Validation of a Molecular Diagnostic Test for Circulating Tumor DNA by Next-Gen Sequencing.

A modified version of the PGDx elioTM Plasma Resolve assay was validated as a laboratory-developed test (LDT) for clinical use in the Molecular Diagnostics Laboratory at Fox Chase Cancer Center. The test detects single nucleotide variants (SNVs) and small insertions and deletions (indels) in 33 target genes using fragmented genomic DNA extracted from plasma. The analytical performance of this assay was assessed with reference standard DNA and 29 samples from cancer patients and detected 66 SNVs and 23 indels. Using 50 ng of input DNA, the sensitivity was 95.5% to detect SNVs at 0.5% allele frequency, and the specificity was 92.3%. The sensitivity to detect indels at 1% allele frequency was 70.4%. A cutoff of 0.25% variant allele frequency (VAF) was set up for diagnostic reporting. An inter-laboratory study of concordance with an orthologous test resulted in a positive percent agreement (PPA) of 91.7%.

Circulating tumor cell and cell-free RNA capture and expression analysis identify platelet-associated genes in metastatic lung cancer.

BACKGROUND: Circulating tumor cells (CTC) and plasma cell-free RNA (cfRNA) can serve as biomarkers for prognosis and treatment response in lung cancer. One barrier to the selected or routine use of CTCs and plasma cfRNA in precision oncology is the limited quantity of both, and CTCs are only seen in metastatic disease. As capture of CTCs and plasma cfRNA presents an opportunity to monitor and assess malignancies without invasive procedures, we compared two methods for CTC capture and identification, and profiled mRNA from CTCs and plasma cfRNA to identify potential tumor-associated biomarkers. METHODS: Peripheral blood was collected from ten patients with small cell lung cancer (SCLC), ten patients with non-small cell lung cancer (NSCLC) and four healthy volunteers. Two methods were used for CTC capture: the standard epithelial cell adhesion molecule (EpCam) CellSearch kit (unicapture) and EpCAM plus HER2, EGFR and MUC-1 specific combined ferrofluid capture (quadcapture). For the quadcapture, anti-cytokeratin 7 (CK7) was additionally used to assist in CTC identification. NanoString analysis was performed on plasma cfRNA and on mRNA from combined ferrofluid isolated CTCs. Expression data was analyzed using STRING and Reactome. RESULTS: Unicapture detected CTCs in 40% of NSCLC and 60% of SCLC; whereas, quadcapture/CK7 identified CTCs in 20% of NSCLC and 80% of SCLC. Bioinformatic analysis of NanoString data identified high expression of a platelet factor 4 (PF4)-related group of transcripts. CONCLUSIONS: Quadcapture ferrofluid reagent did not significantly improve CTC capture efficacy. NanoString analysis based on CTC and plasma cfRNA data highlighted an intriguing PF-4-centric network in patients with metastatic lung cancer.

Mutational studies on single circulating tumor cells isolated from the blood of inflammatory breast cancer patients.

PURPOSE: The molecular characterization of circulating tumor cells (CTCs) is critical to identify the key drivers of cancer metastasis and devising therapeutic approaches, particularly for inflammatory breast cancer (IBC) which is usually diagnosed at advance stages and progresses rapidly. METHODS: Genomic alterations in tumor tissue samples were studied using Foundation One™. Single CTCs were isolated using CellSearch followed by single-cell isolation by DEPArray™. Samples with 20 or more CTCs were chosen to isolate single CTCs using the DEPArray™. RESULTS: Genomic alterations were studied in primary tumor or metastatic sites from 32 IBC patients. Genes with high-frequency mutations were as follows: TP53 (69%), RB1 (16%), PIK3CA (13%), and also ErbB2 (3%). At least once during treatment, CTCs were detected in 26 patients with metastatic IBC, in two patients with locally advanced IBC, and four patients had no detectable CTCs. Per 7.5 mL of blood, fifteen patients (47%) had ≥20 CTCs and six of them were chosen at random to isolate single CTCs. These cells were tested for the presence of TP53, RB1, PIK3CA, and/or ErbB2 mutations previously found in matching tissue biopsies. The isolated CTCs showed the same mutations as primary or metastatic tumor samples. Intra-patient CTC heterogeneity was found by the presence of different CTC subclones, with some CTCs harboring different combinations of mutated and wild-type genes. CONCLUSIONS: Our results indicate that CTCs could represent a non-invasive source of cancer cells from which to determine genetic markers as the disease progresses and identify potential therapeutic targets in IBC patients.

Prognostic Significance of MUC-1 in Circulating Tumor Cells in Patients With Metastatic Pancreatic Adenocarcinoma.

OBJECTIVES: Development of targeted therapies for pancreatic cancer could be enhanced by a reliable method for noninvasive tumor cell assessment. In this pilot study, we isolated and phenotypically characterized circulating tumor cells (CTCs) from patients with metastatic pancreatic cancer and explored their relationship to clinical outcome. METHODS: Peripheral blood from 50 patients was collected at treatment initiation and first disease evaluation for CTC enumeration and phenotyping by CellSearch® system. Expression of human mucin 1 (MUC-1) was performed. RESULTS: Forty-eight and 37 patients had evaluable samples at baseline and first disease evaluation, respectively. The cohort was 62% male, with a median age of 63 years. At least 1 CTC per 7.5 mL was detected in 23 patients (48%) pretreatment and 11 patients (30%) at first disease evaluation. No difference was seen in overall survival between patients with 1 or more CTCs versus no CTC at baseline (P = 0.14). Patients with MUC-1 expressing CTC (n = 10) had shorter median overall survival compared with those with MUC-1 negative CTC (n = 13; 2.7 vs 9.6 m; P = 0.044). CONCLUSIONS: Circulating tumor cell enumeration and phenotypic characterization from metastatic pancreatic cancer patients are feasible. No correlation was found between CTC isolation and survival. However, the presence of MUC-1 expressing CTC demonstrated a trend toward inferior survival.

TP53 mutations detected in circulating tumor cells present in the blood of metastatic triple negative breast cancer patients.

INTRODUCTION: Circulating tumor cells (CTCs) are tumor cells shed from either primary tumors or its metastases that circulate in the peripheral blood of patients with metastatic cancers. The molecular characterization of the CTCs is critical to identifying the key drivers of cancer metastasis and devising therapeutic approaches. However, the molecular characterization of CTCs is difficult to achieve because their isolation is a major technological challenge. METHODS: CTCs from two triple negative breast cancer patients were enriched using CellSearch and single cells selected by DEPArray™. A TP53 R110 fs*13 mutation identified by next generation sequencing in the breast and chest skin biopsies of both patients was studied in single CTCs. RESULTS: From 6 single CTC isolated from one patient, 1 CTC had TP53 R110 delC, 1 CTC showed the TP53 R110 delG mutation, and the remaining 4 single CTCs showed the wild type p53 sequence; a pool of 14 CTCs isolated from the same patient also showed TP53 R110 delC mutation. In the tumor breast tissue of this patient, only the TP53 R110 delG mutation was detected. In the second patient a TP53 R110 delC mutation was detected in the chest wall skin biopsy; from the peripheral blood of this patient, 5 single CTC and 6 clusters of 2 to 6 CTCs were isolated; 3 of the 5 single CTCs showed the TP53 R110 delC mutation and 2 CTCs showed the wild type TP53 allele; from the clusters, 5 showed the TP53 R110 delC mutation, and 1 cluster the wild type TP53 allele. Single white blood cells isolated as controls from both patients only showed the wild type TP53 allele. CONCLUSIONS: We are able to isolate uncontaminated CTCs and achieve single cell molecular analysis. Our studies showed the presence of different CTC sub-clones in patients with metastatic breast cancer. Some CTCs had the same TP53 mutation as their matching tumor samples although others showed either a different TP53 mutation or the wild type allele. Our results indicate that CTCs could represent a non-invasive source of cancer cells from which to determine genetic markers of the disease progression and potential therapeutic targets.