Whole genome deep sequencing analysis of cell-free DNA in samples with low tumour content.

BACKGROUND: Circulating cell-free DNA (cfDNA) in the plasma of cancer patients contains cell-free tumour DNA (ctDNA) derived from tumour cells and it has been widely recognized as a non-invasive source of tumour DNA for diagnosis and prognosis of cancer. Molecular profiling of ctDNA is often performed using targeted sequencing or low-coverage whole genome sequencing (WGS) to identify tumour specific somatic mutations or somatic copy number aberrations (sCNAs). However, these approaches cannot efficiently detect all tumour-derived genomic changes in ctDNA. METHODS: We performed WGS analysis of cfDNA from 4 breast cancer patients and 2 patients with benign tumours. We sequenced matched germline DNA for all 6 patients and tumour samples from the breast cancer patients. All samples were sequenced on Illumina HiSeqXTen sequencing platform and achieved approximately 30x, 60x and 100x coverage on germline, tumour and plasma DNA samples, respectively. RESULTS: The mutational burden of the plasma samples (1.44 somatic mutations/Mb of genome) was higher than the matched tumour samples. However, 90% of high confidence somatic cfDNA variants were not detected in matched tumour samples and were found to comprise two background plasma mutational signatures. In contrast, cfDNA from the di-nucleosome fraction (300 bp-350 bp) had much higher proportion (30%) of variants shared with tumour. Despite high coverage sequencing we were unable to detect sCNAs in plasma samples. CONCLUSIONS: Deep sequencing analysis of plasma samples revealed higher fraction of unique somatic mutations in plasma samples, which were not detected in matched tumour samples. Sequencing of di-nucleosome bound cfDNA fragments may increase recovery of tumour mutations from plasma.

Nanopore sequencing as a scalable, cost-effective platform for analyzing polyclonal vector integration sites following clinical T cell therapy.

BACKGROUND: Analysis of vector integration sites in gene-modified cells can provide critical information on clonality and potential biological impact on nearby genes. Current short-read next-generation sequencing methods require specialized instruments and large batch runs. METHODS: We used nanopore sequencing to analyze the vector integration sites of T cells transduced by the gammaretroviral vector, SFG.iCasp9.2A.ΔCD19. DNA from oligoclonal cell lines and polyclonal clinical samples were restriction enzyme digested with two 6-cutters, NcoI and BspHI; and the flanking genomic DNA amplified by inverse PCR or cassette ligation PCR. Following nested PCR and barcoding, the amplicons were sequenced on the Oxford Nanopore platform. Reads were filtered for quality, trimmed, and aligned. Custom tool was developed to cluster reads and merge overlapping clusters. RESULTS: Both inverse PCR and cassette ligation PCR could successfully amplify flanking genomic DNA, with cassette ligation PCR showing less bias. The 4.8 million raw reads were grouped into 12,186 clusters and 6410 clones. The 3'long terminal repeat (LTR)-genome junction could be resolved within a 5-nucleotide span for a majority of clusters and within one nucleotide span for clusters with ≥5 reads. The chromosomal distributions of the insertional sites and their predilection for regions proximate to transcription start sites were consistent with previous reports for gammaretroviral vector integrants as analyzed by short-read next-generation sequencing. CONCLUSION: Our study shows that it is feasible to use nanopore sequencing to map polyclonal vector integration sites. The assay is scalable and requires minimum capital, which together enable cost-effective and timely analysis. Further refinement is required to reduce amplification bias and improve single nucleotide resolution.

Phase I Trial of Inducible Caspase 9 T Cells in Adult Stem Cell Transplant Demonstrates Massive Clonotypic Proliferative Potential and Long-term Persistence of Transgenic T Cells.

PURPOSE: Inducible caspase 9 (iCasp9) is a cellular safety switch that can make T-cell therapy safer. The purpose of this phase I trial was to investigate the use of iCasp9-transduced T-cell addback in adult patients undergoing haploidentical stem cell transplantation for high-risk hematologic malignancies. PATIENTS AND METHODS: Patients undergoing myeloablative, CD34-selected haploidentical stem cell transplantation were treated with 0.5-1.0 × 106/kg donor-derived iCasp9-transduced T cells on day +25 or 26 post-transplant, with additional doses allowed for disease relapse, infection, or mixed chimerism. RESULTS: Three patients were enrolled. iCasp9-transduced T cells were readily detectable by 4 weeks post-infusion in all patients and remained at high level (114 cells/µL, 11% of T cells) in 1 patient alive at 3.6 years. One patient developed donor-derived Epstein-Barr virus-associated post-transplant lymphoproliferative disease (EBV-PTLD), which was followed by a marked expansion of iCasp9 T cells and cytokine release syndrome (CRS). These iCasp9-transduced T cells infiltrated the affected lymph nodes and secreted IFNγ and IL-10. They peaked at 1,848 cells/µL and were found to be monoclonal by T-cell receptor (TCR) clonotype and oligoclonal by viral integrant analysis, representing a 6-log in vivo expansion of the dominant T-cell clone. These T cells were not autonomous and contracted with the resolution of EBV-PTLD, which did not recur. CONCLUSIONS: iCasp9-transduced T cells could persist long-term. They retained very high in vivo clonotypic proliferative capacity and function, and could cause CRS in response to de novo lymphoma development.

sCNAphase: using haplotype resolved read depth to genotype somatic copy number alterations from low cellularity aneuploid tumors.

Accurate identification of copy number alterations is an essential step in understanding the events driving tumor progression. While a variety of algorithms have been developed to use high-throughput sequencing data to profile copy number changes, no tool is able to reliably characterize ploidy and genotype absolute copy number from tumor samples that contain less than 40% tumor cells. To increase our power to resolve the copy number profile from low-cellularity tumor samples, we developed a novel approach that pre-phases heterozygote germline single nucleotide polymorphisms (SNPs) in order to replace the commonly used 'B-allele frequency' with a more powerful 'parental-haplotype frequency'. We apply our tool-sCNAphase-to characterize the copy number and loss-of-heterozygosity profiles of four publicly available breast cancer cell-lines. Comparisons to previous spectral karyotyping and microarray studies revealed that sCNAphase reliably identified overall ploidy as well as the individual copy number mutations from each cell-line. Analysis of artificial cell-line mixtures demonstrated the capacity of this method to determine the level of tumor cellularity, consistently identify sCNAs and characterize ploidy in samples with as little as 10% tumor cells. This novel methodology has the potential to bring sCNA profiling to low-cellularity tumors, a form of cancer unable to be accurately studied by current methods.

Phenotypic variability of distal 22q11.2 copy number abnormalities.

The availability of microarray technology has led to the recent recognition of copy number abnormalities of distal chromosome 22q11.2 that are distinct from the better-characterized deletions and duplications of the proximal region. This report describes five unrelated individuals with copy number abnormalities affecting distal chromosome 22q11.2. We report on novel phenotypic features including diaphragmatic hernia and uterine didelphys associated with the distal microdeletion syndrome; and frontomedial polymicrogyria and callosal agenesis associated with the distal microduplication syndrome. We describe the third distal chromosome 22q11.2 microdeletion patient with Goldenhar syndrome. Patients with distal chromosome 22q11.2 copy number abnormalities exhibit inter- and intra-familial phenotypic variability, and challenge our ability to draw meaningful genotype-phenotype correlations.