A Delay in Adjuvant Therapy Is Associated With Worse Prognosis Only in Patients With Transitional Circulating Tumor Cells After Resection of Pancreatic Ductal Adenocarcinoma.

OBJECTIVES: The aim of the study was to assess the association of circulating tumor cells (CTCs) with survival as a biomarker in pancreatic ductal adenocarcinoma (PDAC) within the context of a delay in the initiation of adjuvant therapy. BACKGROUND: Outcomes in patients with PDAC remain poor and are driven by aggressive systemic disease. Although systemic therapies improve survival in resected patients, factors such as a delay in the initiation of adjuvant therapy are associated with worse outcomes. CTCs have previously been shown to be predictive of survival. METHODS: A retrospective study was performed on PDAC patients enrolled in the prospective CircuLating tUmor cellS in pancreaTic cancER trial (NCT02974764) on CTC-dynamics at the Johns Hopkins Hospital. CTCs were isolated based on size (isolation by size of epithelial tumor cells; Rarecells) and counted and characterized by subtype using immunofluorescence. The preoperative and postoperative blood samples were used to identify 2 CTC types: epithelial CTCs (eCTCs), expressing pancytokeratin, and transitional CTCs (trCTCs), expressing both pancytokeratin and vimentin. Patients who received adjuvant therapy were compared with those who did not. A delay in the receipt of adjuvant therapy was defined as the initiation of therapy ≥8 weeks after surgical resection. Clinicopathologic features, CTCs characteristics, and outcomes were analyzed. RESULTS: Of 101 patients included in the study, 43 (42.5%) experienced a delay in initiation and 20 (19.8%) did not receive adjuvant therapy. On multivariable analysis, the presence of trCTCs ( P =0.002) and the absence of adjuvant therapy ( P =0.032) were associated with worse recurrence-free survival (RFS). Postoperative trCTC were associated with poorer RFS, both in patients with a delay in initiation (12.4 vs 17.9 mo, P =0.004) or no administration of adjuvant chemotherapy (3.4 vs NR, P =0.016). However, it was not associated with RFS in patients with timely initiation of adjuvant chemotherapy ( P =0.293). CONCLUSIONS: Postoperative trCTCs positivity is associated with poorer RFS only in patients who either experience a delay in initiation or no receipt of adjuvant therapy. This study suggests that a delay in the initiation of adjuvant therapy could potentially provide residual systemic disease (trCTCs) a window of opportunity to recover from the surgical insult. Future studies are required to validate these findings and explore the underlying mechanisms involved.

Persistent Circulating Tumor Cells at 1 Year After Oncologic Resection Predict Late Recurrence in Pancreatic Cancer.

OBJECTIVE: The aim of the study was to assess the association between persistent circulating tumor cells (CTCs) and subsequent recurrence in patients who were clinically recurrence free ~12 months postoperatively. BACKGROUND: Circulating tumor cells have been proposed as biomarkers to predict survival in pancreatic cancer. Some patients demonstrate persistent CTCs postoperatively, which could represent minimal residual disease. METHODS: Patients from previously published prospective circulating tumor cell in pancreatic cancer trial without clinical evidence of recurrence 12 months postoperatively and CTC testing performed 9 to 15 months postoperatively were included. The presence of epithelial and transitional CTCs (trCTCs) was evaluated as predictor of recurrence. Kaplan-Meier curve, log-rank test, and Cox model were used for survival analysis. RESULTS: Thirty-three of 129 eligible patients (circulating tumor cell in pancreatic cancer trial) were included. The trCTC-positive and negative patients were well balanced in clinicopathologic features. Patients with trCTCs had a recurrence rate per-person-month of 10.3% compared with 3.1% in trCTCs-negative patients with a median time to recurrence of 3.9 versus 27.1 months, respectively. On multivariable analysis, trCTCs positivity was associated with higher risk of late recurrence (hazard ratio: 4.7, 95% CI, 1.2-18.3, P =0.024). Fourteen (42.4%) patients recurred during the second postoperative year. One-year postoperative trCTCs positivity was associated with a higher rate of recurrence during the second year (odds ratio:13.1, 95% CI, 1.6-1953.4, P =0.028, area under curve=0.72). Integrating clinicopathologic features with trCTCs increased the area under curve to 0.80. A majority of trCTCs-positive patients (N=5, 62.5%) had multisite recurrence, followed by local-only (N=2, 25.0%) and liver-only (N=1, 12.5%) recurrence. This was in striking contrast to trCTCs-negative patients, where a majority (N=6, 66.7%) had a local-only recurrence, followed by liver-only (N=2, 22.2%) and multisite (N=1, 11.1%) recurrence. CONCLUSIONS: In patients deemed to be clinically disease-free 12 months postoperatively, trCTCs positivity is associated with higher rates of subsequent recurrence with distinct patterns of recurrence. CTCs could be used a putative biomarker to guide patient prognostication and management in pancreatic cancer.

Structured CT reporting of pancreatic ductal adenocarcinoma: impact on completeness of information and interdisciplinary communication for surgical planning.

PURPOSE: With the rise in popularity of structured reports in radiology, we sought to evaluate whether free-text CT reports on pancreatic ductal adenocarcinoma (PDAC) staging at our institute met published guidelines and assess feedback of pancreatic surgeons comparing free-text and structured report styles with the same information content. METHODS: We retrospectively evaluated 298 free-text preoperative CT reports from 2015 to 2017 for the inclusion of key tumor descriptors. Two surgeons independently evaluated 50 free-text reports followed by evaluation of the same reports in a structured format using a 7-question survey to assess the usefulness and ease of information extraction. Fisher's exact test and Chi-square test for independence were utilized for categorical responses and an independent samples t test for comparing mean ratings of report quality as rated on a 5-point Likert scale. RESULTS: The most commonly included descriptors in free-text reports were tumor location (99%), liver lesions (97%), and suspicious lymph nodes (97%). The most commonly excluded descriptors were variant arterial anatomy and peritoneal/omental nodularity, which were present in only 23% and 42% of the reports, respectively. For vascular involvement, a mention of the presence or absence of perivascular disease with the main portal vein was most commonly included (87%). Both surgeons' rating of overall report quality was significantly higher for structured reports (p < 0.001). CONCLUSION: Our results indicate that free-text reports may not include key descriptors for staging PDAC. Surgeons rated structured reports that presented the same information as free-text reports but in a template format superior for guiding clinical management, convenience of use, and overall report quality.

Pancreatic circulating tumor cell detection by targeted single-cell next-generation sequencing.

BACKGROUND AND AIMS: Single-cell next-generation sequencing (scNGS) technology has been widely used in genomic profiling, which relies on whole-genome amplification (WGA). However, WGA introduces errors and is especially less accurate when applied to single nucleotide variant (SNV) analysis. Targeted scNGS for SNV without WGA has not been described. We aimed to develop a method to detect circulating tumor cells (CTCs) with DNA SNVs. METHODS: We tested this targeted scNGS method with three driver mutant genes (KRAS/TP53/SMAD4) on one pancreatic cancer cell line AsPC-1 and then applied it to patients with metastatic PDAC for the validation. RESULTS: All single-cell of AsPC-1 and spiked-in AsPC-1 cells in healthy donor blood, which were isolated by the filtration with size or by flow cytometry, were detected by targeted scNGS method. All blood samples from six patients with metastatic PDAC, for the validation of target scNGS method, showed CTCs with SNVs of KRAS/TP53/SMAD4 and the positive confirmation of immunofluorescent stainings with Pan-CK/Vimentin/CD45. Four patients with early stage disease, one patient with benign pancreatic cyst and a healthy control sample all showed concordant results between targeted scNGS and CTC enumeration. CONCLUSIONS: The novel technique of targeted scNGS for SNV analysis, without pre-amplification, is a promising method for identifying and characterizing circulating tumor cells.

Pattern of Invasion in Human Pancreatic Cancer Organoids Is Associated with Loss of SMAD4 and Clinical Outcome.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by extensive local invasion and systemic spread. In this study, we employed a three-dimensional organoid model of human pancreatic cancer to characterize the molecular alterations critical for invasion. Time-lapse microscopy was used to observe invasion in organoids from 25 surgically resected human PDAC samples in collagen I. Subsequent lentiviral modification and small-molecule inhibitors were used to investigate the molecular programs underlying invasion in PDAC organoids. When cultured in collagen I, PDAC organoids exhibited two distinct, morphologically defined invasive phenotypes, mesenchymal and collective. Each individual PDAC gave rise to organoids with a predominant phenotype, and PDAC that generated organoids with predominantly mesenchymal invasion showed a worse prognosis. Collective invasion predominated in organoids from cancers with somatic mutations in the driver gene SMAD4 (or its signaling partner TGFBR2). Reexpression of SMAD4 abrogated the collective invasion phenotype in SMAD4-mutant PDAC organoids, indicating that SMAD4 loss is required for collective invasion in PDAC organoids. Surprisingly, invasion in passaged SMAD4-mutant PDAC organoids required exogenous TGFß, suggesting that invasion in SMAD4-mutant organoids is mediated through noncanonical TGFß signaling. The Rho-like GTPases RAC1 and CDC42 acted as potential mediators of TGFß-stimulated invasion in SMAD4-mutant PDAC organoids, as inhibition of these GTPases suppressed collective invasion in our model. These data suggest that PDAC utilizes different invasion programs depending on SMAD4 status, with collective invasion uniquely present in PDAC with SMAD4 loss. SIGNIFICANCE: Organoid models of PDAC highlight the importance of SMAD4 loss in invasion, demonstrating that invasion programs in SMAD4-mutant and SMAD4 wild-type tumors are different in both morphology and molecular mechanism.

Pancreatic Nerve Sheath Tumors: a Single Institutional Series and Systematic Review of the Literature.

INTRODUCTION: Improvement in imaging has resulted in frequent diagnosis of benign and premalignant pancreatic tumors. Pancreatic nerve sheath (PNS) tumors are one of the rarest pancreatic tumors. Literature on PNS is limited and their biology is poorly understood. Here, we report the largest series of PNS tumors to date and review the literature to evaluate the current data available on PNS tumors. METHODS: An institutional database was used to identify patients who underwent resection for PNS tumors. Clinicopathological characteristics and outcomes of these patients were reported. Furthermore, a review of literature was performed. RESULTS: From January 1994 through December 2016, seven patients underwent resection for PNS tumors. The median age was 57.7 years (IQR, 44.9-61.9) and the sex was approximately equally distributed (male = 4; 57.1%). Three (42.9%) patients were diagnosed incidentally and six (85.7%) were misdiagnosed as having other pancreatic tumors. The median tumor size was 2.1 (IQR 1.8-3.0) cm and six (85.7%) had no nodal disease. At a median follow-up of 15.5 (IQR 13.7-49.3) months, six patients were alive without evidence of disease and one patient was lost to follow-up. The literature review identified 49 studies reporting 54 patients with PNS tumors. Forty-six were misdiagnosed as having other pancreatic tumors. The median tumor size was 3.6 (range 1-20) cm, nodal disease was present in six patients (22.2%), and no patient had distant metastatic disease. At the time of last follow-up, all patients were free of disease. CONCLUSION: This is the largest single institution series on PNS tumors reported to date. These tumors are rare and are often misdiagnosed, given their radiological characteristics. PNS tumors have a benign course of disease and surgical resection results in favorable long-term outcomes.

Integrated Cheminformatics-Molecular Docking Approach to Drug Discovery Against Viruses.

BACKGROUND: In the current study, we present an integrated in silico cheminformaticsmolecular docking approach to screen and test potential therapeutic compounds against viruses. Fluoroquinolones have been shown to inhibit HCV replication by targeting HCV NS3-helicase. Based on this observation, we hypothesized that natural analogs of fluoroquinolones will have similar or superior inhibitory potential while having potentially fewer adverse effects. METHODS: To screen for natural analogs of fluoroquinolones, we devised an integrated in silico Cheminformatics-Molecular Docking approach. We used 17 fluoroquinolones as bait reference, to screen large databases of natural analogs. 10399 natural compounds and their derivatives were retrieved from the databases. From these compounds, molecules bearing physicochemical similarities with fluoroquinolones were analyzed using a cheminformatics-docking approach. RESULTS: From the 10399 compounds screened using our cheminformatics approach, only 20 compounds were found to share physicochemical similarities with fluoroquinolones, while the remaining 10379 compounds were physiochemically different from fluoroquinolones. Molecular docking analysis showed 32 amino acids in the HCV NS3 active site that were most frequently targeted by fluoroquinolones and their natural analogues, indicating a functional similarity between the two groups of compounds. CONCLUSION: This study describes a speedy and inexpensive approach to complement drug discovery and design against viral agents. The in silico analyses we used here can be employed to shortlist promising compounds/putative drugs that can be further tested in wet-lab.

Application of an integrated cheminformatics-molecular docking approach for discovery for physicochemically similar analogs of fluoroquinolones as putative HCV inhibitors.

BACKGROUND: Hepatitis C Virus (HCV) infection is a major public health concern across the globe. At present, direct-acting antivirals are the treatment of choice. However, the long-term effect of this therapy has yet to be ascertained. Previously, fluoroquinolones have been reported to inhibit HCV replication by targeting NS3 protein. Therefore, it is logical to hypothesize that the natural analogs of fluoroquinolones will exhibit NS3 inhibitory activity with substantially lesser side effects. METHOD: In this study, we tested the application of a recently devised integrated in-silico Cheminformatics-Molecular Docking approach to identify physicochemically similar natural analogs of fluoroquinolones from the available databases (Ambinter, Analyticon, Indofines, Specs, and TimTec). Molecular docking and ROC curve analyses were performed, using PatchDock and Graphpad software, respectively, to compare and analyze drug-protein interactions between active natural analogs, Fluoroquinolones, and HCV NS3 protein. RESULT: In our analysis, we were able to shortlist 18 active natural analogs, out of 10,399, that shared physicochemical properties with the template drugs (fluoroquinolones). These analogs showed comparable binding efficacy with fluoroquinolones in targeting 32 amino acids in the HCV NS3 active site that are crucial for NS3 activity. Our approach had around 80 % sensitivity and 70 % specificity in identifying physicochemically similar analogs of fluoroquinolones. CONCLUSION: Our current data suggest that our approach can be efficiently applied to identify putative HCV drug inhibitors that can be taken for in vitro testing. This approach can be applied to discover physicochemically similar analogs of virtually any drug, thus providing a speedy and inexpensive approach to complement drug discovery and design, which can tremendously economize on time and money spent on the screening of putative drugs.

Circulating Tumor DNA as a Clinical Test in Resected Pancreatic Cancer.

PURPOSE: In research settings, circulating tumor DNA (ctDNA) shows promise as a tumor-specific biomarker for pancreatic ductal adenocarcinoma (PDAC). This study aims to perform analytical and clinical validation of a KRAS ctDNA assay in a Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathology-certified clinical laboratory. EXPERIMENTAL DESIGN: Digital-droplet PCR was used to detect the major PDAC-associated somatic KRAS mutations (G12D, G12V, G12R, and Q61H) in liquid biopsies. For clinical validation, 290 preoperative and longitudinal postoperative plasma samples were collected from 59 patients with PDAC. The utility of ctDNA status to predict PDAC recurrence during follow-up was assessed. RESULTS: ctDNA was detected preoperatively in 29 (49%) patients and was an independent predictor of decreased recurrence-free survival (RFS) and overall survival (OS). Patients who had neoadjuvant chemotherapy were less likely to have preoperative ctDNA than were chemo-naïve patients (21% vs. 69%; P < 0.001). ctDNA levels dropped significantly after tumor resection. Persistence of ctDNA in the immediate postoperative period was associated with a high rate of recurrence and poor median RFS (5 months). ctDNA detected during follow-up predicted clinical recurrence [sensitivity 90% (95% confidence interval (CI), 74%-98%), specificity 88% (95% CI, 62%-98%)] with a median lead time of 84 days (interquartile range, 25-146). Detection of ctDNA during postpancreatectomy follow-up was associated with a median OS of 17 months, while median OS was not yet reached at 30 months for patients without ctDNA (P = 0.011). CONCLUSIONS: Measurement of KRAS ctDNA in a CLIA laboratory setting can be used to predict recurrence and survival in patients with PDAC.

The importance of circulating and disseminated tumor cells in pancreatic cancer.

Pancreatic cancer is a lethal disease in a large part due to the systemic nature at the time of diagnosis. In those patients who undergo a potentially curative resection of pancreatic cancer, the overwhelming majority will have systemic relapse. Circulating tumor cells are an important mediator of the development of metastases. Circulating tumor cells have been identified in patients with clinically localized resectable pancreatic cancer and exist as several phenotypes. Mesenchymal and stem cell-like phenotypes of circulating tumor cells predict early recurrence and worse survival. This review focuses on the current understanding of circulating tumor cells in pancreatic cancer and how this information can be used in developing more effective therapy in the future.

Geographical variation and trends in outcomes of laparoscopic spleen-preserving distal pancreatectomy with or without splenic vessel preservation: A meta-analysis.

BACKGROUND: Distal pancreatectomy (DP) is performed to treat tumors of the pancreatic body and tail. Traditionally, splenectomy is performed with a DP, however, laparoscopic spleen-preserving DP (SPDP) using Warshaw's (splenic vessels ligation) or Kimura's (splenic vessels preservation) techniques have been reported. The clinical benefits of using either technique remain unclear. In this study, we conducted a meta-analysis to compare the clinical outcomes of patients undergoing Warshaw's and Kimura SPDP. This is the first study to evaluate the geographical variation in outcomes of Warshaw's and Kimura SPDP. METHODS: Databases of PubMed, Embase, and Cochrane library were used to identify studies reporting Warshaw's and Kimura SPDP. Clinical outcomes were compared. Pooled odds risk and weighted mean difference with 95% confidence interval were calculated using random effect models. RESULTS: Fourteen non-randomized controlled studies involving 945 patients met our selection criteria. 301 (31.9%) patients underwent Warshaw's SPDP; 644 (68.1%) underwent Kimura SPDP. Compared to Warshaw's SPDP, patients undergoing Kimura SPDP had a lower incidence of post-operative complications including spleen infarction (OR = 9.64, 95% CI = 5.79 to 16.05, P < 0.001) and gastric varices (OR = 11.88, 95% CI = 5.11 to 27.66, P < 0.001). The length of surgery was significantly shorter for Warshaw's SPDP (WMD = -18.12, 95%CI = -26.52 to -9.72, p < 0.001). Decreased blood loss was reported for patients undergoing Warshaw's SPDP (WMD = -59.72, 95%CI = -102.01 to -17.43, p = 0.006). There were no differences between the two groups' rates of conversion to an open procedure (P = 0.35), postoperative pancreatic fistula (P = 0.71), need for reoperation (P = 0.25), and length of hospital stay (P = 0.38). CONCLUSION: Both Warshaw's and Kimura are safe SPDP techniques. These data suggest Kimura SPDP is the preferred technique due to less risk of splenic infarct and gastric varices. Despite evidence of regional variation in volume performed (between Kimura and Warshaw's), there are no statistically significant differences in outcomes between these techniques.