Design of an immunohistochemistry biomarker panel for diagnosis of pancreatic adenocarcinoma.

BACKGROUND: Challenges still exist in differentiating pancreatic adenocarcinoma from benign disease. The use of adjuvant testing of tissue biopsies has demonstrated potential diagnostic value. We designed a proof of concept study to first validate four individual immunohistochemistry biomarkers and then combine them into a panel to boost overall diagnostic sensitivity. METHODS: Malignant and benign pancreas from 27 pancreaticoduodenectomy specimens underwent immunohistochemistry staining with VHL, IMP3, S100A4, S100P. Using ROC curve analysis, threshold criteria for number of cells staining were chosen for each biomarker. Biomarkers were then evaluated as a panel for their ability to discriminate malignant from benign specimens. RESULTS: Diagnostic sensitivity of VHL, IMP3, S100A4, and S100P were 75.0%, 79.2%, 45.8%, and 0%. When VHL, IMP3, and S100A4 were grouped into a panel, they were able to distinguish cancer from normal tissue with a sensitivity of 100% and a specificity of 96%. CONCLUSIONS: The high diagnostic value of an IHC panel consisting of VHL, IMP3, and S100A4 on surgical specimens suggests the need for future prospective studies of these biomarkers on biopsy specimens.

Peritoneal carcinomatosis index predicts survival in colorectal patients undergoing HIPEC using oxaliplatin: a retrospective single-arm cohort study.

BACKGROUND: Peritoneal carcinomatosis (PC) from colorectal cancer is associated with poor prognosis. Cytoreductive surgery (CRS) combined with hyperthermic intraperitoneal chemotherapy (HIPEC) has improved survival for patients with colorectal peritoneal carcinomatosis. However, standardization of HIPEC protocols, including which chemotherapeutic agent to use, is lacking in the literature. Therefore, we sought to report survival outcomes from colorectal cancer patients undergoing CRS/oxaliplatin-based HIPEC at our institution over the last 10 years. METHODS: Colorectal PC patients treated with CRS/oxaliplatin-based HIPEC 2004-2015 were included. Demographic, clinical, and oncologic data were abstracted from the medical record. Overall (OS) and disease-free survival (DFS) were calculated using Kaplan-Meier analysis. Univariate/multivariate Cox regression analysis was done. RESULTS: Laparotomy was performed in 113 patients for colorectal PC; 91 completed a curative intent CRS/HIPEC. At 3 and 5 years, OS for the CRS/HIPEC cohort was 75% and 55%, and DFS was 50% and 25%, respectively. On multivariate analysis, incremental increases in peritoneal carcinomatosis index (PCI) were associated with worse OS (p = 0.0001) and DFS (p = 0.0001). Grade III/IV complications were also associated with worse OS. CONCLUSIONS: A standardized regimen of CRS and oxaliplatin-based HIPEC for colorectal PC is effective with favorable OS and DFS and acceptable complication rates.

Liposomal bupivacaine reduces narcotic use and time to flatus in a retrospective cohort of patients who underwent laparotomy.

INTRODUCTION: Sustained release liposomal bupivacaine (LB) is a new pain control option that can reduce opioid use after laparotomy, which is known to prolong ileus, length of stay. METHODS: Sixty-one consecutive patients undergoing laparotomy were treated with a standardized multi-modal therapy (MMT) consisting of IV tylenol, toradol, and morphine/dilaudid PCA. Thirty-one of those patients were additionally treated with LB infiltrated during fascial closure. Endpoints were opioid use, time to flatus, length of stay, and complications. RESULTS: Overall opioid use for 72 h was 78 mg of morphine for the MMT + LB group and 112 mg in the MMT control group (p = 0.04). During 0-24 h s PCA use was similar. However, during 24-48 h PCA use was decreased by 46% in the MMT + LB group (p = 0.038), and decreased by 55% during the 48-72 h period (p = 0.019). Time to flatus was decreased by 1.0 days in the MMT + LB group (p = 0.005). CONCLUSION: Use of LB in laparotomy patients decreases opioid use, time to flatus, and should be considered as a component of post-operative pain control.

A Review of the Long-Term Oncologic Outcomes of Robotic Surgery Versus Laparoscopic Surgery for Colorectal Cancer.

The goal of this review was to compare long-term oncologic outcomes of robotic versus laparoscopic resection of colorectal cancer. A literature search was performed using PubMed, EMBASE, Cochrane, and Medline (2002-2014). Search terms: laparoscopic, robotic, rectal, colon, surgery, oncologic, and outcomes. Studies comparing overall and disease free survival of robotic versus laparoscopic surgery for colorectal cancer were included. Meta-analysis was performed using OpenMeta[Analyst] for Windows 8. Five studies were identified reporting on overall survival, disease free survival, lymph node extraction, and distal and circumferential resection margin. Three hundred and seventeen patients underwent robotic resection and 368 underwent laparoscopic resection, with similar demographics. Operative times were longer with robotic resections, with no difference in estimated blood loss (EBL) or length of stay. The disease stage was distributed similarly in both groups. Similar numbers underwent neo-adjuvant therapy. Laparoscopic resection was associated with 3.2 mm larger distal resection margins (p = 0.04) and 2.2 more lymph nodes removed (p = 0.001), but with equivalent circumferential resection margin status. Disease-free and overall survival was equivalent. Robotic and laparoscopic surgery for colorectal cancer offer comparable overall and disease free survival. Laparoscopic surgery offered a slight advantage in operative time, distal margin, and lymph node yield. Larger, prospective trials are needed to confirm the equivalence of these approaches.

Erratum to: A Review of the Long-Term Oncologic Outcomes of Robotic Surgery Versus Laparoscopic Surgery for Colorectal Cancer.

[This corrects the article DOI: 10.1007/s12262-015-1375-8.].

Cost-effectiveness of the evaluation of a suspicious biliary stricture.

BACKGROUND: Biliary stricture without mass presents diagnostic and therapeutic challenges because the poor sensitivity of the available tests and significant mortality and cost with operation. METHODS: A decision model was developed to analyze costs and survival for 1) investigation first with endoscopic ultrasound (EUS) and fine needle aspiration, 2) investigation first with endoscopic retrograde cholangiopancreatography (ERCP) and brushing, or 3) surgery on every patient. The average age of someone with a biliary stricture was found to be 62-y-old and the rate of cancer was 55%. Incremental cost-effectiveness ratios (ICER) were calculated based on the change in quality adjusted life years (QALYs) and costs (US$) between the different options, with a threshold of $150,000 to determine the most cost-effective strategy. One-way, two-way, and probabilistic-sensitivity analysis were performed to validate the model. RESULTS: ERCP results in 9.05 QALYs and a cost of $34,685.11 for a cost-effectiveness ratio of $3832.33. EUS results in an incremental increase in 0.13 QALYs and $2773.69 for an ICER of $20,840.28 per QALY gained. Surgery resulted in a decrease of 1.37 QALYs and increased cost of $14,323.94 (ICER-$10,490.53). These trends remained within most sensitivity analyses; however, ERCP and EUS were dependent on the test sensitivity. CONCLUSIONS: In patients with a biliary stricture with no mass, the most cost-effective strategy is to investigate the patient before operation. The choice between EUS and ERCP should be institutionally dependent, with EUS being more cost-effective in our base case analysis.

Sensitivity of alternative testing for pancreaticobiliary cancer: a 10-y review of the literature.

BACKGROUND: Biliary strictures present a diagnostic challenge to differentiate benign disease from hepatopancreaticobiliary (HPB) malignancies. Endoscopic retrograde cholangiopancreatography cytology is commonly performed in these patients; however, its sensitivity for diagnosis of HPB malignancy is poor (41.6%). Many adjunctive tests have been investigated to improve the sensitivity of HPB biopsies. To determine the best tests available, however, we reviewed the literature and performed a comparative analysis of all recently investigated tests and their sensitivities. METHODS: A PubMed search identified articles published between 2003 and 2014, describing alternate methods for diagnosing HPB malignancies, reported sensitivity, final pathology, and had data available online. Meta-analysis was conducted for tests with multiple articles. Tests with the highest sensitivity and specificities were reported. RESULTS: A total of 77 studies were identified. Meta-analysis was performed on the sensitivity of EUS-FNA (74.2%), fluorescence in situ hybridization (54.2%), immunostain of insulin-like growth factor 2 mRNA-binding Protein 3 (IMP3; 80.4%), IMP3 + cytology (86.4%), K homology domain containing protein overexpressed in cancer (KOC; 85.9%), S100P (77.8%), serum CA19-9 (69.3%), and K-ras mutations (47.0%) to detect malignancy. Ultimately, 12 tests were identified with superior sensitivity (85.3%-100%) and specificities (81.6%-100%) including stricture scrapping, brush sectioning, IMP3 stain + cytology, IMP3+S100A4, bile carcinoembryonic cell adhesion molecule 6 protein (±CA19-9), bile micro RNA (miRNA)-135b, serum miRNA-RNU2-1f, serum miRNA-21 (+CA19-9), peripheral blood mononuclear cells miRNA-27a-3p (+CA19-9), serum miRNA-16 + miRNA-196a (+CA19-9), peripheral blood mononuclear cells mRNAs h-TERT + CK20 + CEA + C-MET. CONCLUSIONS: We recommend immunostaining with a panel of IMP3+KOC + S100A4 + cytology to achieve maximum sensitivity and specificity from HPB biopsies. One biliary protein (carcinoembryonic cell adhesion molecule 6) and several RNAs (bile and blood) offer exceptional sensitivity and specificity and should be tested prospectively in larger populations. Overall, this review identifies several tests to improve the sensitivity of diagnostic algorithms to identify HPB malignancies.

Sensitivity of endoscopic retrograde cholangiopancreatography standard cytology: 10-y review of the literature.

BACKGROUND: Biliary strictures present a unique diagnostic challenge to clinicians as they can be caused by both benign and malignant conditions. With the high mortalities associated with hepatopancreaticobiliary malignancies, accurate and rapid tissue diagnosis is imperative and typically done before initiation of treatment. However, the exact sensitivity of standard cytology from endoscopic retrograde cholangiopancreatography (ERCP) to diagnose malignancy remains unclear because of wide distribution of reported values in the literature. Furthermore, the use of radical surgery to obtain tissue when cytology is indeterminate has led to questions about the role of ERCP in patients with biliary strictures. METHODS: A PubMed search was conducted using the terms ERCP, cytology, and brushings. Articles reviewed were published between 2002 and 2012, had patient population with biliary stricture, and had ERCP brushing results and final pathology available for review. The cytology and pathology data were abstracted from each study, and the combined overall sensitivity was calculated. RESULTS: Sixteen studies were identified, with sensitivities ranging from 6%-64% and 99% confidence intervals (CIs) ranging from ±6% to ±32%. A combined total of 1556 patients were included, with positive ERCP cytology results in 358 cases. On final pathology, however, 861 patients were positive for malignancy. When the data were combined, we found an overall sensitivity of 41.6% ± 3.2% (99% CI) with a negative predictive value of 58.0% ± 3.2% (99% CI). CONCLUSIONS: ERCP brushings suffer from low sensitivity and negative predictive value. This study questions the utility of ERCP to change the surgical management of these diseases in patients with radiographic evidence of a neoplasm or high suspicion of a malignancy.

Human metapneumovirus nucleoprotein and phosphoprotein interact and provide the minimal requirements for inclusion body formation.

Human metapneumovirus (HMPV) is a recently discovered paramyxovirus of the subfamily Pneumovirinae, which also includes avian pneumovirus and human respiratory syncytial virus (HRSV). HMPV is an important cause of respiratory disease worldwide. To understand early events in HMPV replication, cDNAs encoding the HMPV nucleoprotein (N), phosphoprotein (P), matrix protein (M), M2-1 protein and M2-2 protein were cloned from cells infected with the genotype A1 HMPV wild-type strain TN/96-12. HMPV N and P were shown to interact using a variety of techniques: yeast two-hybrid assays, co-immunoprecipitation and fluorescence resonance energy transfer (FRET). Confocal microscopy studies showed that, when expressed individually, fluorescently tagged HMPV N and P exhibited a diffuse expression pattern in the host-cell cytoplasm of uninfected cells but were recruited to cytoplasmic viral inclusion bodies in HMPV-infected cells. Furthermore, when HMPV N and P were expressed together, they also formed cytoplasmic inclusion-like complexes, even in the absence of viral infection. FRET microscopy revealed that HMPV N and P interacted directly within cytoplasmic inclusion-like complexes. Moreover, it was shown by yeast two-hybrid analysis that the N-terminal 28 aa are required for the recruitment to and formation of cytoplasmic inclusions, but are dispensable for binding to HMPV P. This work showed that HMPV N and P proteins provide the minimal viral requirements for HMPV inclusion body formation, which may be a distinguishing characteristic of members of the subfamily Pneumovirinae.

APOBEC3G multimers are recruited to the plasma membrane for packaging into human immunodeficiency virus type 1 virus-like particles in an RNA-dependent process requiring the NC basic linker.

APOBEC3G is an endogenous host restriction factor that inhibits human immunodeficiency virus (HIV) replication. The antiviral activity of APOBEC3G is dependent upon its incorporation into the virus particle. The mechanisms governing incorporation of APOBEC3G into virus particles are not completely understood. In particular, some investigators have reported that APOBEC3G interacts directly with the nucleocapsid (NC) subunit of Gag, while others have found that an RNA intermediate is required for Gag-APOBEC3G interactions. In this study, we confirmed the RNA dependence of APOBEC3G packaging and performed detailed mapping of the determinants within NC that are required for virion incorporation. Surprisingly, APOBEC3G packaging did not correlate well with the presence of the N-terminal "I," or interaction, domain within NC. Specifically, Gag constructs containing only the N-terminal region of NC packaged minimal amounts of APOBEC3G, while significant levels of APOBEC3G packaging were achieved with Gag constructs containing the basic linker region of NC. Furthermore, membrane-binding experiments revealed that the basic linker region was essential for the membrane association of APOBEC3G in a Gag-APOBEC3G complex. Fluorescence resonance energy transfer was detected between labeled APOBEC3G in cells and in particles, indicating that APOBEC3G is packaged as a multimer that is bound to packaged RNA. Regions of APOBEC3G-Gag colocalization at the plasma membrane were detected that were distinct from the punctate cytoplasmic bodies where APOBEC3G accumulates within the cell. Together, our results indicate that APOBEC3G multimerizes in an RNA-dependent fashion and that RNA-APOBEC3G multimers are recruited to the plasma membrane and subsequently into virion particles by Gag.

AP-3 directs the intracellular trafficking of HIV-1 Gag and plays a key role in particle assembly.

Gag proteins direct the process of retroviral particle assembly and form the major protein constituents of the viral core. The matrix region of the HIV-1 Gag polyprotein plays a critical role in the transport of Gag to the plasma membrane assembly site. Recent evidence indicates that Gag trafficking to late endosomal compartments, including multivesicular bodies, occurs prior to viral particle budding from the plasma membrane. Here we demonstrate that the matrix region of HIV-1 Gag interacts directly with the delta subunit of the AP-3 complex, and that this interaction plays an important functional role in particle assembly. Disruption of this interaction eliminated Gag trafficking to multivesicular bodies and diminished HIV particle formation. These studies illuminate an early step in retroviral particle assembly and provide evidence that the trafficking of Gag to late endosomes is part of a productive particle assembly pathway.