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Circulating tumour cells as a biomarker for diagnosis and staging in pancreatic cancer

Background:Current diagnosis and staging of pancreatic ductal adenocarcinoma (PDAC) has important limitations and better biomarkers are needed to guide initial therapy. We investigated the performance of circulating tumour cells (CTCs) as an adjunctive biomarker at the time of disease presentation.Methods:Venous blood (VB) was collected prospectively from 100 consecutive, pre-treatment patients with PDAC. Utilising the microfluidic NanoVelcro CTC chip, samples were evaluated for the presence and number of CTCs. KRAS mutation analysis was used to compare the CTCs with primary tumour tissue. CTC enumeration data was then evaluated as a diagnostic and staging biomarker in the setting of PDAC.Results:We found 100% concordance for KRAS mutation subtype between primary tumour and CTCs in all five patients tested. Evaluation of CTCs as a diagnostic revealed the presence of CTCs in 54/72 patients with confirmed PDAC (sensitivity=75.0%, specificity=96.4%, area under the curve (AUROC)=0.867, 95% CI=0.798–0.935, and P<0.001). Furthermore, a cut-off of ⩾3 CTCs in 4 ml VB was able to discriminate between local/regional and metastatic disease (AUROC=0.885; 95% CI=0.800–0.969; and P<0.001).Conclusion:CTCs appear to function well as a biomarker for diagnosis and staging in PDAC.

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.

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.

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

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.

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.

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

BackgroundSequencing 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.MethodsSingle 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.ResultsSingle-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.ConclusionThe 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.

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 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.

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.

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

BackgroundEndocrine 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.MethodsWe 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.ResultsEndocrine 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.ConclusionsEstablishment 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.