Assessing the Quality of Rectal Cancer Pathology Reports in National Surgical Adjuvant Breast and Bowel Project Protocol R-04/NRG Oncology.

BACKGROUND: Accurate and comprehensive surgical pathology reports are integral to the quality of cancer care. Despite guidelines from the College of American Pathologists, variations in reporting quality continue to exist. OBJECTIVE: The aim of this study was to evaluate the quality of rectal cancer pathology reports and to identify areas of deficiency and potential sources of reporting variations. DESIGN: This is a retrospective analysis of prospectively obtained pathology reports. SETTING: This study is based at the hospitals participating in the National Surgical Adjuvant Breast and Bowel Project Protocol R-04 study. PATIENTS: Patients with rectal cancer undergoing surgical resection between July 2004 and August 2010 were included. MAIN OUTCOME MEASURES: The primary outcomes measured were the adherence to the College of American Pathologists guidelines and the impact of synoptic reporting, academic status, rural/urban setting, and hospital bed size on reporting quality. RESULTS: We identified 1004 surgical pathology reports for rectal cancer surgery from 383 hospitals and 755 pathologists. The overall adherence rate to the College of American Pathologists guidelines was 73.3%. Notable reporting deficiencies were found in several key pathology characteristics, including tumor histologic grade (reporting rate 77.8%), radial margin (84.6%), distance from the closest margin (47.9%), treatment effect (47.1%), and lymphovascular (73.1%)/perineural invasions (35.4%). Synoptic reporting use and urban hospital settings were associated with better adherence rates, whereas academic status and hospital bed size had no impact. Reporting variations existed not only between institutions, but also within individual hospitals and pathologists. There was a trend for improved adherence over time (2005 = 65.7% vs 2010 = 82.3%, p < 0.001), which coincided with the increased adoption of synoptic reporting by pathologists (2005 vs 2010, 9.4% vs 25.3%, p < 0.001). LIMITATIONS: Data were obtained from a restricted setting (ie, hospitals participating in a randomized clinical trial). CONCLUSIONS: Wide variations in the quality of pathology reporting are observed for rectal cancer. The National Accreditation Program for Rectal Cancer mandates that programs meet strict quality standards for surgical pathology reporting. Further improvement is needed in this key aspect of oncology care for patients with rectal cancer. See Video Abstract at http://links.lww.com/DCR/B238.ClinicalTrials.gov registration: NCT00058 EVALUACIÓN DE LA CALIDAD DE LOS INFORMES DE PATOLOGÍA QUIRÚRGICA EN CASOS DE CÁNCER DE RECTO DEL NSABP R-04/ ONCOLOGÍA DEL NRG: Un informe de patología quirúrgica preciso y completo es fundamental en la calidad de atención de pacientes con cáncer. A pesar de las normas establecidas por el Colegio Americano de Patología, la variabilidad en la calidad de los informes es evidente.Evaluar la calidad de los informes de patología en casos de cáncer de recto para así identificar las áreas con deficiencias y las posibles fuentes variables en los mencionados informes.Análisis retrospectivo de informes de patología quirúrgica obtenidos prospectivamente.Hospitales que participan del Protocolo del Estudio Nacional R-04 como Adyuvantes Quirúrgicos de Mama e Intestino.Todos aquellos pacientes con cáncer de recto sometidos a resección quirúrgica entre Julio 2004 y Agosto 2010.Cumplimiento de las normas del Colegio Americano de Patología, del impacto de los informes sinópticos, del estado académico, del entorno rural / urbano y el número de camas hospitalarias en en la calidad de los informes.Identificamos 1,004 informes de patología quirúrgica en casos de cirugía en cáncer de recto en 383 hospitales y 755 patólogos. La tasa general de adherencia a las directivas del Colegio Americano de Patología fue del 73.3%. Se encontraron deficiencias notables en los informes en varias características patológicas clave incluidos, el grado histológico del tumor (tasa de informe 77.8%), margenes radiales (84.6%), distancia del margen más cercano (47.9%), efecto del tratamiento (47.1%) invasión linfovascular (73.1 %) / invasion perineural (35.4%). El uso de informes sinópticos y los entornos hospitalarios urbanos se asociaron con mejores tasas de adherencia, mientras que el estado académico y el número de camas hospitalarias no tuvieron ningún impacto. Hubo variaciones en los informes no solo entre instituciones, sino también dentro de hospitales y patólogos individuales. Hubo una tendencia a una mejor adherencia a lo largo del tiempo (2005 = 65.7% v 2010 = 82.3%, p < 0.001), que coincidió con la mayor adopción de informes sinópticos por parte de los patólogos (2005 v 2010, 9.4% v 25.3%, p < 0.001)Datos obtenidos de un entorno restringido (es decir, hospitales que participan en un ensayo clínico aleatorizado).Se observaron grandes variaciones en la calidad de los informes de patología quirúrgica en casos de cáncer de recto. El Programa Nacional de Acreditación para Cáncer de Recto exige que los programas cumplan con estrictos estándares de calidad para los informes de patología quirúrgica. Se necesita una mejoría adicional en este aspecto clave de la atención oncológica para pacientes con cáncer de recto. Video Resumen en http://links.lww.com/DCR/B238.Registro de Clinical Trials.gov: NCT00058.

Operative Incision and Drainage for Perirectal Abscesses: What Are Risk Factors for Prolonged Length of Stay, Reoperation, and Readmission?

BACKGROUND: Perirectal abscess is a common problem. Despite a seemingly simple disease to manage, clinical outcomes of perirectal abscesses can vary significantly given the wide array of patients who are susceptible to this disease. OBJECTIVE: Our aims were to evaluate the outcomes after operative incision and drainage for perirectal abscess and to examine factors associated with length of stay, reoperations, and readmissions. DESIGN: This was a retrospective analysis of the National Surgical Quality Improvement Program database. SETTINGS: The study was conducted with hospitals participating in the surgical database. PATIENTS: Adult patients undergoing outpatient perirectal abscess procedures from 2011 through 2016 were included. MAIN OUTCOME MEASURES: Study outcomes were length of stay, reoperation, and readmission. RESULTS: We identified 2358 patients undergoing incision and drainage for perirectal abscesses. Approximately 35% of patients required hospital stay. Reoperations occurred in 3.4%, with median time to reoperation of 15.5 days. The majority of reoperations (79.7%) were performed for additional incision and drainage. Readmissions rate was 3.0%, with median time to readmission of 10.5 days. Common indications for readmissions included recurrent/persistent abscess (41.4%) and fever/sepsis (8.6%). Risk factors for hospitalization in multivariable analysis were preoperative sepsis, bleeding disorder, and non-Hispanic black and Hispanic races. For reoperations, risk factors included morbid obesity, preoperative sepsis, and dependent functional status. Lastly, for readmissions, female sex, steroid/immunosuppression, and dependent functional status were significant risk factors. LIMITATIONS: The study was limited by its retrospective analysis and potential selection bias in decisions on hospital stay, reoperation, and readmission. CONCLUSIONS: Suboptimal outcomes after outpatient operative incision and drainage for perirectal abscesses are not uncommon in the United States. In the era of value-based care, additional work is needed to optimize use outcomes for high-risk patients undergoing perirectal incision and drainage. Strategies to prevent inadequate drainage at the time of the initial operative incision and drainage (ie, use of imaging modalities and thorough examination under anesthesia) are warranted to improve patient outcomes. See Video Abstract at http://links.lww.com/DCR/B229. INCISIÓN Y DRENAJE QUIRÚRGICOS DE ABSCESOS PERIRRECTALES: CUALES SON LOS FACTORES DE RIESGO PARA UNA ESTADÍA PROLONGADA, REINTERVENCIÓN Y READMISION?: Los abscesos perirrectales son un problema frecuente. A pesar que parecen ser una afección aparentemente simple de manejar, los resultados clínicos de la incisión y drenaje quirúrgicos pueden variar significativamente dada la amplia variedad de pacientes susceptibles de sufrir esta afección.Evaluar los resultados después de la incisión y el drenaje quirúrgicos de un absceso perirrectal y analizar los factores asociados con la duración de la hospitalización, la reoperación y la readmisión.Análisis retrospectivo de la base de datos del Programa Americano de Mejora de la Calidad Quirúrgica.Hospitales que participan en la base de datos quirúrgica.Pacientes adultos sometidos a incisión y drenaje quirúrgico ambulatorio de un absceso perirrectal desde 2011 hasta 2016.Los resultados del estudio fueron la duración de la hospitalización, la reoperación y el reingreso.Fueron estudiados 2,358 pacientes sometidos a incisión y drenaje por abscesos perirrectales. Aproximadamente el 35% de los pacientes requirieron hospitalización. Las reoperaciones ocurrieron en 3.4% con una mediana de tiempo de reoperación de 15.5 días. La mayoría de las reoperaciones (79.7%) se realizaron para una incisión y drenaje adicionales. La tasa de reingreso fue del 3.0% con una mediana de tiempo de reingreso de 10.5 días. Las indicaciones comunes para los reingresos incluyeron abscesos recurrentes / persistentes (41.4%) y fiebre / sepsis (8.6%). Los factores de riesgo para la hospitalización en el análisis multivariable fueron sepsis preoperatoria, trastorno hemorrágico, raza negra no hispánica y raza hispana. Para las reoperaciones, los factores de riesgo incluyeron obesidad mórbida, sepsis preoperatoria y estado funcional dependiente. Por último, para los reingresos, el sexo femenino, uso de corticoides / inmunosupresores y un estadío funcional dependiente fueron factores de riesgo significativos.Análisis retrospectivo y posible sesgo de selección en las decisiones sobre hospitalización, reoperación y reingreso.Un resultado poco satisfactorio después de la incisión quirúrgica el drenaje de abscesos perirrectales ambulatoriamente no son infrecuentes en los Estados Unidos. En la era de la atención basada en los resultados, se necesita mucho más trabajo para optimizar los mismos en pacientes de alto riesgo sometidos a incisión y drenaje perirrectales. Las estrategias para prevenir el drenaje inadecuado en el momento de la incisión quirúrgica inicial y el drenaje (es decir, el uso de modalidades de imágenes, un examen completo bajo anestesia) son una garantía para mejorar los resultados en estos pacientes. Consulte Video Resumen en http://links.lww.com/DCR/B229.

Biochemical and Skeletal Outcomes of Parathyroidectomy for Normocalcemic (Incipient) Primary Hyperparathyroidism.

BACKGROUND: Normocalcemic (incipient) primary hyperparathyroidism (PHPT) is characterized by inappropriately elevated parathyroid hormone (PTH) levels in the setting of normal serum calcium. The biochemical and skeletal outcomes after parathyroidectomy for normocalcemic PHPT are not well-described. METHODS: All patients who underwent parathyroidectomy for normocalcemic PHPT at a single institution were retrospectively reviewed (2006-2016). Pre- and postoperative calcium, PTH, and bone mineral density (BMD) were compared between patients with normalized versus persistently elevated PTH levels > 6 months after parathyroidectomy. Multivariable Cox regression was used to identify risk factors associated with persistently elevated PTH levels after parathyroidectomy. RESULT: Parathyroidectomy was performed in 71 patients with normocalcemic PHPT, of whom 38 (53.5%) had multi-gland disease. No patients became hypercalcemic, with a median follow-up of 23.1 months. Persistently elevated PTH levels were noted in 33 (46.5%) patients. In multivariable analysis, preoperative PTH > 100 pg/mL was associated with persistently elevated PTH levels after parathyroidectomy. In 38 patients with available pre- and postoperative BMD measurements, the mean preoperative BMD improved + 5.6% (p < 0.01) in patients with normalized PTH, while no significant change was observed in patients with persistently elevated PTH levels (- 2.2%, p = 0.47). CONCLUSIONS: Elevated PTH levels are common after parathyroidectomy for normocalcemic PHPT. Improvements in BMD may be predicated on long-term normalized PTH levels following surgery.

A Prognostic Scoring System for the Prediction of Metastatic Recurrence Following Curative Resection of Pancreatic Neuroendocrine Tumors.

BACKGROUND: Patients with early-stage pancreatic neuroendocrine tumors (PNETs) may develop metastatic recurrences despite undergoing potentially curative pancreas resections. We sought to identify factors predictive of metastatic recurrences and develop a prognostication strategy to predict recurrence-free survival (RFS) in resected PNETs. METHODS: Patients with localized PNETs undergoing surgical resection between 1989 and 2015 were identified. Univariate and multivariate analysis were used to identify potential predictors of post-resection metastasis. A score-based prognostication system was devised using the identified factors. The bootstrap model validation methodology was utilized to estimate the external validity of the proposed prognostication strategy. RESULTS: Of the 140 patients with completely resected early-stage PNETs, overall 5- and 10-year RFS were 84.6% and 67.1%, respectively. The median follow-up was 56 months. Multivariate analysis identified tumor size > 5 cm, Ki-67 index 8-20%, lymph node involvement, and high histologic grade (G3, or Ki-67 > 20%) as independent predictors of post-resection metastatic recurrence. A scoring system based on these factors stratified patients into three prognostic categories with distinct 5-year RFS: 96.9%, 54.8%, and 33.3% (P < 0.0001). The bootstrap model validation methodology projected our proposed prognostication strategy to retain a high predictive accuracy even when applied in an external dataset (validated c-index of 0.81). CONCLUSIONS: The combination of tumor size, LN status, grade, and Ki-67 was identified as the most highly predictive indicators of metastatic recurrences in resected PNETs. The proposed prognostication strategy may help stratify patients for adjuvant therapies, enhanced surveillance protocols and future clinical trials.

A novel multimarker assay for the phenotypic profiling of circulating tumor cells in hepatocellular carcinoma.

Current clinicopathologic staging systems and serum biomarkers poorly discriminate tumor biology in hepatocellular carcinoma (HCC), with high recurrence rates following curative-intent surgical resection and liver transplantation (LT). Identification of accurate biomarkers for improved prognostication and treatment selection is a critical unmet need. We sought to develop a novel "liquid-biopsy" assay capable of detecting HCC circulating tumor cells (CTCs) and characterizing phenotypic subpopulations with prognostic significance. Using HCC cell lines, a tissue microarray, and human blood samples, an antibody cocktail targeting the cell-surface markers asialoglycoprotein receptor (ASGPR), glypican-3, and epithelial cell adhesion molecule was optimized for HCC CTC capture using the NanoVelcro CTC Assay. The ability of HCC CTCs and vimentin (VIM)-positive CTCs (a subpopulation expressing an epithelial-to-mesenchymal phenotype) to accurately discriminate tumor stage, recurrence, progression, and overall survival (OS) was evaluated in a prospective study of 80 patients. Multimarker capture detected greater numbers of CTCs than any individual antibody alone for both cell line and patient samples (P < 0.001). HCC CTCs were identified in 59/61 (97%) patients, and HCC (median, 6 CTCs) and non-HCC patients (median, 1 CTC; area under the receiver operating characteristic curve [AUROC] = 0.92; P < 0.001; sensitivity = 84.2%; specificity = 88.5%) were accurately discriminated. VIM-positive CTCs accurately discriminated early-stage, LT eligible patients (median, 0 CTCs) from locally advanced/metastatic, LT ineligible patients (median, 6 CTCs; AUROC = 0.89; P = 0.001; sensitivity = 87.1%; specificity = 90.0%), and predicted OS for all patients (hazard ratio [HR], 2.21; P = 0.001), and faster recurrence after curative-intent surgical or locoregional therapy in potentially curable early-stage HCC (HR, 3.14; P = 0.002). In conclusion, we developed a novel multimarker CTC enrichment assay that detects HCC CTCs with high efficiency and accuracy. A phenotypic subpopulation of VIM-positive CTCs appears to signify the presence of aggressive underlying disease and occult metastases and may have important implications for treatment selection. Liver Transplantation 24 946-960 2018 AASLD.

Circulating Tumor Cells Predict Occult Metastatic Disease and Prognosis in Pancreatic Cancer.

BACKGROUND: Occult metastatic tumors, below imaging thresholds, are a limitation of staging systems that rely on cross-sectional imaging alone and are a cause of the routine understaging of pancreatic ductal adenocarcinomas (PDACs). We investigated circulating tumor cells (CTCs) as a preoperative predictor of occult metastatic disease and as a prognostic biomarker for PDAC patients. EXPERIMENTAL DESIGN: A total of 126 patients (100 with cancer, 26 with benign disease) were enrolled in our study and CTCs were identified and enumerated from 4 mL of venous blood using the microfluidic NanoVelcro assay. CTC enumeration was correlated with clinicopathologic variables and outcomes following both surgical and systemic therapies. RESULTS: CTCs were identified in 78% of PDAC patients and CTC counts correlated with increasing stage (ρ = 0.42, p < 0.001). Of the 53 patients taken for potentially curative surgery, 13 (24.5%) had occult metastatic disease intraoperatively. Patients with occult disease had significantly more CTCs than patients with local disease only (median 7 vs. 1 CTC, p < 0.0001). At a cut-off of three or more CTCs/4 mL, CTCs correctly identified patients with occult metastatic disease preoperatively (area under the receiver operating characteristic curve 0.82, 95% confidence interval (CI) 0.76-0.98, p < 0.0001). CTCs were a univariate predictor of recurrence-free survival following surgery [hazard ratio (HR) 2.36, 95% CI 1.17-4.78, p = 0.017], as well as an independent predictor of overall survival on multivariate analysis (HR 1.38, 95% CI 1.01-1.88, p = 0.040). CONCLUSIONS: CTCs show promise as a prognostic biomarker for PDAC patients at all stages of disease being treated both medically and surgically. Furthermore, CTCs demonstrate potential as a preoperative biomarker for identifying patients at high risk of occult metastatic disease.

Risk Factors Associated With Reoperation and Disease-Specific Mortality in Patients With Medullary Thyroid Carcinoma.

Importance: The association of initial neck dissection with recurrence in medullary thyroid carcinoma (MTC) has not been evaluated on a population level to date. Objective: To elucidate risk factors associated with reoperation in MTC and disease-specific mortality. Design, Setting, and Participants: A retrospective analysis was performed of hospital data obtained from the California Cancer Registry and the Office of Statewide Health Planning and Development from January 1, 1999, through December 31, 2012. The dates of the analysis were January 1, 1999, to December 31, 2012. A population-based sample of 953 patients with MTC was identified. Patients who underwent thyroid surgery and had a minimum postoperative follow-up of 2 years (n = 609) were included in the analysis. Exposure: Initial neck dissection in MTC. Main Outcomes and Measures: Recurrent MTC leading to reoperation and disease-specific mortality. Results: Of the 609 patients with MTC who underwent thyroid surgery, the mean (SD) patient age at diagnosis was 52.6 (17.5) years, and 60.8% (n = 370) of the patients were female. The mean (SD) tumor size was 2.8 (2.0) cm. Although initial central neck dissection is recommended by published MTC guidelines, only 35.5% (216 of 609) of patients underwent central neck dissection at the time of the initial thyroidectomy. The rate of reoperation was 16.3% (99 of 609), and the median time to reoperation was 6.4 months. The presence of lymph node metastasis increased the risk of reoperation (hazard ratio [HR], 3.43; 95% CI, 2.00-5.90), while central and lateral neck dissection performed at the initial operation was protective (HR, 0.53; 95% CI, 0.30-0.93). In patients who underwent reoperation, 45.5% (45 of 99) were disease free at a median follow-up of 7.7 years. Five-year disease-specific mortality for the entire cohort was 13.5% (82 of 609). Independent risk factors for disease-specific mortality included older age (HR, 1.36 per decade; 95% CI, 1.17-1.59), tumor size greater than 2 cm (HR, 2.83; 95% CI, 1.08-7.44 for >2 to 4 cm and HR, 2.89; 95% CI, 1.09-7.71 for >4 cm), and regional (HR, 4.77; 95% CI, 2.29-9.94) and metastatic (HR, 21.08; 95% CI, 9.90-44.89) disease. Reoperation was not associated with increased mortality. Conclusions and Relevance: Lymph node dissection may decrease recurrence leading to reoperation for patients with MTC. Reoperation is a viable strategy to achieve long-term disease-free survival in appropriately selected patients. Central neck dissection remains underused.

Factors Associated With Discordance Between Preoperative Parathyroid 4-Dimensional Computed Tomographic Scans and Intraoperative Findings During Parathyroidectomy.

IMPORTANCE: Parathyroid 4-dimensional computed tomographic scans (4D-CTs) have emerged as an accurate and cost-effective initial localization study for patients with primary hyperparathyroidism. However, potential limitations and factors affecting the accuracy of preoperative 4D-CTs remain poorly defined. OBJECTIVES: To characterize factors associated with missed parathyroid lesions on preoperative 4D-CTs and to investigate patterns of commonly observed errors. DESIGN, SETTING, AND PARTICIPANTS: A prospectively accrued patient database was analyzed from September 1, 2011, through October 31, 2016. The study was performed in a tertiary referral center. Consecutive patients with primary hyperparathyroidism undergoing preoperative 4D-CTs and subsequent parathyroidectomy were included in the study. MAIN OUTCOMES AND MEASURES: Discordance between preoperative 4D-CTs and intraoperative findings in the number and location of abnormal parathyroid lesions. RESULTS: Of 411 patients studied (mean [SD] age, 59 [14] years; 325 [79.1%] female), 123 (29.9%) had discordance between preoperative 4D-CTs and intraoperative findings. Among the 411 patients, 75 (18.2%) had major discordance, including incorrectly localized adenoma on the contralateral side of the neck, missed double adenoma, and absence of any abnormal lesion detected on 4D-CTs. Compared with concordant cases, discordant cases had higher frequencies of multigland disease (66.7% [82 of 123] vs 24.3% [70 of 288], P < .001) and multinodular goiter or thyroid nodule (40.7% [50 of 123] vs 29.2% [84 of 288], P = .02). Missed parathyroid lesions were smaller (mean [SD], 0.86 [0.29] vs 1.24 [0.50] cm; P < .001) and were more likely to be in the inferior position (65.4% [87 of 133] vs 38.1% [177 of 465], P < .001). Parathyroid lesion size of 10 mm or less (odds ratio [OR], 4.37; 95% CI, 2.24-8.54), multigland disease (OR, 7.63; 95% CI, 3.49-16.69), multinodular goiter or thyroid nodule (OR, 1.82; 95% CI, 1.01-3.28), and parathyroid lesion in the inferior position (OR, 6.82; 95% CI, 3.10-14.99) were independently associated with discordant 4D-CT results. CONCLUSIONS AND RELEVANCE: Multigland disease was most strongly associated with discordance between preoperative 4D-CTs and intraoperative findings, followed by parathyroid lesion in the inferior position and parathyroid lesion size of 10 mm or less. Awareness of these potential pitfalls may allow surgeons to better leverage this new localization technique in preoperative planning and intraoperative troubleshooting.

A prognostic mutation panel for predicting cancer recurrence in stages II and III colorectal cancer.

BACKGROUND AND OBJECTIVES: Approximately 20-40% of stage II/III colorectal cancer (CRC) patients develop relapse. Clinicopathological factors alone are limited in detecting these patients, resulting in potential under/over-treatment. We sought to identify a prognostic tumor mutational profile that could predict CRC recurrence. METHODS: Whole-exome sequencing data were obtained for 207 patients with stage II/III CRC from The Cancer Genome Atlas. Mutational landscape in relapse-free versus relapsed cohort was compared using Fisher's exact test, followed by multivariate Cox regression to identify genes associated with cancer recurrence. Bootstrap-validation was used to examine internal/external validity. RESULTS: We identified five prognostic genes (APAF1, DIAPH2, NTNG1, USP7, and VAV2), which were combined to form a prognostic mutation panel. Patients with ≥1 mutation(s) within this five-gene panel had worse prognosis (3-yr relapse-free survival [RFS]: 53.0%), compared to patients with no mutation (3-yr RFS: 84.3%). In multivariate analysis, the five-gene panel remained prognostic for cancer recurrence independent of stage and high-risk features (hazard ratio 3.63, 95%CI [1.93-6.83], P < 0.0001). Furthermore, its prognostic accuracy was superior to the American Joint Commission on Cancer classification (concordance-index: 0.70 vs 0.54). CONCLUSIONS: Our proposed mutation panel identifies CRC patients at high-risk for recurrence, which may help guide adjuvant therapy and post-operative surveillance protocols.

Precision oncology using a limited number of cells: optimization of whole genome amplification products for sequencing applications.

BACKGROUND: Sequencing analysis of circulating tumor cells (CTCs) enables "liquid biopsy" to guide precision oncology strategies. However, this requires low-template whole genome amplification (WGA) that is prone to errors and biases from uneven amplifications. Currently, quality control (QC) methods for WGA products, as well as the number of CTCs needed for reliable downstream sequencing, remain poorly defined. We sought to define strategies for selecting and generating optimal WGA products from low-template input as it relates to their potential applications in precision oncology strategies. METHODS: Single pancreatic cancer cells (HPAF-II) were isolated using laser microdissection. WGA was performed using multiple displacement amplification (MDA), multiple annealing and looping based amplification (MALBAC) and PicoPLEX. Quality of amplified DNA products were assessed using a multiplex/RT-qPCR based method that evaluates for 8-cancer related genes and QC-scores were assigned. We utilized this scoring system to assess the impact of de novo modifications to the WGA protocol. WGA products were subjected to Sanger sequencing, array comparative genomic hybridization (aCGH) and next generation sequencing (NGS) to evaluate their performances in respective downstream analyses providing validation of the QC-score. RESULTS: Single-cell WGA products exhibited a significant sample-to-sample variability in amplified DNA quality as assessed by our 8-gene QC assay. Single-cell WGA products that passed the pre-analysis QC had lower amplification bias and improved aCGH/NGS performance metrics when compared to single-cell WGA products that failed the QC. Increasing the number of cellular input resulted in improved QC-scores overall, but a resultant WGA product that consistently passed the QC step required a starting cellular input of at least 20-cells. Our modified-WGA protocol effectively reduced this number, achieving reproducible high-quality WGA products from ≥5-cells as a starting template. A starting cellular input of 5 to 10-cells amplified using the modified-WGA achieved aCGH and NGS results that closely matched that of unamplified, batch genomic DNA. CONCLUSION: The modified-WGA protocol coupled with the 8-gene QC serve as an effective strategy to enhance the quality of low-template WGA reactions. Furthermore, a threshold number of 5-10 cells are likely needed for a reliable WGA reaction and product with high fidelity to the original starting template.

Sustained Growth of a University-Based Endocrine Surgery Program Over 10 Years.

BACKGROUND: Endocrine surgery continues to mature as a subspecialty field. We describe the clinical performance of an academic endocrine surgery program (ESP) over its first 10 years. METHODS: We examined all endocrine procedures performed during the 10-year period (2006-2015) following the inception of the ESP. Institutional and state-level data on case volume, patient geographic origin, and hospital-side costs were obtained. RESULTS: Endocrine case volume increased by approximately ninefold over the study period (from 102 cases in 2006 to 919 cases in 2015). The rate of growth remained approximately linear, and was driven by geographic expansion of referral regions coupled with transitioning low- to moderate-acuity operations to venues outside of the main tertiary care hospital. Market share across the eight-county Southern California region grew by more than twofold over the study period. Increased utilization of outpatient surgery led to cost reductions, averaging 11.1% per case by 2015. CONCLUSIONS: Establishment of an academic ESP can lead to sustained clinical growth and a fundamental shift in regional referral patterns. The nation's continued need for skilled high-volume endocrine surgeons represents opportunities for medical centers to institute their own dedicated endocrine surgery programs.

Digital PCR Improves Mutation Analysis in Pancreas Fine Needle Aspiration Biopsy Specimens.

Applications of precision oncology strategies rely on accurate tumor genotyping from clinically available specimens. Fine needle aspirations (FNA) are frequently obtained in cancer management and often represent the only source of tumor tissues for patients with metastatic or locally advanced diseases. However, FNAs obtained from pancreas ductal adenocarcinoma (PDAC) are often limited in cellularity and/or tumor cell purity, precluding accurate tumor genotyping in many cases. Digital PCR (dPCR) is a technology with exceptional sensitivity and low DNA template requirement, characteristics that are necessary for analyzing PDAC FNA samples. In the current study, we sought to evaluate dPCR as a mutation analysis tool for pancreas FNA specimens. To this end, we analyzed alterations in the KRAS gene in pancreas FNAs using dPCR. The sensitivity of dPCR mutation analysis was first determined using serial dilution cell spiking studies. Single-cell laser-microdissection (LMD) was then utilized to identify the minimal number of tumor cells needed for mutation detection. Lastly, dPCR mutation analysis was performed on 44 pancreas FNAs (34 formalin-fixed paraffin-embedded (FFPE) and 10 fresh (non-fixed)), including samples highly limited in cellularity (100 cells) and tumor cell purity (1%). We found dPCR to detect mutations with allele frequencies as low as 0.17%. Additionally, a single tumor cell could be detected within an abundance of normal cells. Using clinical FNA samples, dPCR mutation analysis was successful in all preoperative FNA biopsies tested, and its accuracy was confirmed via comparison with resected tumor specimens. Moreover, dPCR revealed additional KRAS mutations representing minor subclones within a tumor that were not detected by the current clinical gold standard method of Sanger sequencing. In conclusion, dPCR performs sensitive and accurate mutation analysis in pancreas FNAs, detecting not only the dominant mutation subtype, but also the additional rare mutation subtypes representing tumor heterogeneity.

Single-incision retroperitoneoscopic adrenalectomy: a North American experience.

BACKGROUND: Endoscopic adrenalectomy is currently performed using multiple ports placed either transabdominally or retroperitoneally. We report our initial experience with single-incision retroperitoneoscopic adrenalectomy (SIRA). METHODS: A prospective database of patients undergoing adrenalectomy from December 2013 through March 2016 was analyzed. We adopted conventional retroperitoneoscopic adrenalectomy (CORA) in December 2013 and transitioned to SIRA in March 2015. SIRA was performed using three trocars placed through a single 2-cm incision below the 12th rib. Clinical characteristics and outcomes were compared between patients undergoing SIRA and CORA. RESULTS: One hundred and five adrenalectomies were performed in 102 patients: 34 laparoscopic transperitoneal, 24 CORA, 37 SIRA and 7 open. The SIRA and CORA groups were similar with respect to clinical characteristics (SIRA vs. CORA: mean BMI 27.0 vs. 28.8 kg/m2, maximum BMI 38.9 vs. 44.3 kg/m2; mean nodule size 3.2 vs. 3.2 cm, maximum nodule size 8.0 vs. 6.0 cm). One patient undergoing SIRA required placement of an additional 5-mm port because of extensive adhesions. No patients who underwent SIRA or CORA required conversion to open adrenalectomy. There were no deaths, and blood loss remained <10 mL for all cases. Operative length was similar between SIRA and CORA (105 vs. 92 min, P = 0.26). In multivariable linear regression analysis, nodule size > 5 cm (effect = 1.75, P < 0.001) and pheochromocytoma (effect = 1.30, P = 0.05) were significant predictors of increased operative length for SIRA. BMI and laterality (right vs. left) did not affect operative length. Rates of postoperative temporary abdominal wall relaxation, length of stay and postoperative pain medication use were similar between the two groups. CONCLUSIONS: SIRA is safe and feasible to implement as a refinement of CORA and may be applied to technically challenging cases involving obese patients or large nodules. The use of three ports allows for two-handed dissection, which may shorten the learning curve for many surgeons.

Reality of Single Circulating Tumor Cell Sequencing for Molecular Diagnostics in Pancreatic Cancer.

To understand the potential and limitations of circulating tumor cell (CTC) sequencing for molecular diagnostics, we investigated the feasibility of identifying the ubiquitous KRAS mutation in single CTCs from pancreatic cancer (PC) patients. We used the NanoVelcro/laser capture microdissection CTC platform, combined with whole genome amplification and KRAS Sanger sequencing. We assessed both KRAS codon-12 coverage and the degree that allele dropout during whole genome amplification affected the detection of KRAS mutations from single CTCs. We isolated 385 single cells, 163 from PC cell lines and 222 from the blood of 12 PC patients, and obtained KRAS sequence coverage in 218 of 385 single cells (56.6%). For PC cell lines with known KRAS mutations, single mutations were detected in 67% of homozygous cells but only 37.4% of heterozygous single cells, demonstrating that both coverage and allele dropout are important causes of mutation detection failure from single cells. We could detect KRAS mutations in CTCs from 11 of 12 patients (92%) and 33 of 119 single CTCs sequenced, resulting in a KRAS mutation detection rate of 27.7%. Importantly, KRAS mutations were never found in the 103 white blood cells sequenced. Sequencing of groups of cells containing between 1 and 100 cells determined that at least 10 CTCs are likely required to reliably assess KRAS mutation status from CTCs.

Circulating Tumor Cells in Gastrointestinal Cancer: Current Practices and Future Directions.

GI cancers are the leading cause of cancer-related death worldwide primarily due to a combination of late presentation and aggressive biology. The lack of adequate biomarkers for screening, diagnosis, staging, and prognosis confounds clinical decision-making and delays potentially effective therapies. Circulating tumor cells (CTCs) are a new biomarker with particular promise in GI cancers, potentially offering clinicians and researchers real-time access to tumor tissue in a reliable, safe, and cost-effective manner. Preliminary studies have investigated the potential clinical utility of CTCs for all GI cancer types with promising results. Furthermore, advances in single cell analytics have been successfully applied to CTCs, allowing for exciting new clinical and research applications. In this chapter, we will review the current state of CTC research in GI cancers as well as the potential future applications that are currently being developed.

A novel scoring system to predict the development of necrotizing enterocolitis totalis in premature infants.

BACKGROUND/PURPOSE: Necrotizing enterocolitis totalis (NEC-totalis) is the severest form of NEC, with mortality rate of almost 100% even in the busiest neonatal centers. Despite such a prognosis, its risk factors remain elusive. We seek to identify clinical and laboratory parameters that differentiate NEC-totalis from NEC, and to use these factors to develop a scoring system to identify patients at risk for NEC-totalis upon presentation. METHOD: NEC patients were identified from our electronic medical record using ICD9 code. Diagnosis of NEC-totalis was based on operative and autopsy reports. Patients were divided into 2 groups: NEC-but-no-totalis and NEC-totalis. Clinical/laboratory data were obtained for each group. T-test, multivariate logistic regression and backward stepwise regression analysis were performed to identify risk factors for NEC-totalis and these risk factors were formulated into a "Totalis Score." RESULT: Among 157 NEC patients, 13 had NEC-totalis. NEC-totalis patients, compared to NEC alone, had fewer platelets, older age at diagnosis of NEC and greater phosphorus and creatinine levels. A 0-5 point scale "Totalis Score" based on these risk factors had sensitivity of 92% and a specificity of 78% for the diagnosis of NEC-totalis. CONCLUSION: Low platelet, high phosphorus, high creatinine and older age at diagnosis of NEC were associated with a greater risk of developing NEC-totalis.

Intestinal epithelial Toll-like receptor 4 regulates goblet cell development and is required for necrotizing enterocolitis in mice.

BACKGROUND & AIMS: Little is known about factors that regulate intestinal epithelial differentiation; microbial recognition receptors such as Toll-like receptor (TLR)4 might be involved. We investigated whether intestinal TLR4 regulates epithelial differentiation and is involved in development of necrotizing enterocolitis (NEC) of the immature intestine. METHODS: Mice with conditional disruption of TLR4 in the intestinal epithelium and TLR4 knockout (TLR4(-/-)) mice were generated by breeding TLR4(loxp/loxp) mice with villin-cre and Ella-cre, respectively. Enterocytes that did not express or overexpressed TLR4 were created by lentiviral or adenoviral transduction. Intestinal organoids were cultured on tissue matrices. Bile acids were measured by colorimetric assays, and microbial composition was determined by 16S pyrosequencing. NEC was induced in 7- to 10-day-old mice by induction of hypoxia twice daily for 4 days. RESULTS: TLR4(-/-) mice and mice with enterocyte-specific deletion of TLR4 were protected from NEC; epithelial differentiation into goblet cells was increased via suppressed Notch signaling in the small intestinal epithelium. TLR4 also regulates differentiation of goblet cells in intestinal organoid and enterocyte cell cultures; differentiation was increased on deletion of TLR4 and restored when TLR4 was expressed ectopically. TLR4 signaling via Notch was increased in intestinal tissue samples from patients with NEC, and numbers of goblet cells were reduced. 16S pyrosequencing revealed that wild-type and TLR4-deficient mice had similar microbial profiles; increased numbers of goblet cells were observed in mice given antibiotics. TLR4 deficiency reduced levels of luminal bile acids in vivo, and addition of bile acids to TLR4-deficient cell cultures prevented differentiation of goblet cells. CONCLUSIONS: TLR4 signaling and Notch are increased in intestinal tissues of patients with NEC and required for induction of NEC in mice. TLR4 prevents goblet cell differentiation, independently of the microbiota. Bile acids might initiate goblet cell development.