Genome-wide profiling of circulating tumor DNA depicts landscape of copy number alterations in pancreatic cancer with liver metastasis.
ABSTRACT: Cell-free DNA (cfDNA) offers an alternative to tissue biopsies for genomic profiling in tumors. Here, we sought to evaluate copy number alterations in PDAC through whole-genome sequencing (WGS) of cfDNA and determine their clinical significance. Using shallow WGS across 90 plasma samples from 70 pancreatic cancer patients, we detected somatic copy number alterations (CNAs) in 34 subjects (48.6%). Additionally, a higher tumor fraction (TFx) was associated with increased carbohydrate antigen 19-9 (CA19-9), metastasis, and a worse prognosis. Serial cfDNA analysis suggested that CNAs were highly concordant even for progressive disease after chemotherapy. TFx dynamics were largely in line with changed CA19-9 levels and tumor burden following chemotherapy. Notably, patients with more abundant, baseline CNAs exhibited a better response to chemotherapy. In conclusion, shallow WGS for cfDNA enables a high-throughput characterization of CNAs and an estimation of tumor burden in metastatic pancreatic cancer. These findings reinforce our understanding of the genomic evolution of metastatic PDAC and might have clinical relevance for guiding treatment.
Project description:Liquid biopsy may assist in the management of cancer patients, which can be particularly applicable in pancreatic ductal adenocarcinoma (PDAC). In this study, we investigated the utility of circulating cell-free DNA (cfDNA)-based markers as prognostic tools in metastatic PDAC. Plasma was obtained from 61 metastatic PDAC patients, and cfDNA levels and fragmentation were determined. BEAMing technique was used for quantitative determination of RAS mutation allele fraction (MAF) in cfDNA. We found that the prognosis was more accurately predicted by RAS mutation detection in plasma than in tissue. RAS mutation status in plasma was a strong independent prognostic factor for both overall survival (OS) and progression-free survival (PFS). Moreover, RAS MAF in cfDNA was also an independent risk factor for poor OS, and was strongly associated with primary tumours in the body/tail of the pancreas and liver metastases. Higher cfDNA levels and fragmentation were also associated with poorer OS and shorter PFS, body/tail tumors, and hepatic metastases, whereas cfDNA fragmentation positively correlated with RAS MAF. Remarkably, the combination of CA19-9 with MAF, cfDNA levels and fragmentation improved the prognostic stratification of patients. Furthermore, dynamics of RAS MAF better correlated with patients' outcome than standard CA19-9 marker. In conclusion, our study supports the use of cfDNA-based liquid biopsy markers as clinical tools for the non-invasive prognosis and monitoring of metastatic PDAC patients.
Project description:Purpose The aqueous humor (AH) liquid biopsy enables in vivo evaluation of tumor-derived cell-free DNA (cfDNA) from retinoblastoma (RB) eyes. Herein, we test our hypothesis that longitudinal dynamics of AH cfDNA—including tumor fraction (TFx) and somatic copy number alteration (SCNA) amplitude—correspond to therapeutic response. Methods Eyes with ?3 AH extractions during intravitreal chemotherapy (IVM) or at secondary enucleation between 2015 to 2019 were included. AH cfDNA was sequenced to assess RB SCNA amplitude; ichorCNA software was used to estimate TFx. Eyes without SCNAs or with TFx < 0.10 across all samples were excluded. Therapeutic responses for each eye were determined from clinical records. Statistical analyses included Mann-Whitney U and Pearson correlation tests. Results Twenty eyes of 20 patients underwent ?3 AH extractions; 6 eyes lacked SCNAs or had TFx < 0.10 throughout sampling and were excluded. Clinical progression was associated with significantly higher SCNA amplitudes and TFx values than regression (P ? 0.04). Relative increases in TFx (?TFx 1.86 ± 2.22) were associated with disease progression, whereas relative decreases in TFx (?TFx 0.53 ± 0.36) were associated with disease regression (P < 0.00001). A ?15% increase in TFx relative to baseline during treatment was associated with an over 90-fold increased likelihood of clinical progression (odds ratio = 90.67, 95% confidence interval = 8.30–990.16, P = 0.0002). TFx and SCNA amplitude were significantly positively correlated throughout sampling (P ? 0.002). Conclusions Longitudinal changes in AH-derived cfDNA TFx and SCNA amplitude are concordant with clinical responses of intraocular RB during active therapy. Translational Relevance Longitudinal evaluation of AH cfDNA may provide an objective, quantitative way to monitor therapeutic response and disease burden in RB patients.
Project description:BACKGROUND:Tumor content in circulating cell-free DNA (cfDNA) is a promising biomarker, but longitudinal dynamics of tumor-derived and non-tumor-derived cfDNA through multiple courses of therapy have not been well described. METHODS:CfDNA from 663 plasma samples from 140 patients with castration-resistant prostate cancer (CRPC) was subject to sparse whole genome sequencing. Tumor fraction (TFx) estimated using the computational tool ichorCNA was correlated with clinical features and responses to therapy. RESULTS:TFx associated with the number of bone metastases (median TFx = 0.014 with no bone metastases, 0.047 with 1-3 bone metastases, 0.190 for 4+ bone metastases; P < 0.0001) and with visceral metastases (P < 0.0001). In multivariable analysis, TFx remained associated with metastasis location (P = 0.042); TFx was positively correlated with alkaline phosphatase (P = 0.0227) and negatively correlated with hemoglobin (Hgb) (P < 0.001), but it was not correlated with prostate specific antigen (PSA) (P = 0.75). Tumor-derived and non-tumor-derived cfDNA track together and do not increase with generalized tissue damage from chemotherapy or radiation at the time scales examined. All new treatments that led to ?30% PSA decline at 6 weeks were associated with TFx decline when baseline TFx was >7%; however, TFx in patients being subsequently maintained on secondary hormonal therapy was quite dynamic. CONCLUSION:TFx correlates with clinical features associated with overall survival in CRPC, and TFx decline is a promising biomarker for initial therapeutic response. TRIAL REGISTRATION:Dana-Farber/Harvard Cancer Center (DF/HCC) protocol no. 18-135. FUNDING:Wong Family Award in Translational Oncology, Dana Farber Cancer Institute Medical Oncology grant, Gerstner Family Foundation, Janssen Pharmaceuticals Inc., and Koch Institute Support (core) grant P30-CA14051 from the National Cancer Institute (NCI).
Project description:The aim of this observational study was to test whether ABO blood type was a prognostic factor for pancreatic ductal adenocarcinoma (PDAC) patients and whether other risk factors could influence pancreatic cancer patients' survival. This study included 610 patients who were diagnosed as pancreatic cancer and had undergone radical surgery. Patients' characteristics included age, gender, tumor stage, tumor grade, adenosquamous carcinoma (ASC) status, preoperative serum carbohydrate antigen 19-9 (CA19-9) levels, preoperative serum carcinoembryonic antigen (CEA) levels, ABO blood type, smoking status, and drinking status were analyzed in this study. Cox proportional hazards regression model and Kaplan-Meier method were used to evaluate the role of prognostic factors. For pancreatic cancer patients undergoing radical surgery, the overall survival was worse for ASC patients than PDAC patients (Log-rank?=?11.315, P?<?.001). Compared with ASC patients (Log-rank?<?0.001, P?=?.996), PDAC patients can benefit from chemotherapy (Log-rank?=?17.665, P?<?.001). For PDAC patients, O blood type had better overall survival than non-O blood type (Log-rank?=?4.153, P?=?.042). Moreover, the group with higher serum levels of CA19-9 had poor prognosis compared to another group with low serum CA19-9 (Log-rank?=?4.122, P?=?.042). Higher CEA levels indicated poor prognosis (Log-rank?=?13.618, P?<?.001). In conclusion, ASC status was associated with overall survival of pancreatic cancer patients and cannot benefit from postoperative chemotherapy. Non-O blood type was a prognostic factor for PDAC patients.
Project description:CA19-9, which is currently in clinical use as a pancreatic ductal adenocarcinoma (PDAC) biomarker, has limited performance in detecting early-stage disease. We and others have identified protein biomarker candidates that have the potential to complement CA19-9. We have carried out sequential validations starting with 17 protein biomarker candidates to determine which markers and marker combination would improve detection of early-stage disease compared with CA19-9 alone.Candidate biomarkers were subjected to enzyme-linked immunosorbent assay based sequential validation using independent multiple sample cohorts consisting of PDAC cases (n?=?187), benign pancreatic disease (n?=?93), and healthy controls (n?=?169). A biomarker panel for early-stage PDAC was developed based on a logistic regression model. All statistical tests for the results presented below were one-sided.Six out of the 17 biomarker candidates and CA19-9 were validated in a sample set consisting of 75 PDAC patients, 27 healthy subjects, and 19 chronic pancreatitis patients. A second independent set of 73 early-stage PDAC patients, 60 healthy subjects, and 74 benign pancreatic disease patients (combined validation set) yielded a model that consisted of TIMP1, LRG1, and CA19-9. Additional blinded testing of the model was done using an independent set of plasma samples from 39 resectable PDAC patients and 82 matched healthy subjects (test set). The model yielded areas under the curve (AUCs) of 0.949 (95% confidence interval [CI] = 0.917 to 0.981) and 0.887 (95% CI?=?0.817 to 0.957) with sensitivities of 0.849 and 0.667 at 95% specificity in discriminating early-stage PDAC vs healthy subjects in the combined validation and test sets, respectively. The performance of the biomarker panel was statistically significantly improved compared with CA19-9 alone (P < .001, combined validation set; P = .008, test set).The addition of TIMP1 and LRG1 immunoassays to CA19-9 statistically significantly improves the detection of early-stage PDAC.
Project description:Currently the diagnosis of pancreatic ductal adenocarcinoma (PDAC) relies on CA19-9 and radiological means, whereas some patients do not have elevated levels of CA19-9 secondary to pancreatic cancer. The purpose of this study was to identify potential serum biomarkers for CA19-9 negative PDAC.A total of 114 serum samples were collected from 3 groups: CA19-9 negative PDAC patients (n?=?34), CA19-9 positive PDAC patients (n?=?44), and healthy volunteers (n?=?36), whereas the first 12 samples from each group were used for isobaric tags for relative and absolute quantitation (iTRAQ) analysis. Thereafter, candidate biomarkers were selected for validation by enzyme-linked immunosorbent assay (ELISA) with the rest specimens.Using the iTRAQ approach, a total of 5 proteins were identified as significantly different between CA19-9 negative PDAC patients and healthy subjects according to our defined criteria. Apolipoprotein A-I (APOA-I) and transferrin (TF) were selected to validate the proteomic results by ELISA in a further 78 serum specimens. It revealed that TF significantly correlated with the degree of histological differentiation (P?=?0.042), and univariate and multivariate analyses indicated that TF is an independent prognostic factor for survival (hazard ratio, 0.302; 95% confidence interval, 0.118-0.774; P?=?0.013) of patients with PDAC after curative surgery.ITRAQ-based quantitative proteomics revealed that APOA-I and TF may be potential CA19-9 negative PDAC serum markers.
Project description:PURPOSE:The CA19-9 biomarker is elevated in a substantial group of patients with pancreatic ductal adenocarcinoma (PDAC), but not enough to be reliable for the detection or diagnosis of the disease. We hypothesized that a glycan called sTRA (sialylated tumor-related antigen) is a biomarker for PDAC that improves upon CA19-9. EXPERIMENTAL DESIGN:We examined sTRA and CA19-9 expression and secretion in panels of cell lines, patient-derived xenografts, and primary tumors. We developed candidate biomarkers from sTRA and CA19-9 in a training set of 147 plasma samples and used the panels to make case-control calls, based on predetermined thresholds, in a 50-sample validation set and a blinded, 147-sample test set. RESULTS:The sTRA glycan was produced and secreted by pancreatic tumors and models that did not produce and secrete CA19-9. Two biomarker panels improved upon CA19-9 in the training set, one optimized for specificity, which included CA19-9 and 2 versions of the sTRA assay, and another optimized for sensitivity, which included 2 sTRA assays. Both panels achieved statistical improvement (P < 0.001) over CA19-9 in the validation set, and the specificity-optimized panel achieved statistical improvement (P < 0.001) in the blinded set: 95% specificity and 54% sensitivity (75% accuracy), compared with 97%/30% (65% accuracy). Unblinding produced further improvements and revealed independent, complementary contributions from each marker. CONCLUSIONS:sTRA is a validated serological biomarker of PDAC that yields improved performance over CA19-9. The new panels may enable surveillance for PDAC among people with elevated risk, or improved differential diagnosis among patients with suspected pancreatic cancer.
Project description:Targeted deep sequencing across broad genomic regions has been used to detect circulating tumor DNA (ctDNA) in pancreatic ductal adenocarcinoma (PDAC) patients. However, since most PDACs harbor a mutation in KRAS, sequencing of broad regions needs to be systemically compared to analyzing only KRAS mutations for PDAC. Using capture-based targeted deep sequencing, we detected somatic tumor mutations in 17 fine needle aspiration biopsy and 69 longitudinal cell-free DNA (cfDNA) samples from 17 PDAC patients. KRAS mutations were detected in 10 out of 17 pretreatment patient plasma samples. Next, interrogation of genetic alterations in matched primary tumor samples detected ctDNA in 12 of 17 pretreatment plasma samples and cfDNA sequencing across the 83 target genes identified ctDNA in 15 of 17 cases (88.2% sensitivity). This improved sensitivity of ctDNA detection resulted in enhanced tumor burden monitoring when we analyzed longitudinal plasma samples. We found that cfDNA sequencing detected the lowest mutant allelic fractions and number of variants when complete response or partial response to chemotherapy was achieved. We demonstrated that ctDNA levels measured by targeted deep sequencing sensitively indicate the presence of cancer and correlate well with clinical responses to therapy and disease progression in PDAC patients.
Project description:Markers are needed to facilitate early detection of pancreatic ductal adenocarcinoma (PDAC), which is often diagnosed too late for effective therapy. Starting with a PDAC cell reprogramming model that recapitulated the progression of human PDAC, we identified secreted proteins and tested a subset as potential markers of PDAC. We optimized an enzyme-linked immunosorbent assay (ELISA) using plasma samples from patients with various stages of PDAC, from individuals with benign pancreatic disease, and from healthy controls. A phase 1 discovery study (n = 20), a phase 2a validation study (n = 189), and a second phase 2b validation study (n = 537) revealed that concentrations of plasma thrombospondin-2 (THBS2) discriminated among all stages of PDAC consistently. The receiver operating characteristic (ROC) c-statistic was 0.76 in the phase 1 study, 0.84 in the phase 2a study, and 0.87 in the phase 2b study. The plasma concentration of THBS2 was able to discriminate resectable stage I cancer as readily as stage III/IV PDAC tumors. THBS2 plasma concentrations combined with those for CA19-9, a previously identified PDAC marker, yielded a c-statistic of 0.96 in the phase 2a study and 0.97 in the phase 2b study. THBS2 data improved the ability of CA19-9 to distinguish PDAC from pancreatitis. With a specificity of 98%, the combination of THBS2 and CA19-9 yielded a sensitivity of 87% for PDAC in the phase 2b study. A THBS2 and CA19-9 blood marker panel measured with a conventional ELISA may improve the detection of patients at high risk for PDAC.