Interactive enhancer hubs (iHUBs) mediate transcriptional reprogramming and adaptive resistance in pancreatic cancer.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) displays a remarkable propensity towards therapy resistance. However, molecular epigenetic and transcriptional mechanisms enabling this are poorly understood. In this study, we aimed to identify novel mechanistic approaches to overcome or prevent resistance in PDAC. DESIGN: We used in vitro and in vivo models of resistant PDAC and integrated epigenomic, transcriptomic, nascent RNA and chromatin topology data. We identified a JunD-driven subgroup of enhancers, called interactive hubs (iHUBs), which mediate transcriptional reprogramming and chemoresistance in PDAC. RESULTS: iHUBs display characteristics typical for active enhancers (H3K27ac enrichment) in both therapy sensitive and resistant states but exhibit increased interactions and production of enhancer RNA (eRNA) in the resistant state. Notably, deletion of individual iHUBs was sufficient to decrease transcription of target genes and sensitise resistant cells to chemotherapy. Overlapping motif analysis and transcriptional profiling identified the activator protein 1 (AP1) transcription factor JunD as a master transcription factor of these enhancers. JunD depletion decreased iHUB interaction frequency and transcription of target genes. Moreover, targeting either eRNA production or signaling pathways upstream of iHUB activation using clinically tested small molecule inhibitors decreased eRNA production and interaction frequency, and restored chemotherapy responsiveness in vitro and in vivo. Representative iHUB target genes were found to be more expressed in patients with poor response to chemotherapy compared with responsive patients. CONCLUSION: Our findings identify an important role for a subgroup of highly connected enhancers (iHUBs) in regulating chemotherapy response and demonstrate targetability in sensitisation to chemotherapy.

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.

Neoadjuvant Chemotherapy Switch in Borderline Resectable/Locally Advanced Pancreatic Cancer.

BACKGROUND: Neoadjuvant chemotherapy (NAC) is an integral part of preoperative treatment for patients with borderline resectable/locally advanced (BR/LA) pancreatic ductal adenocarcinoma (PDAC). The identification of a chemotherapeutic regimen that is both effective and tolerable is critical for NAC to be of oncologic benefit. After initial first-line (FL) NAC, some patients have lack of response or therapeutic toxicities precluding further treatment with the same regimen; optimal decision making regarding this patient population is unclear. Chemotherapy switch (CS) may allow for a larger proportion of patients to undergo curative-intent resection after NAC. METHODS: We reviewed our surgical database for patients undergoing combinatorial NAC for BR/LA PDAC. Variant histologic exocrine carcinomas, intraductal papillary mucinous neoplasm-associated PDAC, and patients without research consent were excluded. RESULTS: Overall, 468 patients with BR/LA PDAC receiving FL chemotherapy were reviewed, of whom 70% (329/468) continued with FL chemotherapy followed by surgical resection. The remaining 30% (139/468) underwent CS, with 72% (100/139) of CS patients going on to curative-intent surgical resection. Recurrence-free survival (RFS) and overall survival (OS) were not significantly different between the resected FL and CS cohorts (30.0 vs. 19.1 months, p = 0.13, and 41.4 vs. 36.4 months, p = 0.94, respectively) and OS was significantly worse in those undergoing CS without subsequent resection (19 months, p < 0.0001). On multivariable analysis, carbohydrate antigen (CA) 19-9 and pathologic treatment responses were predictors of RFS and OS. CONCLUSION: CS in patients undergoing NAC for BR/LA pancreatic cancer does not incur oncologic detriment. The incorporation of CS into NAC treatment sequencing may allow a greater proportion of patients to proceed to curative-intent surgery.

FDG-PET Predicts Neoadjuvant Therapy Response and Survival in Borderline Resectable/Locally Advanced Pancreatic Adenocarcinoma.

BACKGROUND: Neoadjuvant therapy (NAT) is used in borderline resectable/locally advanced (BR/LA) pancreatic ductal adenocarcinoma (PDAC). Anatomic imaging (CT/MRI) poorly predicts response, and biochemical (CA 19-9) markers are not useful (nonsecretors/nonelevated) in many patients. Pathologic response highly predicts survival post-NAT, but is only known postoperatively. Because metabolic imaging (FDG-PET) reveals primary tumor viability, this study aimed to evaluate our experience with preoperative FDG-PET in patients with BR/LA PDAC in predicting NAT response and survival. METHODS: We reviewed all patients with resected BR/LA PDAC who underwent NAT with FDG-PET within 60 days of resection. Pre- and post-NAT metabolic (FDG-PET) and biochemical (CA 19-9) responses were dichotomized in addition to pathologic responses. We compared post-NAT metabolic and biochemical responses as preoperative predictors of pathologic responses and recurrence-free survival (RFS) and overall survival (OS). RESULTS: We identified 202 eligible patients. Post-NAT, 58% of patients had optimization of CA 19-9 levels. Major metabolic and pathologic responses were present in 51% and 38% of patients, respectively. Median RFS and OS times were 21 and 48.7 months, respectively. Metabolic response was superior to biochemical response in predicting pathologic response (area under the curve, 0.86 vs 0.75; P<.001). Metabolic response was the only univariate preoperative predictor of OS (odds ratio, 0.25; 95% CI, 0.13-0.40), and was highly correlated (P=.001) with pathologic response as opposed to biochemical response alone. After multivariate adjustment, metabolic response was the single largest independent preoperative predictor (P<.001) for pathologic response (odds ratio, 43.2; 95% CI, 16.9-153.2), RFS (hazard ratio, 0.37; 95% CI, 0.2-0.6), and OS (hazard ratio, 0.21; 95% CI, 0.1-0.4). CONCLUSIONS: Among patients with post-NAT resected BR/LA PDAC, FDG-PET highly predicts pathologic response and survival, superior to biochemical responses alone. Given the poor ability of anatomic imaging or biochemical markers to assess NAT responses in these patients, FDG-PET is a preoperative metric of NAT efficacy, thereby allowing potential therapeutic alterations and surgical treatment decisions. We suggest that FDG-PET should be an adjunct and recommended modality during the NAT phase of care for these patients.

Patency rates of hepatic arterial resection and revascularization in locally advanced pancreatic cancer.

BACKGROUND: Arterial resection (AR) for pancreatic adenocarcinoma is increasingly considered at specialized centers. We aimed to examine the incidence, risk factors, and outcomes of hepatic artery (HA) occlusion after revascularization. METHODS: We included patients undergoing HA resection with interposition graft (IG) or primary end-to-end anastomoses (EE). Complete arterial occlusion (CAO) was defined as "early" (EO) or "late" (LO) before/after 90 days respectively. Kaplan-Meier and change-point analysis for CAO was performed. RESULTS: HA resection was performed in 108 patients, IG in 61% (66/108) and EE in 39% (42/108). An equal proportion (50%) underwent HA resection alone or in combination with celiac and/or superior mesenteric artery. CAO was identified in 18% of patients (19/108) with arterial IG least likely to occlude (p=0.019). Hepatic complications occurred in 42% (45/108) and correlated with CAO, symptomatic patients, venous resection, and postoperative portal venous patency. CAO-related operative mortality was 4.6% and significantly higher in EO vs LO (p = 0.046). Median CAO occlusion was 126 days. With change-point analysis, CAO was minimal beyond postoperative day 158. CONCLUSION: CAO can occur in up to 18% of patients and the first 5-month post-operative period is critical for surveillance. LO is associated with better outcomes compared to EO unless there is inadequate portal venous inflow.

The use of patient factors to improve the prediction of operative duration using laparoscopic cholecystectomy.

BACKGROUND: Reliable prediction of operative duration is essential for improving patient and care team satisfaction, optimizing resource utilization and reducing cost. Current operative scheduling systems are unreliable and contribute to costly over- and underestimation of operative time. We hypothesized that the inclusion of patient-specific factors would improve the accuracy in predicting operative duration. METHODS: We reviewed all elective laparoscopic cholecystectomies performed at a single institution between 01/2007 and 06/2013. Concurrent procedures were excluded. Univariate analysis evaluated the effect of age, gender, BMI, ASA, laboratory values, smoking, and comorbidities on operative duration. Multivariable linear regression models were constructed using the significant factors (p < 0.05). The patient factors model was compared to the traditional surgical scheduling system estimates, which uses historical surgeon-specific and procedure-specific operative duration. External validation was done using the ACS-NSQIP database (n = 11,842). RESULTS: A total of 1801 laparoscopic cholecystectomy patients met inclusion criteria. Female sex was associated with reduced operative duration (-7.5 min, p < 0.001 vs. male sex) while increasing BMI (+5.1 min BMI 25-29.9, +6.9 min BMI 30-34.9, +10.4 min BMI 35-39.9, +17.0 min BMI 40 + , all p < 0.05 vs. normal BMI), increasing ASA (+7.4 min ASA III, +38.3 min ASA IV, all p < 0.01 vs. ASA I), and elevated liver function tests (+7.9 min, p < 0.01 vs. normal) were predictive of increased operative duration on univariate analysis. A model was then constructed using these predictive factors. The traditional surgical scheduling system was poorly predictive of actual operative duration (R 2 = 0.001) compared to the patient factors model (R 2 = 0.08). The model remained predictive on external validation (R 2 = 0.14).The addition of surgeon as a variable in the institutional model further improved predictive ability of the model (R 2 = 0.18). CONCLUSION: The use of routinely available pre-operative patient factors improves the prediction of operative duration during cholecystectomy.

Impact of single-incision laparoscopic cholecystectomy (SILC) versus conventional laparoscopic cholecystectomy (CLC) procedures on surgeon stress and workload: a randomized controlled trial.

INTRODUCTION: Single-incision laparoscopic cholecystectomy (SILC) may lead to higher patient satisfaction; however, SILC may expose the surgeon to increased workload. The goal of this study was to compare surgeon stress and workload between SILC and conventional laparoscopic cholecystectomy (CLC). METHODS: During a double-blind randomized controlled trial comparing patient outcomes for SILC versus CLC (NCT0148943), surgeon workload was assessed by four measures: surgery task load index questionnaire (Surg-TLX), maximum heart rate, salivary cortisol level, and instruments usability survey. The maximum heart rate and salivary cortisol levels were sampled from the surgeon before the random assignment of the surgical procedure, intraoperatively after the cystic duct was clipped, and at skin closure. After each procedure, the surgeon completed the Surg-TLX and an instrument usability survey. Student's t tests, Wilcoxon rank sum test, and Kruskal-Wallis nonparametric ANOVAs on the dependent variables by the technique (SILC vs. CLC) were performed with α = 0.05. RESULTS: Twenty-three SILC and 25 CLC procedures were included in the intent-to-treat analysis. No significant differences were observed between SILC and CLC for patient demographics and procedure duration. SILC had significantly higher post-surgery surgeon maximum heart rates than CLC (p < 0.05). SILC also had significantly higher mean change in the maximum heart rate between during and post-procedure (p < 0.05) than CLC. Salivary cortisol level was significantly higher during SILC than CLC (p < 0.01). Awkward manipulation of the instruments and limited fine motions were reported significantly more frequently with SILC than CLC (p < 0.01). In the surgeon-reported Surg-TLX, subscale of physical demand was significantly more demanding for SILC than CLC (p < 0.05). CONCLUSIONS: Surgeon heart rate, salivary cortisol level, instrument usability, and Surg-TLX ratings indicate that SILC is significantly more stressful and physically demanding than the CLC. Surgeon stress and workload may impact patients' outcomes; thus, ergonomic improvement on SILC is necessary.

Baseline Characteristics and Use of Pretherapeutic 18 F-Fluorodeoxyglucose-PET for Pancreatic Cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal malignancy. Surgical resection is the only curative modality combined with neoadjuvant chemotherapy to improve survival. Given the limitations of traditional responses such as cross-sectional imaging (CT/MRI) or tumor markers, carbohydrate antigen 19-9 (CA19-9), the 2023 National Comprehensive Cancer Network guidelines included 18 F-fluorodeoxyglucose (FDG)-PET as an adjunct to assess response to neoadjuvant chemotherapy. There are common misconceptions on the metabolic activity (tumor avidity) in PDAC so we aimed to describe the baseline characteristics and use of FDG-PET in a cohort of treatment-naive patients with PDAC. STUDY DESIGN: A single-center retrospective study was conducted capturing all biopsy-proven, treatment-naive patients with PDAC who underwent either baseline FDG-PET/CT or FDG-PET/MRI imaging between 2008 and 2023. Baseline FDG-PET characteristics were collected, including primary tumors' maximum standardized uptake value defined as metabolic activity (FDG uptake) of tumor compared with surrounding pancreatic parenchymal background, and the identification of extrapancreatic metastatic disease. RESULTS: We identified 1,095 treatment-naive patients with PDAC who underwent baseline FDG-PET imaging at diagnosis. CA19-9 was elevated in 76% of patients. Overall, 96.3% (1,054) of patients had FDG-avid tumors with a median maximum standardized uptake value of 6.4. FDG-PET also identified suspicious extrapancreatic metastatic lesions in 50% of patients, with a higher proportion (p < 0.001) in PET/MRI (59.9%) vs PET/CT (44.3%). After controlling for CA19-9 elevation, PET/MRI was superior in detection of extrapancreatic lesions compared with PET/CT. CONCLUSIONS: FDG-PET has significant use in PDAC as a baseline imaging modality earlier neoadjuvant therapy given the majority of tumors are FDG-avid. FDG-PET can identify additional extrapancreatic suspicious lesions allowing for optimal initial staging, with PET/MRI having increased sensitivity over PET/CT.

Metabolome-wide association identifies altered metabolites and metabolic pathways in the serum of patients with cholangiocarcinoma.

Background & Aims: Metabolomic and lipidomic analyses provide an opportunity for novel biological insights. Cholangiocarcinoma (CCA) remains a highly lethal cancer with limited response to systemic, targeted, and immunotherapeutic approaches. Using a global metabolomics and lipidomics platform, this study aimed to discover and characterize metabolomic variations and associated pathway derangements in patients with CCA. Methods: Leveraging a biospecimen collection, including samples from patients with digestive diseases and normal controls, global serum metabolomic and lipidomic profiling was performed on 213 patients with CCA and 98 healthy controls. The CCA cohort of patients included representation of intrahepatic, perihilar, and distal CCA tumours. Metabolome-wide association studies utilizing multivariable linear regression were used to perform case-control comparisons, followed by pathway enrichment analysis, CCA subtype analysis, and disease stage analysis. The impact of biliary obstruction was evaluated by repeating analyses in subsets of patients only with normal bilirubin levels. Results: Of the 420 metabolites that discriminated patients with CCA from controls, decreased abundance of cysteine-glutathione disulfide was most closely associated with CCA. Additional conjugated bile acid species were found in increased abundance even in the absence of clinically relevant biliary obstruction denoted by elevated serum bilirubin levels. Pathway enrichment analysis also revealed alterations in caffeine metabolism and mitochondrial redox-associated pathways in the serum of patients with CCA. Conclusions: The presented metabolomic and lipidomic profiling demonstrated multiple alterations in the serum of patients with CCA. These exploratory data highlight novel metabolic pathways in CCA and support future work in therapeutic targeting of these pathways and the development of a precision biomarker panel for diagnosis. Impact and implications: Cholangiocarcinoma (CCA) is a highly lethal hepatobiliary cancer with limited treatment response, highlighting the need for a better understanding of the disease biology. Using a global metabolomics and lipidomics platform, we characterized distinct changes in the serum of 213 patients with CCA compared with healthy controls. The results of this study elucidate novel metabolic pathways in CCA. These findings benefit stakeholders in both the clinical and research realms by providing a foundation for improved disease diagnostics and identifying novel targets for therapeutic design.

Combined Antitumor Effect of the Serine Protease Urokinase Inhibitor Upamostat and the Sphingosine Kinase 2 Inhibitor Opaganib on Cholangiocarcinoma Patient-Derived Xenografts.

Upamostat is an orally available small-molecule serine protease inhibitor that is a highly potent inhibitor of trypsin 1, trypsin 2, trypsin 3 (PRSS1/2/3), and the urokinase-type plasminogen activator (uPA). These enzymes are expressed in many cancers, especially during tissue remodeling and subsequent tumor cell invasion. Opaganib (ABC294640), a novel, orally available small molecule is a selective inhibitor of the phosphorylation of sphingosine to sphingosine-1-phosphate (S-1-P) by sphingosine kinase 2 (SPHK2). Both sphingosine kinase 1 (SPHK1) and SPHK2 are known to regulate the proliferation-inducing compound S-1-P. However, SPHK2 is more critical in cancer pathogenesis. The goal of this project was to investigate the potential antitumor effects of upamostat and opaganib, individually and in combination, on cholangiocarcinoma (CCA) xenografts in nude mice. PAX165, a patient-derived xenograft (PDX) from a surgically resected CCA, expresses substantial levels of SPHK2, PRSS1, PRSS2, and PRSS3. Four groups of 18 mice each were treated with upamostat, opaganib, both, or vehicle. Mouse weights and PAX165 tumor volumes were measured. Tumor volumes in the upamostat, opaganib, and upamostat plus opaganib groups were significantly decreased compared to the control group.

Cancer "Avatars": Patient-Derived Xenograft Growth Correlation with Postoperative Recurrence and Survival in Pancreaticobiliary Cancer.

BACKGROUND: Pancreaticobiliary (PB) cancers are a diverse group of cancers with poor prognoses and high rates of recurrence after resection. Patient-derived xenografts (PDX), created from surgical specimens, provide a reliable preclinical research platform and high-fidelity cancer model from which to study these malignancies with consistent recapitulation of their original patient tumors in vivo. However, the relationship between PDX engraftment success (growth or no growth) and patient oncologic outcomes has not been well studied. We sought to evaluate the correlation between successful PDX engraftment and survival in several PB exocrine carcinomas, including the pancreatic and biliary tract. STUDY DESIGN: In accordance with IRB and Institutional Animal Care and Use Committee protocols and with appropriate consent and approval, excess tumor tissue obtained from surgical patients was implanted into immunocompromised mice. Mice were monitored for tumor growth to determine engraftment success. PDX tumors were verified to recapitulate their tumors of origin by a hepatobiliary pathologist. Xenograft growth was correlated with clinical recurrence and overall survival data. RESULTS: A total of 384 PB xenografts were implanted. The successful engraftment rate was 41% (158/384). We found that successful PDX engraftment was highly associated with both recurrence-free survival (p < 0.001) and overall survival (p < 0.001) outcomes. Successful PDX tumor generation occurs significantly in advance of clinical recurrences in their corresponding patients (p < 0.001). CONCLUSIONS: Successful PB cancer PDX models predict recurrence and survival across tumor types and may provide critical lead time to alter patients' surveillance or treatment plans before cancer recurrence.

Therapeutic Efficacy of Temsirolimus in a Patient-derived Model of Metastatic Fibrolamellar Hepatocellular Carcinoma.

BACKGROUND/AIM: Fibrolamellar hepatocellular carcinoma (FLHCC) is a rare tumor presenting in younger patients without chronic liver disease. Up to 80-100% develop recurrent disease, necessitating additional surgery or systemic treatment. Systemic options and pre-clinical treatment studies are lacking. We previously described patient-derived xenograft (PDX) development, allowing for pre-clinical studies. Herein, we develop FLHCC PDX models and utilize these to define tumor characteristics and determine the efficacy of systemic agents. MATERIALS AND METHODS: Primary and lymph node metastatic tumor tissues were obtained at the time of FLHCC resection in two patients. Tumor lysates were screened for protein upregulation. Cell lines were generated from metastatic and primary tumor tissue. The viability of the cell lines was assessed after treatment with temsirolimus, gemcitabine/oxaliplatin, and FOLFIRINOX. Two PDX models were developed from metastatic tissue. For in vivo studies, tumor-bearing mice were treated with temsirolimus, FOLFIRINOX, and Gemcitabine/oxaliplatin. RESULTS: PDX models were successfully generated from metastatic FLHCC, which closely recapitulated the original tumor. Upregulation of mTOR was seen in metastatic tissue compared to primary tumors. Cell lines from metastatic tissue demonstrated significant sensitivity to temsirolimus. In vivo testing of PDX models demonstrated a significant response to single-agent temsirolimus with minimal toxicity. CONCLUSION: Herein, we demonstrate the feasibility of developing PDX models that closely recapitulate FLHCC. Upregulation of mTOR was seen in metastatic tissue compared to primary tissue. The efficacy of mTOR inhibition with temsirolimus treatment suggests that the upregulation of the mTOR pathway may be a significant mechanism for growth in metastatic lesions and a potential target for therapeutics.

Technical Outcomes of Porto-Mesenteric Venous Reconstruction in Pancreatic Resection Using Autologous Left Renal Vein Graft as Conduit.

BACKGROUND: Portal or superior mesenteric vein (PV-SMV) resection and reconstruction is sometimes required during pancreatic tumor resection. In patients requiring segmental venous resection with interposition grafting, the left renal vein (LRV) is an accessible autologous solution. However, long-term patency outcomes of the LRV as an interposition conduit in this setting have not been analyzed. STUDY DESIGN: We conducted a retrospective analysis of patients undergoing pancreatic resection with PV-SMV reconstruction using LRV between 2002 and 2022. The primary outcome was PV-SMV patency at last follow-up, assessed with postoperative CT scans and analyzed using Kaplan-Meier survival methods that account for variation in follow-up duration. Development of any postoperative acute kidney injury within 7 days of surgery and morbidity were secondary outcomes. RESULTS: The study cohort includes 65 patients who underwent LRV harvest; 60 (92%) ultimately underwent successful reconstruction with harvested LRV graft. Kaplan-Meier 2-year estimated patency rate of the LRV graft was 88%, with no cases of complete occlusion. Six (10%) patients experienced graft stenosis. Nine of 61 (15%) patients experienced grade II or III acute kidney injury, 6 of 9 returning to normal renal function before discharge. No difference in median serum creatinine was observed at baseline, 6 and 12 months from surgery. LRV remnant thrombosis was seen in 7 of 65 (11%) patients. Only 3 of 61 (5%) patients had persistent acute kidney injury caused by complications unrelated to LRV harvesting. CONCLUSIONS: Autologous LRV graft was a reliable conduit for segmental PV-SMV reconstruction, resulting in a high patency rate and marginal impact on renal function. LRV harvest is a safe and potentially ideal surgical option for PV-SMV reconstruction in pancreatic surgery.

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

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.

Mixed Acinar Neuroendocrine Carcinoma of the Pancreas: Comparative Population-Based Epidemiology of a Rare and Fatal Malignancy in The United States.

Mixed acinar neuroendocrine carcinoma of the pancreas (MANEC-P) is an extremely rare malignancy with a poor prognosis. However, epidemiological estimates of MANEC-P remain unknown. This study aimed to estimate and compare the incidence, prevalence, and cancer-specific survival (CSS) of MANEC-P in the United States (US). Patients with MANEC-P were identified through the Surveillance, Epidemiology, and End Results (SEER) and National Program of Cancer Registries databases between 2000-2017. The primary outcomes included age-adjusted incidence rate, limited-duration prevalence, and CSS. A total of 630 patients were identified for the incidence analysis and 149 for the prevalence and CSS analyses. The MANEC-P incidence rate was 0.011 per 100,000 individuals, which was the lowest among pancreatic cancer histologic subtypes. The incidence rate was significantly higher in men and Black races and peaked at 75-79 years of age. The incidence rate was the lowest in the midwestern region (0.009) and the highest in the northeastern US (0.013). The 17-year prevalence was 0.00005%, indicating that 189 patients were alive in the United States at the beginning of 2018. The median CSS of MANEC-P was estimated to be 41 (23, 69) months. In conclusion, MANEC-P is very rare, and its incidence rate has been steady in the US over the last two decades. MANEC-P has a poor prognosis and is the 5th leading cause of pancreatic cancer-related death in the US.

Identification of a ΔNp63-Dependent Basal-Like A Subtype-Specific Transcribed Enhancer Program (B-STEP) in Aggressive Pancreatic Ductal Adenocarcinoma.

A major hurdle to the application of precision oncology in pancreatic cancer is the lack of molecular stratification approaches and targeted therapy for defined molecular subtypes. In this work, we sought to gain further insight and identify molecular and epigenetic signatures of the Basal-like A pancreatic ductal adenocarcinoma (PDAC) subgroup that can be applied to clinical samples for patient stratification and/or therapy monitoring. We generated and integrated global gene expression and epigenome mapping data from patient-derived xenograft models to identify subtype-specific enhancer regions that were validated in patient-derived samples. In addition, complementary nascent transcription and chromatin topology (HiChIP) analyses revealed a Basal-like A subtype-specific transcribed enhancer program in PDAC characterized by enhancer RNA (eRNA) production that is associated with more frequent chromatin interactions and subtype-specific gene activation. Importantly, we successfully confirmed the validity of eRNA detection as a possible histologic approach for PDAC patient stratification by performing RNA-ISH analyses for subtype-specific eRNAs on pathologic tissue samples. Thus, this study provides proof-of-concept that subtype-specific epigenetic changes relevant for PDAC progression can be detected at a single-cell level in complex, heterogeneous, primary tumor material. IMPLICATIONS: Subtype-specific enhancer activity analysis via detection of eRNAs on a single-cell level in patient material can be used as a potential tool for treatment stratification.

Microsurgical skills training course and impact on trainee confidence and workload.

INTRODUCTION: Our objective was to measure the impact of a basic microsurgery training course on trainees' confidence and workload in performing microsurgery. METHODS: A prospective study of participants in an accredited 5-day microsurgery course over a 3-month period. The confidence and workload of the participants were assessed after the first and final day. The workload was assessed using the validated NASA Task Load Index composed of 6 subscales scored on a 20-point visual analog scale (VAS). Confidence was assessed over 5 dimensions on a 5-point VAS for anastomosis performance, vessels preparation, knot tying, training effectiveness, and future practice of microsurgery. RESULTS: A total of 31 participants completed the study with 55% reporting some previous microsurgery experience. All confidence dimensions improved significantly after completing the course, regardless of prior experience (p<0.01). Those with prior experience started and finished the course at higher confidence levels in anastomosis performance and vessel preparation than the non-experienced group (p<0.05). Overall workload showed a downward trend (improvement) at the end of the course, but no significant changes in the experienced and non-experienced groups (p>0.05). Most participants scored above the 50% "sustainability threshold" for mental demand, both before (71%) and after the course (73%), however, perceived physical demand significantly reduced, p = 0.01. CONCLUSION: The microsurgery course teaches fundamental skills and principles; therefore, it has merit in those who will utilize these skills in their future practice. Although there is increased confidence in skill acquisition, the impact on perceived workload during a short 5-day basic microsurgery course did not significantly change.

"Answers in hours": A prospective clinical study using nanopore sequencing for bile duct cultures.

BACKGROUND: Surgical site infection is a major source of morbidity in patients undergoing pancreatic head resection and is often from organisms in intraoperative bile duct cultures. As such, many institutions use prolonged prophylactic antibiotics and tailor based on bile duct cultures. However, standard cultures take days, leaving many patients unnecessarily on prolonged antibiotics. Nanopore sequencing can provide data in hours and, thus, has the potential to improve antibiotic stewardship. The present study investigates the feasibility of nanopore sequencing in intraoperative bile samples. METHODS: Patients undergoing pancreatic head resection were included. Intra-operative bile microbial profiles were determined with standard cultures and nanopore sequencing. Antibiotic recommendations were generated, and time-to-results determined for both methods. Organism yields, resistance patterns, antibiotic recommendations, and costs were compared. RESULTS: Out of 42 patients, 22 (52%) had samples resulting in positive standard cultures. All positive standard cultures had microbes detected using nanopore sequencing. All 20 patients with negative standard cultures had negative nanopore sequencing. Nanopore sequencing detected more bacterial species compared to standard cultures (10.5 vs 4.4, p < 0.05) and more resistance genotypes (10.3 vs 2.7, p < 0.05). Antimicrobial recommendations based on nanopore sequencing provided coverage for standard cultures in 27 out of 44 (61%) samples, with broader coverage recommended by nanopore sequencing in 13 out of 27 (48%) of these samples. Nanopore sequencing results were faster (8 vs 98 hours) than standard cultures but had higher associated costs ($165 vs $38.49). CONCLUSION: Rapid microbial profiling with nanopore sequencing is feasible with broader organism and resistance profiling compared to standard cultures. Nanopore sequencing has perfect negative predictive value and can potentially improve antibiotic stewardship; thus, a randomized control trial is under development.

Intraoperative bile duct cultures in patients undergoing pancreatic head resection: Prospective comparison of bile duct swab versus bile duct aspiration.

BACKGROUND: Postoperative surgical site infection is a major source of morbidity after pancreatic head resections, and data suggest bacterobilia as a leading cause. Some centers use intraoperative bile duct cultures to guide postoperative antimicrobial prophylaxis. This prospective study evaluates culture differences between traditional bile duct swab versus bile duct aspiration intraoperative samples. METHODS: Prospective patients undergoing pancreatic head resection with both bile duct swab and bile duct aspiration were included. Cultures were reviewed for organism characteristics. Any growth of organisms was considered a positive culture. Bile duct swab yield and characteristics were compared with bile duct aspiration. Postoperative surgical site infection complications were compared to bile duct culture results. RESULTS: Fifty patients were included. Bile duct aspiration resulted in a significantly higher median number of organisms compared to bile duct swab (6 vs 3; P < .001). There were no differences in the number of patients (37 vs 33) having positive bile duct aspiration and bile duct swab cultures (P = .385). Anaerobic cultures (not possible with bile duct swab) were positive in 21 patients with bile duct aspiration. A total of 37 (74%) patients had preoperative biliary stenting, which highly associated (P < .001) with positive cultures. Bile duct culture organisms correlated with postoperative surgical site infection in 12/17 (71%) patients. CONCLUSION: Use of bile duct aspiration improves intraoperative bile duct culture organism yield over bile duct swab and may improve tailoring of antibiotics in patients undergoing pancreatic head resection.

Molecular Peritoneal Staging for Pancreatic Ductal Adenocarcinoma Using Mutant KRAS Droplet-Digital Polymerase Chain Reaction: Results of a Prospective Clinical Trial.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with predilection for peritoneal dissemination. Accurate peritoneal staging is imperative for treatment recommendations, as one-third of patients develop peritoneal recurrence after resection. Because >90% of PDAC tumors harbor mutant KRAS (mKRAS), we sought to determine feasibility of mKRAS DNA detection in peritoneal lavage (PL) fluid using droplet-digital polymerase chain reaction (ddPCR) via a prospective trial. STUDY DESIGN: Patients with nonmetastatic PDAC undergoing staging laparoscopy with PL were included. PL fluid was sent for cytologic examination, CA19-9/CEA levels, and mKRAS ddPCR assay. Clinically positive laparoscopy was defined as gross metastases or positive cytology. PL mKRAS status was compared with gross findings, cytology, and CA19-9/CEA levels. RESULTS: There were 136 patients enrolled; 70 of 136 (51%) patients received neoadjuvant therapy before PL, and 32 of 136 (24%) patients had clinically positive laparoscopy. Cytology was positive in 17 of 136 (13%) patients, and 22 of 136 (16%) patients had gross metastases. Of patients with gross metastases, only 8 of 22 (36%) had positive cytology; 97 of 136 (71%) patients had mKRAS in PL. PL mKRAS was present in 27 of 32 (84%) clinically positive laparoscopies, with higher mean copy number in clinically positive patients (643 vs 10, p = 0.02). Peritoneal mKRAS was positive in an additional 70 clinically negative patients. CONCLUSIONS: This prospective study establishes the feasibility of PL mKRAS detection. Clinically positive disease was identified in 1 in 4 staging laparoscopies. Although PL mKRAS was highly associated with clinically positive findings, many clinically negative laparoscopies had detectable PL mKRAS, suggesting that standard staging may be inadequate. Longer follow-up will elucidate utility of this promising molecular assay.