Targeted therapies and immunotherapies have transformed melanoma care, extending median survival from ∼9 to over 25 months, but nevertheless most patients still die of their disease. The aim of ...precision medicine is to tailor care for individual patients and improve outcomes. To this end, we developed protocols to facilitate individualized treatment decisions for patients with advanced melanoma, analyzing 364 samples from 214 patients. Whole exome sequencing (WES) and targeted sequencing of circulating tumor DNA (ctDNA) allowed us to monitor responses to therapy and to identify and then follow mechanisms of resistance. WES of tumors revealed potential hypothesis-driven therapeutic strategies for BRAF wild-type and inhibitor-resistant BRAF-mutant tumors, which were then validated in patient-derived xenografts (PDX). We also developed circulating tumor cell-derived xenografts (CDX) as an alternative to PDXs when tumors were inaccessible or difficult to biopsy. Thus, we describe a powerful technology platform for precision medicine in patients with melanoma.
Although recent developments have revolutionized melanoma care, most patients still die of their disease. To improve melanoma outcomes further, we developed a powerful precision medicine platform to monitor patient responses and to identify and validate hypothesis-driven therapies for patients who do not respond, or who develop resistance to current treatments.
Abstract
Motivation
Circulating-free DNA (cfDNA) profiling by sequencing is an important minimally invasive protocol for monitoring the mutation profile of solid tumours in cancer patients. Since the ...concentration of available cfDNA is limited, sample library generation relies on multiple rounds of PCR amplification, during which the accumulation of errors results in reduced sensitivity and lower accuracy.
Results
We present PCR Error Correction (PEC), an algorithm to identify and correct errors in short read sequencing data. It exploits the redundancy that arises from multiple rounds of PCR amplification. PEC is particularly well suited to applications such as single-cell sequencing and circulating tumour DNA (ctDNA) analysis, in which many cycles of PCR are used to generate sufficient DNA for sequencing from small amounts of starting material. When applied to ctDNA analysis, PEC significantly improves mutation calling accuracy, achieving similar levels of performance to more complex strategies that require additional protocol steps and access to calibration DNA datasets.
Availability and implementation
PEC is available under the GPL-v3 Open Source licence, and is freely available from: https://github.com/CRUKMI-ComputationalBiology/PCR_Error_Correction.git.
Supplementary information
Supplementary data are available at Bioinformatics online.
SCLC accounts for approximately 250,000 deaths worldwide each year. Acquisition of adequate tumor biopsy samples is challenging, and liquid biopsies present an alternative option for patient ...stratification and response monitoring.
We applied whole genome next-generation sequencing to circulating free DNA (cfDNA) from 39 patients with limited-stage (LS) SCLC and 30 patients with extensive-stage SCLC to establish genome-wide copy number aberrations and also performed targeted mutation analysis of 110 SCLC associated genes. Quantitative metrics were calculated for copy number aberrations, including percent genome amplified (PGA the percentage of genomic regions amplified), Z-score (a measure of standard deviation), and Moran’s I (a measure of spatial autocorrelation). In addition CellSearch, an epitope-dependent enrichment platform, was used to enumerate circulating tumor cells (CTCs) from a parallel blood sample.
Genome-wide and targeted cfDNA sequencing data identified tumor-related changes in 94% of patients with LS SCLC and 100% of patients with extensive-stage SCLC. Parallel analysis of CTCs based on at least 1 CTC/7.5 mL of blood increased tumor detection frequencies to 95% for LS SCLC. Both CTC counts and cfDNA readouts correlated with disease stage and overall survival.
We demonstrate that a simple cfDNA genome-wide copy number approach provides an effective means of monitoring patients through treatment and show that targeted cfDNA sequencing identifies potential therapeutic targets in more than 50% of patients. We are now incorporating this approach into additional studies and trials of targeted therapies.
Circulating tumour cells (CTCs) have potential utility as minimally-invasive biomarkers to aid cancer treatment decision making. However, many current CTC technologies enrich CTCs using specific ...surface epitopes that do not necessarily reflect CTC heterogeneity. Here we evaluated the epitope-independent Parsortix system which enriches CTCs based on size and rigidity using both healthy normal volunteer blood samples spiked with tumour cells and blood samples from patients with small cell lung cancer (SCLC). Blood samples were maintained unfractionated at room temperature for up to 4 days followed by plasma removal for circulating free DNA (cfDNA) isolation and direct application of the remaining cell component to the Parsortix system. For tumour cells expressing the EpCAM cell surface marker the numbers of spiked cells retained using the Parsortix system and by EpCAM-positive selection using CellSearch® were not significantly different, whereas only the Parsortix system showed strong enrichment of cells with undetectable EpCAM expression. In a pilot clinical study we banked both enriched CTCs as well as plasma from SCLC patient blood samples. Upon retrieval of the banked Parsortix cellular samples we could detect cytokeratin positive CTCs in all 12 SCLC patients tested. Interestingly, processing parallel samples from the same patients by EpCAM enrichment using CellSearch® revealed only 83% (10/12) with cytokeratin positive CTCs indicating the Parsortix system is enriching for EpCAM negative SCLC CTCs. Our combined results indicate the Parsortix system is a valuable tool for combined cfDNA isolation and CTC enrichment that enables CTC analysis to be extended beyond dependence on surface epitopes.
Owing to major advances in the field of radiation oncology, patients with lung cancer can now receive technically individualized radiotherapy treatments. Nevertheless, in the era of precision ...oncology, radiotherapy-based treatment selection needs to be improved as many patients do not benefit or are not offered optimum therapies. Cost-effective robust biomarkers can address this knowledge gap and lead to individuals being offered more bespoke treatments leading to improved outcome. This narrative review discusses some of the current achievements and challenges in the realization of personalized radiotherapy delivery in patients with lung cancer.
The adoptive transfer of chimeric antigen receptor (CAR)-expressing T cells is a relatively new but promising approach in the field of cancer immunotherapy. This therapeutic strategy is based on the ...genetic reprogramming of T cells with an artificial immune receptor that redirects them against targets on malignant cells and enables their destruction by exerting T cell effector functions. There has been an explosion of interest in the use of CAR T cells as an immunotherapy for cancer. In the pre-clinical setting, there has been a considerable focus upon optimizing the structural and signaling potency of the CAR while advances in bio-processing technology now mean that the clinical testing of these gene-modified T cells has become a reality. This review will summarize the concept of CAR-based immunotherapy and recent clinical trial activity and will further discuss some of the likely future challenges facing CAR-modified T cell therapies.
Molecular profiling from liquid biopsy, in particular cell-free DNA (cfDNA), represents an attractive alternative to tissue biopsies for the detection of actionable targets and tumor monitoring. In ...addition to PCR-based assays, Next Generation Sequencing (NGS)-based cfDNA assays are now commercially available and are being increasingly adopted in clinical practice. However, the validity of these products as well as the clinical utility of cfDNA in the management of patients with cancer has yet to be proven. Within framework of the Innovative Medicines Initiative (IMI) program CANCER-ID we evaluated the use of commercially available reference materials designed for ctDNA testing and cfDNA derived from Diagnostic Leukaphereses (DLA) for inter- and intra-assay as well as intra- and inter-laboratory comparisons. In three experimental setups, a broad range of assays including ddPCR, MassARRAY and various NGS-based assays were tested. We demonstrate that both reference materials with predetermined VAFs and DLA samples are extremely useful for the performance assessment of mutation analysis platforms. Moreover, our data indicate a substantial variability of NGS assays with respect to sensitivity and specificity highlighting the importance of extensive validation of the test performance before offering these tests in clinical routine practice.
Next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) supports blood-based genomic profiling but is not yet routinely implemented in the setting of a phase I trials clinic. TARGET is a ...molecular profiling program with the primary aim to match patients with a broad range of advanced cancers to early phase clinical trials on the basis of analysis of both somatic mutations and copy number alterations (CNA) across a 641 cancer-associated-gene panel in a single ctDNA assay. For the first 100 TARGET patients, ctDNA data showed good concordance with matched tumor and results were turned round within a clinically acceptable timeframe for Molecular Tumor Board (MTB) review. When a 2.5% variant allele frequency (VAF) threshold was applied, actionable mutations were identified in 41 of 100 patients, and 11 of these patients received a matched therapy. These data support the application of ctDNA in this early phase trial setting where broad genomic profiling of contemporaneous tumor material enhances patient stratification to novel therapies and provides a practical template for bringing routinely applied blood-based analyses to the clinic.
Molecular information obtained from cancer patients' blood is an emerging and powerful research tool with immense potential as a companion diagnostic for patient stratification and monitoring. Blood, ...which can be sampled routinely, provides a means of inferring the current genetic status of patients' tumours via analysis of circulating tumour cells (CTCs) or circulating tumour DNA (ctDNA). However, accurate assessment of both CTCs and ctDNA requires all blood cells to be maintained intact until samples are processed. This dictates for ctDNA analysis EDTA blood samples must be processed with 4 h of draw, severely limiting the use of ctDNA in multi-site trials. Here we describe a blood collection protocol that is amenable for analysis of both CTCs and ctDNA up to four days after blood collection. We demonstrate that yields of circulating free DNA (cfDNA) obtained from whole blood CellSave samples are equivalent to those obtained from conventional EDTA plasma processed within 4 h of blood draw. Targeted and genome-wide NGS revealed comparable DNA quality and resultant sequence information from cfDNA within CellSave and EDTA samples. We also demonstrate that CTCs and ctDNA can be isolated from the same patient blood sample, and give the same patterns of CNA enabling direct analysis of the genetic status of patients' tumours.
In summary, our results demonstrate the utility of a simple approach that enabling robust molecular analysis of CTCs and cfDNA for genotype-directed therapies in multi-site clinical trials and represent a significant methodological improvement for clinical benefit.
•Demonstrated collection of blood in CellSave tubes preserves cfDNA up to 96 h post draw.•No significant difference between standard EDTA cfDNA and preserved cfDNA.•Preserved cfDNA applicable to both targeted and genome wide NGS analysis.•CTCs and cfDNA can be isolated from same CellSave blood sample.•Comparable copy number aberrations seen in both CTCs and ctDNA from same blood sample.
The spontaneous adsorption of biomolecules onto the surface of nanoparticles (NPs) in complex physiological biofluids has been widely investigated over the last decade. Characterisation of the ...protein composition of the 'biomolecule corona' has dominated research efforts, whereas other classes of biomolecules, such as nucleic acids, have received no interest. Scarce, speculative statements exist in the literature about the presence of nucleic acids in the biomolecule corona, with no previous studies attempting to describe the contribution of genomic content to the blood-derived NP corona. Herein, we provide the first experimental evidence of the interaction of circulating cell-free DNA (cfDNA) with lipid-based NPs upon their incubation with human plasma samples, obtained from healthy volunteers and ovarian carcinoma patients. Our results also demonstrate an increased amount of detectable cfDNA in patients with cancer. Proteomic analysis of the same biomolecule coronas revealed the presence of histone proteins, suggesting an indirect, nucleosome-mediated NP-cfDNA interaction. The finding of cfDNA as part of the NP corona, offers a previously unreported new scope regarding the chemical composition of the 'biomolecule corona' and opens up new possibilities for the potential exploitation of the biomolecule corona for the enrichment and analysis of blood-circulating nucleic acids.
The biomolecule corona spontaneously adsorbed onto lipid-based nanoparticles (NPs), upon incubation with human plasma, contains circulating cell-free DNA (cfDNA).
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