Colin M. Court

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.

Improving pancreatic cancer diagnosis using circulating tumor cells: prospects for staging and single-cell analysis

Pancreatic cancer (PC) is the fourth most common cause of cancer-related death in the USA, primarily due to late presentation coupled with an aggressive biology. The lack of adequate biomarkers for diagnosis and staging confound clinical decision-making and delay potentially effective therapies. Circulating tumor cells (CTCs) are a promising new biomarker in PC. Preliminary studies have demonstrated their potential clinical utility, and newer CTC isolation platforms have the potential to provide clinicians access to tumor tissue in a reliable, real-time manner. Such a ‘liquid biopsy’ has been demonstrated in several cancers, and small studies have demonstrated its potential applications in PC. This article reviews the available literature on CTCs as a biomarker in PC and presents the latest innovations in CTC research as well as their potential applications in PC.

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.

Propensity Matched Analysis of Patients with Mixed Hepatocellular‐Cholangiocarcinoma and Hepatocellular Carcinoma Undergoing Liver Transplantation

Mixed hepatocellular‐cholangiocarcinomas (HCC‐CCAs) are rare tumors with both hepatocellular and biliary differentiation. While liver transplantation (LT) is the gold standard treatment for patients with unresectable hepatocellular carcinoma (HCC), it is contraindicated in known HCC‐CCA because of concerns of poor prognosis. We sought to compare posttransplant oncologic outcomes for HCC‐CCA and a matched cohort of HCC LT recipients. A retrospective, single‐center analysis (1984‐2015) identified 12 patients with mixed HCC‐CCA who were matched 1:3 to patients with HCC on both pretransplant (radiologic diameter and alpha‐fetoprotein) and explant (pathologic diameter, grade/differentiation, and vascular invasion) tumor characteristics. Compared with HCC patients matched on pretransplant characteristics (n = 36), HCC‐CCA had higher explant tumor grade, more poorly differentiated tumors, but similar T stage and vascular invasion. HCC‐CCA recipients trended toward inferior recurrence‐free survival at 5 years (28% versus 61%; P = 0.12) and greater recurrence (HCC‐CCA: 50%, median time to recurrence 297 days versus HCC: 22%, median time to recurrence 347 days; P = 0.07). However, when matched to a separate HCC cohort with similar explant pathology, HCC‐CCA had similar 5‐year recurrence‐free survival (42% versus 44%; P = 0.45) and posttransplant recurrence (50% versus 27%; P = 0.13). All 6 HCC‐CCA recurrences occurred with poorly differentiated tumors (median survival 21.3 months), without a single recurrence in 5 of the 12 HCC‐CCA patients with well‐moderately differentiated tumors (median survival 60.2 months). Mixed HCC‐CCA tumors are more likely poorly differentiated tumors compared with HCC with similar pretransplant characteristics. However, compared with HCC with similar pathologic characteristics, they display similar recurrence‐free survival and are not inherently more aggressive tumors. Low‐grade, well‐moderately differentiated HCC‐CCAs have excellent survival with a low risk for post‐LT recurrence, and they should not be excluded from LT. Improved pretransplant identification of pathologic characteristics in HCC‐CCA may allow for successful utilization of LT in this subset of patients.

Determination of hepatocellular carcinoma grade by needle biopsy is unreliable for liver transplant candidate selection

The objective of this article is to evaluate the utility of preoperative needle biopsy (PNB) grading of hepatocellular carcinoma (HCC) as a biomarker for liver transplantation (LT) candidate selection. Given the prognostic significance of HCC tumor grade, PNB grading has been proposed as a biomarker for LT candidate selection. Clinicopathologic characteristics of HCC LT recipients (1989‐2014) with a PNB were analyzed, and the concordance of PNB grade to explant grade and vascular invasion was assessed to determine whether incorporation of PNB grade to accepted transplant criteria improved candidate selection. Of 965 patients undergoing LT for HCC, 234 (24%) underwent PNB at a median of 280 days prior to transplant. Grade by PNB had poor concordance to final explant pathology (κ = 0.22; P = 0.003), and low sensitivity (29%) and positive predictive value (35%) in identifying poorly differentiated tumors. Vascular invasion was predicted by explant pathologic grade (rs = 0.24; P < 0.001) but not PNB grade (rs= −0.05; P = 0.50). Increasing explant pathology grade (P = 0.02), but not PNB grade (P = 0.65), discriminated post‐LT HCC recurrence risk. The incorporation of PNB grade to the established radiologic Milan criteria (MC) did not result in improved prognostication of post‐LT recurrence (net reclassification index [NRI] = 0%), whereas grade by explant pathology resulted in significantly improved reclassification of risk (NRI = 19%). Preoperative determination of HCC grade by PNB has low concordance with explant pathologic grade and low sensitivity and positive predictive value in identifying poorly differentiated tumors. PNB grade did not accurately discriminate post‐LT HCC recurrence and had no utility in improving prognostication compared with the MC alone. Incorporation of PNB to guide transplant candidate selection appears unjustified. Liver Transplantation 23 1123–1132 2017 AASLD.

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.