophagic flux. Correspondingly, abemaciclib potentiated ribonucleotide reductase catalytic subunit M1 reduction, resulting in sensitization to gemcitabine.Conclusions: As such, these data provide robust preclinical mechanistic evidence of synergy between gemcitabine/cisplatin and CDK4/6 inhibitors and delineate a path forward for translation of these findings to preliminary clinical studies in advanced BTC patients.</p

Rituximab Decreases Lymphoproliferative Tumor Formation in Hepatopancreaticobiliary and Gastrointestinal Cancer Patient-Derived Xenografts

High engraftment rates are critical to any patient-derived xenograft (PDX) program and the loss of PDX models due to the development of lymphoproliferative tumors (LTs) is costly and inefficient. We hypothesized that routine injection of rituximab, an anti-CD20 antibody, at the time of implantation would reduce the incidence of LTs. Rituximab injection was added to the standard PDX engraftment protocol. Univariate analysis and multivariate logistic regression were used to determine the significance of various factors. A total of 811 generations of PDX were implanted with 406 receiving rituximab with implantation. On multivariable analysis, rituximab was an independent factor for decreased LT formation across the entire cohort (OR 0.465, 95% CI 0.271-0.797, p = 0.005). Hepatocellular carcinomas (OR 0.319, 95% CI 0.107-0.949, p = 0.040) and cholangiocarcinomas (OR 0.185, 95% CI 0.049-0.696, p = 0.113) were the specific malignant histologic subtypes that demonstrated the greatest benefit. The frequency of LTs decreased across the entire cohort with rituximab administration and PDX tumors that are traditionally associated with higher rates of LT formation, HCCs and CCAs, appear to benefit the most from rituximab treatment. Routine use of rituximab at the time of tumor implantation may have significant programmatic benefits for laboratories that utilize PDX models

Successful Secondary Engraftment of Pancreatic Ductal Adenocarcinoma and Cholangiocarcinoma Patient-Derived Xenografts After Previous Failed Primary Engraftment

BACKGROUND: Patient-derived xenografts (PDX) provide histologically accurate cancer models that recapitulate patient malignant phenotype and allow for highly correlative oncologic in-vivo downstream translational studies. Primary PDX engraftment failure has significant negative consequences on programmatic efficiency and resource utilization and is due to either no tumor growth or development of lymphoproliferative tumors. We aimed to determine if secondary engraftment of previously cryopreserved patient tumor tissues would allow salvage of PDX models that failed previous primary engraftment and increase overall engraftment efficiency. METHODS: Patient hepatobiliary and pancreatic cancers that failed primary engraftment were identified. Previously cryopreserved primary patient cancerous tissues were implanted into immunodeficient mice (NOD/SCID). Mice were monitored, growth metrics calculated, and secondary engraftment outcomes were recorded. Established PDX were verified and compared to original patient tissue through multiple generations by a GI pathologist. RESULTS: We identified 55 patient tumors that previously failed primary engraftment: no tumor growth (n = 46, 84%) or lymphoproliferative tumor (LT) (n = 9, 16%). After secondary implantation using cryopreserved patient tissues, 29 new histologically validated PDX models were generated with an overall secondary engraftment rate of 53% for all tumor types with greatest yield in pancreatic and biliary tract cancers. Of the secondary engraftment failures (n = 26), 21 (38%) were due to no growth and 5 (9%) developed LT. CONCLUSION: Secondary PDX engraftment using cryopreserved primary cancerous is feasible after previous failed engraftment attempts and can result in a 50% increase in overall engraftment efficiency with decreases in LT formation. This technique allows for salvage of critical patient PDX models that would otherwise not exist. SYNOPSIS: Patient-derived xenografts have many important translational applications however can be limited by engraftment failure. We demonstrate optimized methodology utilizing cryopreservation of primary tumor tissue that allows for subsequent successful secondary engraftment and creation of PDX models that failed previous primary engraftment and allowed salvage of patient PDX models that would otherwise not exist

Successful Secondary Engraftment of Pancreatic Ductal Adenocarcinoma and Cholangiocarcinoma Patient-Derived Xenografts After Previous Failed Primary Engraftment

BACKGROUND: Patient-derived xenografts (PDX) provide histologically accurate cancer models that recapitulate patient malignant phenotype and allow for highly correlative oncologic in-vivo downstream translational studies. Primary PDX engraftment failure has significant negative consequences on programmatic efficiency and resource utilization and is due to either no tumor growth or development of lymphoproliferative tumors. We aimed to determine if secondary engraftment of previously cryopreserved patient tumor tissues would allow salvage of PDX models that failed previous primary engraftment and increase overall engraftment efficiency. METHODS: Patient hepatobiliary and pancreatic cancers that failed primary engraftment were identified. Previously cryopreserved primary patient cancerous tissues were implanted into immunodeficient mice (NOD/SCID). Mice were monitored, growth metrics calculated, and secondary engraftment outcomes were recorded. Established PDX were verified and compared to original patient tissue through multiple generations by a GI pathologist. RESULTS: We identified 55 patient tumors that previously failed primary engraftment: no tumor growth (n = 46, 84%) or lymphoproliferative tumor (LT) (n = 9, 16%). After secondary implantation using cryopreserved patient tissues, 29 new histologically validated PDX models were generated with an overall secondary engraftment rate of 53% for all tumor types with greatest yield in pancreatic and biliary tract cancers. Of the secondary engraftment failures (n = 26), 21 (38%) were due to no growth and 5 (9%) developed LT. CONCLUSION: Secondary PDX engraftment using cryopreserved primary cancerous is feasible after previous failed engraftment attempts and can result in a 50% increase in overall engraftment efficiency with decreases in LT formation. This technique allows for salvage of critical patient PDX models that would otherwise not exist. SYNOPSIS: Patient-derived xenografts have many important translational applications however can be limited by engraftment failure. We demonstrate optimized methodology utilizing cryopreservation of primary tumor tissue that allows for subsequent successful secondary engraftment and creation of PDX models that failed previous primary engraftment and allowed salvage of patient PDX models that would otherwise not exist

Patient-Derived Xenografts Can Be Reliably Generated from Patient Clinical Biopsy Specimens

BACKGROUND: Patient-derived xenografts (PDX) are clinically relevant human cancer models that can be used to guide individualized medicine. We aimed to generate PDX models from clinically obtained biopsy specimens (surgical or image-guided) hypothesizing that low volume biopsy specimens could provide sufficient viable tissue to successfully engraft PDX models from patients with unresectable or metastatic disease. MATERIALS AND METHODS: We maintain a prospective high volume gastrointestinal malignancy PDX program. With informed consent and institutional approval, biopsy specimens (surgical or image-guided) were obtained from patients with unresectable or metastatic tumors: pancreatic adenocarcinoma (PDAC), cholangiocarcinoma, gastric and gallbladder carcinoma. Biopsies were implanted into immunodeficient mice. Tumor growth was monitored, viable tumor was passed into subsequent generations, and histopathology was confirmed. RESULTS: In this study, biopsy specimens from 29 patients were used for PDX engraftment. Successful PDX engraftment was variable with highest engraftment rates in gastric and gallbladder carcinoma specimens (100%) compared to engraftment rates of 33% and 29% in PDAC and cholangiocarcinoma respectively. PDX models created from metastasis biopsies compared to unresectable primary tumor tissue demonstrated higher engraftment rates (69% versus 15.4%, p = 0.001). PDX models demonstrated higher engraftment rates when biopsies were obtained during surgical operations (n = 15) compared to image-guided (n = 14) (73% versus 14%, p = 0.003). Patient age, pretreatment status, or ischemic time was not different between biopsy methods. CONCLUSIONS: PDX models can be successfully created from clinical biopsy specimens in patients with metastatic or unresectable GI cancers. The use of clinical biopsy specimens for PDX engraftment can expand the repertoire of stage-specific PDX models for downstream basic/translational research

In vivo label-free optical signatures of chemotherapy response in human pancreatic ductal adenocarcinoma patient-derived xenografts

Abstract Pancreatic cancer is a devastating disease often detected at later stages, necessitating swift and effective chemotherapy treatment. However, chemoresistance is common and its mechanisms are poorly understood. Here, label-free multi-modal nonlinear optical microscopy was applied to study microstructural and functional features of pancreatic tumors in vivo to monitor inter- and intra-tumor heterogeneity and treatment response. Patient-derived xenografts with human pancreatic ductal adenocarcinoma were implanted into mice and characterized over five weeks of intraperitoneal chemotherapy (FIRINOX or Gem/NabP) with known responsiveness/resistance. Resistant and responsive tumors exhibited a similar initial metabolic response, but by week 5 the resistant tumor deviated significantly from the responsive tumor, indicating that a representative response may take up to five weeks to appear. This biphasic metabolic response in a chemoresistant tumor reveals the possibility of intra-tumor spatiotemporal heterogeneity of drug responsiveness. These results, though limited by small sample size, suggest the possibility for further work characterizing chemoresistance mechanisms using nonlinear optical microscopy