Reconstitution of human PDAC using primary cells reveals oncogenic transcriptomic features at tumor onset.

Animal studies have demonstrated the ability of pancreatic acinar cells to transform into pancreatic ductal adenocarcinoma (PDAC). However, the tumorigenic potential of human pancreatic acinar cells remains under debate. To address this gap in knowledge, we expand sorted human acinar cells as 3D organoids and genetically modify them through introduction of common PDAC mutations. The acinar organoids undergo dramatic transcriptional alterations but maintain a recognizable DNA methylation signature. The transcriptomes of acinar organoids are similar to those of disease-specific cell populations. Oncogenic KRAS alone do not transform acinar organoids. However, acinar organoids can form PDAC in vivo after acquiring the four most common driver mutations of this disease. Similarly, sorted ductal cells carrying these genetic mutations can also form PDAC, thus experimentally proving that PDACs can originate from both human acinar and ductal cells. RNA-seq analysis reveal the transcriptional shift from normal acinar cells towards PDACs with enhanced proliferation, metabolic rewiring, down-regulation of MHC molecules, and alterations in the coagulation and complement cascade. By comparing PDAC-like cells with normal pancreas and PDAC samples, we identify a group of genes with elevated expression during early transformation which represent potential early diagnostic biomarkers.

Association between SMAD4 Mutations and GATA6 Expression in Paired Pancreatic Ductal Adenocarcinoma Tumor Specimens: Data from Two Independent Molecularly-Characterized Cohorts.

Several molecular biomarkers have been identified to guide induction treatment selection for localized pancreatic ductal adenocarcinoma (PDAC). SMAD4 alterations and low GATA6 expression/modified "Moffitt" basal-like phenotype have each been associated with inferior survival uniquely for patients receiving 5-FU-based therapies. SMAD4 may directly regulate the expression of GATA6 in PDAC, pointing to a common predictive biomarker. To evaluate the relationship between SMAD4 mutations and GATA6 expression in human PDAC tumors, patients with paired SMAD4 mutation and GATA6 mRNA expression data in the TCGA and CPTAC were identified. In 321 patients (TCGA: n = 180; CPTAC: n = 141), the rate of SMAD4 alterations was 26.8%. The rate of SMAD4 alteration did not vary per tertile of normalized GATA6 expression (TCGA: p = 0.928; CPTAC: p = 0.828). In the TCGA, SMAD4 alterations and the basal-like phenotype were each associated with worse survival (log rank p = 0.077 and p = 0.080, respectively), but their combined presence did not identify a subset with uniquely inferior survival (p = 0.943). In the CPTAC, the basal-like phenotype was associated with significantly worse survival (p < 0.001), but the prognostic value was not influenced by the combined presence of SMAD4 alterations (p = 0.960). SMAD4 alterations were not associated with poor clinico-pathological features such as poor tumor grade, advanced tumor stage, positive lymphovascular invasion (LVI), or positive perineural invasion (PNI), compared with SMAD4-wildtype. Given that SMAD4 mutations were not associated with GATA6 expression or Moffitt subtype in two independent molecularly characterized PDAC cohorts, distinct biomarker-defined clinical trials are necessary.

Genomic Biomarkers Associated with Response to Induction Chemotherapy in Patients with Localized Pancreatic Ductal Adenocarcinoma.

PURPOSE: There is increasing use of neoadjuvant chemotherapy in the management of localized pancreatic ductal adenocarcinoma (PDAC), yet there are few validated biomarkers to guide therapy selection. We aimed to determine whether somatic genomic biomarkers predict response to induction FOLFIRINOX or gemcitabine/nab-paclitaxel. EXPERIMENTAL DESIGN: This single-institution cohort study included consecutive patients (N = 322) with localized PDAC (2011-2020) who received at least one cycle of FOLFIRINOX (N = 271) or gemcitabine/nab-paclitaxel (N = 51) as initial treatment. We assessed somatic alterations in four driver genes (KRAS, TP53, CDKN2A, and SMAD4) by targeted next-generation sequencing, and determined associations between these alterations and (1) rate of metastatic progression during induction chemotherapy, (2) surgical resection, and (3) complete/major pathologic response. RESULTS: The alteration rates in driver genes KRAS, TP53, CDKN2A, and SMAD4 were 87.0%, 65.5%, 26.7%, and 19.9%, respectively. For patients receiving first-line FOLFIRINOX, SMAD4 alterations were uniquely associated with metastatic progression (30.0% vs. 14.5%; P = 0.009) and decreased rate of surgical resection (37.1% vs. 66.7%; P < 0.001). For patients receiving induction gemcitabine/nab-paclitaxel, alterations in SMAD4 were not associated with metastatic progression (14.3% vs. 16.2%; P = 0.866) nor decreased rate of surgical resection (33.3% vs. 41.9%; P = 0.605). Major pathologic response was rare (6.3%) and not associated with type of chemotherapy regimen. CONCLUSIONS: SMAD4 alterations were associated with more frequent development of metastasis and lower probability of reaching surgical resection during neoadjuvant FOLFIRINOX but not gemcitabine/nab-paclitaxel. Confirmation in a larger, diverse patient cohort will be important before prospective evaluation of SMAD4 as a genomic biomarker to guide treatment selection.

Local Therapy for Oligoprogression or Consolidation in High Mutational Burden Stage 4 Colorectal Cancer Treated With PD-1 or PD-L1 Blockade.

BACKGROUND: Immune checkpoint blockade (ICI) of programmed cell death protein 1 (PD-1) or PD-1 ligand (PD-L1) can induce durable responses in patients who have colorectal cancer (CRC) with a high tumor mutational burden (TMB). Two recurring clinical dilemmas show how to manage oligoprogressive disease and stable disease after ICI. METHODS: A cohort study was conducted to analyze patients with metastatic CRC who underwent PD-1 or PD-L1 blockade. Tumors were mismatch repair (MMR) deficient or had more than 25 mutations per megabase. Patients were identified who had local therapy (surgery, ablation, or radiotherapy) for one to three sites of progressive disease (PD) or surgery to consolidate SD. The study evaluated clinical and biologic factors associated with patient selection, outcomes, and pathologic response rates. RESULTS: From 2014 to 2020, treatment was administered to 111 patients with ICI. Of these 111 patients, 19 (17%) survived fewer than 6 months, whereas to date, 50 have not had progression of disease. The remaining 42 patients experienced PD, and 16 (38%) were treated with local therapy for oligoprogression. Selection for local therapy was associated with response to ICI. The 2-year progression-free survival (PFS) after local therapy was 62%. Finally, 6 of the 50 patients without PD had consolidation of SD, and 5 had complete or near complete pathologic responses. CONCLUSIONS: Oligoprogression, a frequent pattern of failure after ICI, can be managed effectively with local therapy. In contrast, it may not be necessary to consolidate SD for selected patients. Further research is essential to define management algorithms better and to explore heterogeneity in response patterns.

Validation of the Memorial Sloan Kettering Gastric Cancer Post-Resection Survival Nomogram: Does It Stand the Test of Time?

BACKGROUND: The Memorial Sloan Kettering Cancer Center (MSK) nomogram combined both gastroesophageal junction (GEJ) and gastric cancer patients and was created in an era from patients who generally did not receive neoadjuvant chemotherapy. We sought to reevaluate the MSK nomogram in the era of multidisciplinary treatment for GEJ and gastric cancer. STUDY DESIGN: Using data on patients who underwent R0 resection for GEJ or gastric cancer between 2002 and 2016, the C-index of prediction for disease-specific survival (DSS) was compared between the MSK nomogram and the American Joint Committee on Cancer (AJCC) 8th edition staging system after segregating patients by tumor location (GEJ or gastric cancer) and neoadjuvant treatment. A new nomogram was created for the group for which both systems poorly predicted prognosis. RESULTS: During the study period, 886 patients (645 gastric and 241 GEJ cancer) underwent up-front surgery, and 999 patients (323 gastric and 676 GEJ) received neoadjuvant treatment. Compared with the AJCC staging system, the MSK nomogram demonstrated a comparable C-index in gastric cancer patients undergoing up-front surgery (0.786 vs 0.753) and a better C-index in gastric cancer patients receiving neoadjuvant treatment (0.796 vs 0.698). In GEJ cancer patients receiving neoadjuvant chemotherapy, neither the MSK nomogram nor the AJCC staging system performed well (C-indices 0.647 and 0.646). A new GEJ nomogram was created based on multivariable Cox regression analysis and was validated with a C-index of 0.718. CONCLUSIONS: The MSK gastric cancer nomogram's predictive accuracy remains high. We developed a new GEJ nomogram that can effectively predict DSS in patients receiving neoadjuvant treatment.

Alterations in Somatic Driver Genes Are Associated with Response to Neoadjuvant FOLFIRINOX in Patients with Localized Pancreatic Ductal Adenocarcinoma.

BACKGROUND: There is increased use of neoadjuvant fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX) in the management of localized pancreatic ductal adenocarcinoma (PDAC), yet there are few validated biomarkers of treatment response. STUDY DESIGN: Consecutive patients (n = 196) with resectable, borderline resectable or locally advanced PDAC (2012-2019) receiving FOLFIRINOX as initial treatment and with targeted sequencing of a pretreatment biopsy were identified in a prospective institutional database. Genomic alterations were determined in the 4 driver mutations (KRAS, TP53, CDKN2A, SMAD4), and associations between genomic alterations and clinical outcomes were assessed. RESULTS: Alterations in KRAS (n = 172, 87.8%) and TP53 (n = 131, 66.8%) were common; alterations in CDKN2A (n = 49, 25.0%) and SMAD4 (n = 36, 18.4%) were less frequently observed. A total of 105 patients (53.6%) were able to undergo resection, of whom 8 (7.6%) had a complete/near-complete pathologic response. There were no somatic alterations associated with major pathologic response. Alterations in SMAD4 were associated with a lower rate of surgical resection (27.8% vs 59.4%, p < 0.001); this was additionally observed in a multivariable regression model accounting for resectability status (OR 0.35, 95% confidence interval [CI] 0.15-0.85). Thirty-three patients (16.8%) developed metastatic disease while on neoadjuvant therapy. SMAD4 alterations were associated with a significant risk of metastatic progression on therapy when controlling for resectability status (OR 3.31, 95% CI 1.44-7.60). CONCLUSIONS: SMAD4 alterations are associated with more frequent development of metastasis during neoadjuvant FOLFIRINOX and lower probability of reaching surgical resection. Evaluation of alternative chemotherapy regimens in patients with SMAD4 alterations will be important to distinguish whether this represents a prognostic or predictive biomarker.

Genomic Stratification of Resectable Colorectal Liver Metastasis Patients and Implications for Adjuvant Therapy and Survival.

OBJECTIVE: To determine whether genomic risk groups identified by somatic mutation testing of colorectal liver metastasis (CRLM) can be used for "molecularly-guided" selection for adjuvant systemic chemotherapy and hepatic artery infusion of FUDR (SYS+HAI-FUDR). BACKGROUND: Several genomic biomarkers have been associated with clinical phenotype and survival for patients with resectable CRLM. It is unknown whether prognostication afforded by genomic stratification translates into enhanced patient selection for adjuvant hepatic artery infusion therapy. METHODS: Consecutive patients with resected CRLM and available mutational characterization via Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets were reviewed from a prospective institutional database. Patients were stratified into three genomic risk groups based on previously defined alterations in SMAD4, EGFR and the RAS/RAF pathway. The association between SYS+HAI-FUDR and overall survival, relative to adjuvant chemotherapy alone (SYS), was evaluated in each genomic risk group by Cox proportional hazard regression and propensity score matched analyses. RESULTS: A total of 334 patients (SYS+HAI-FUDR 204; SYS 130) were identified; the rates of RAS/RAF alterations and SMAD4 inactivation were 47.4% and 11.7%, respectively. After a median follow-up of 58 months, adjuvant SYS+HAI-FUDR was independently associated with a reduced risk of death (HR 0.50, 95%CI 0.26-0.98, P = 0.045) in the low-risk genomic group, but not in the moderate-risk (HR 1.07, 95%CI 0.5-2.07, P = 0.749) or high-risk (HR 1.62, 95%CI 0.29-9.12, P = 0.537) cohorts. Following propensity score matching, adjuvant SYS+HAI-FUDR remained associated with significant improvements in long-term survival selectively in the low-risk genomic cohort (5-year actuarial survival: 89% vs. 68%, P = 0.019). CONCLUSIONS: Genomic alterations in RAS/RAF, SMAD4, and EGFR may be useful to guide treatment selection in resectable CRLM patients and warrant external validation and integration in future clinical trial design.

Hepatocellular Carcinoma-Circulating Tumor Cells Expressing PD-L1 Are Prognostic and Potentially Associated With Response to Checkpoint Inhibitors.

Hepatocellular carcinoma (HCC) is a leading cause of mortality. Checkpoint inhibitors of programmed cell death protein-1 (PD-1) and programmed death-ligand 1 (PD-L1) have shown great efficacy, but lack biomarkers that predict response. Circulating tumor cells (CTCs) have promise as a liquid-biopsy biomarker; however, data on HCC CTCs expressing PD-L1 have not been reported. We sought to detect PD-L1-expressing HCC-CTCs and investigated their role as a prognostic and predictive biomarker. Using an antibody-based platform, CTCs were enumerated/phenotyped from a prospective cohort of 87 patients with HCC (49 early-stage, 22 locally advanced, and 16 metastatic), 7 patients with cirrhosis, and 8 healthy controls. Immunocytochemistry identified total HCC CTCs (4',6-diamidino-2-phenylindole-positive [DAPI+]/cytokeratin-positive [CK+]/clusters of differentiation 45-negative [CD45-]) and a subpopulation expressing PD-L1 (DAPI+/CK+/PD-L1+/CD45-). PD-L1+ CTCs were identified in 4 of 49 (8.2%) early-stage patients, but 12 of 22 (54.5%) locally advanced and 15 of 16 (93.8%) metastatic patients, accurately discriminating early from locally advanced/metastatic HCC (sensitivity = 71.1%, specificity = 91.8%, area under the receiver operating characteristic curve = 0.807; P < 0.001). Compared to patients without PD-L1+ CTCs, patients with PD-L1+ CTCs had significantly inferior overall survival (OS) (median OS = 14.0 months vs. not reached, hazard ratio [HR] = 4.0, P = 0.001). PD-L1+ CTCs remained an independent predictor of OS (HR = 3.22, P = 0.010) even after controlling for Model for End-Stage Liver Disease score (HR = 1.14, P < 0.001), alpha-fetoprotein (HR = 1.55, P < 0.001), and overall stage/tumor burden (beyond University of California, San Francisco, HR = 7.19, P < 0.001). In the subset of 10 patients with HCC receiving PD-1 blockade, all 5 responders demonstrated PD-L1+ CTCs at baseline, compared with only 1 of 5 nonresponders, all of whom progressed within 4 months of starting treatment. Conclusion: We report a CTC assay for the phenotypic profiling of HCC CTCs expressing PD-L1. PD-L1+ CTCs are predominantly found in advanced-stage HCC, and independently prognosticate OS after controlling for Model for End-Stage Liver Disease, alpha-fetoprotein, and tumor stage. In patients with HCC receiving anti-PD-1 therapy, there was a strong association with the presence of PD-L1+ CTCs and favorable treatment response. Prospective validation in a larger cohort will better define the utility of PD-L1+ CTCs as a prognostic and predictive biomarker in HCC.

Somatic copy number profiling from hepatocellular carcinoma circulating tumor cells.

Somatic copy number alterations (SCNAs) are important genetic drivers of many cancers. We investigated the feasibility of obtaining SCNA profiles from circulating tumor cells (CTCs) as a molecular liquid biopsy for hepatocellular carcinoma (HCC). CTCs from ten HCC patients underwent SCNA profiling. The Cancer Genome Atlas (TCGA) SCNA data were used to develop a cancer origin classification model, which was then evaluated for classifying 44 CTCs from multiple cancer types. Sequencing of 18 CTC samples (median: 4 CTCs/sample) from 10 HCC patients using a low-resolution whole-genome sequencing strategy (median: 0.88 million reads/sample) revealed frequent SCNAs in previously reported HCC regions such as 8q amplifications and 17p deletions. SCNA profiling revealed that CTCs share a median of 80% concordance with the primary tumor. CTCs had SCNAs not seen in the primary tumor, some with prognostic implications. Using a SCNA profiling model, the tissue of origin was correctly identified for 32/44 (73%) CTCs from 12/16 (75%) patients with different cancer types.

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.

Pancreatic cancer circulating tumor cells: applications for personalized oncology.

INTRODUCTION: Pancreatic cancer (PC) is a highly lethal disease, in part because of early metastasis, late diagnosis, and limited treatment options. Circulating tumor cells (CTCs) are cancer cells that have achieved the metastatic step of intravasation, and are thus a unique source of biomarkers with potential applications in the staging, prognostication, and treatment of PC. Areas covered: This review describes the use of CTCs in PC, including isolation methods, the significance of CTC enumeration, and studies examining phenotypic and molecular characteristics of CTCs. We also speculate on future directions for PC CTC research such as single-cell analysis and CTC culture. Expert commentary: CTCs represent a potential unique serial source of cancer tissue via a convenient and minimally invasive blood draw. Recent development of isolation methods that allow for the release of viable CTCs with unaltered molecular characteristics has set the stage for single-cell analysis and ex vivo culture. Although there is significant potential for CTCs as a biomarker to impact PC from diagnosis to therapy, there still remain a number of challenges to the routine implementation of CTCs in the clinical management of PC.

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.

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.

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.

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.

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.