Search Results - EGA European Genome-Phenome Archive

WGS of MAPKi acquired resistant samples from patients and PDX models

Blocking cancer genomic instability, which generates diverse variants that enable rapid adaptations, may prevent tumor escape from therapies. We show that, after MAPK inhibitor (MAPKi) therapy in patients and patient-derived xenografts, acquired-resistant genomes of metastatic cutaneous melanoma accumulate amplicons of resistance-driver, non-homologous end-joining (NHEJ), and homologous recombination repair (HRR) genes within complex genomic rearrangements (CGRs) and extrachromosomal circular DNAs (ecDNAs). Also, chromothriptic genomic regions enrich for mutations and may engender ecDNAs and CGRs. Overall, somatic mutations within ecDNA- and CGR-amplicons enrich for HRR signatures, and, in acquired resistance, ecDNA (versus CGR) mutations enrich for APOBEC3 signatures. Breakpoint-sequence analysis suggests NHEJ as critical to double-stranded DNA break repair underlying CGR and ecDNA formation. In vivo, NHEJ-targeting by a DNA-PKCS inhibitor prevented acquired MAPKi-resistance by reducing the size of ecDNAs and CGRs early on combination treatment. Thus, targeting the cause(s), as opposed to the consequences, of genomic instability prevents acquired resistance.

Study EGAS00001006874

Comparison of sequencing assays for sensitive detection of circulating tumour DNA in stage IA-IV breast cancer

This study compared different assays for the detection of circulating tumour DNA (ctDNA) in serial plasma from stage IA-IV breast cancer patients, targeting structural variants (SVs), single nucleotide variants (SNVs) and/or somatic copy-number aberrations (SCNAs). SV-multiplex PCR, SNV-/SV-hybrid capture, and different depths of whole-genome sequencing (WGS) were used to evaluate ctDNA levels, demonstrating concordant results. SNV-hybrid capture targeting 1,347-7,491 mutations was the most sensitive assay, detecting 67% (36/54) of samples down to an allele fraction (AF) of 0.00024%. SV-multiplex PCR, targeting 21-47 mutations, detected 63% (34/54) of samples down to 0.00047% AF and has potential as a clinical assay.

Dataset EGAD00001008589

Whole genome and Whole exome sequencing of patient-derived xenograft models of endometrial cancer

Dataset EGAD00001008326

Chemotherapy induces canalization of cell state in childhood B-cell precursor acute lymphoblastic leukemia

Comparison of intratumor genetic heterogeneity in cancer at diagnosis and relapse suggests that chemotherapy induces bottleneck selection of subclonal genotypes. However, evolutionary events subsequent to chemotherapy could also explain changes in clonal dominance seen at relapse. We therefore investigated the mechanisms of selection in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) during induction chemotherapy where maximal cytoreduction occurs. To distinguish stochastic versus deterministic events, individual leukemias were transplanted into multiple xenografts and chemotherapy administered. Analyses of the immediate post-treatment leukemic residuum at single-cell resolution revealed that chemotherapy has little impact on genetic heterogeneity. Rather, it acts on extensive, previously unappreciated, transcriptional and epigenetic heterogeneity in BCP-ALL, dramatically reducing the spectrum of cell states represented, leaving a genetically polyclonal but phenotypically uniform population, with hallmark signatures relating to developmental stage, cell cycle and metabolism. Hence, canalization of the cell state accounts for a significant component of bottleneck selection during induction chemotherapy.

Study EGAS00001004407

Analysis of Donor Pancreata Defines the Transcriptomic Signature and Microenvironment of Early Neoplastic Pancreatic Lesions

The adult healthy human pancreas has been poorly studied given lack of indication to obtain tissue from the pancreas in the absence of disease and rapid postmortem degradation. We obtained pancreata from brain dead donors thus avoiding any warm ischemia time. We found that neoplastic lesions occur frequently in healthy organs, in donors as young as in their 3rd decade of life. Using the 10X Genomics Platform, we performed single cell RNA sequencing on 7 donor pancreata, 6 of which we separately sequenced tissue from the pancreas head and tail, for a total of 11 sequencing runs. Of note, because one of our runs (containing sequencing from pancreas head and tail) did not pass qc, our final analysis in our study utilized 6 donor pancreata, 5 of which contained pancreas head and tail sequenced separately. This is a robust single-cell dataset of healthy, nonpathologic pancreas tissue and includes acinar, ductal, and non-epithelial cells (myeloid cells, fibroblasts, endothelial cells, lymphocytes).Briefly, the donor pancreas was dissected by a clinically-trained pancreatobiliary surgeon. The organ was dissected into head, body, and tail, with portions of each placed into DMEM media with 1% BSA/10µM Y27632. Tissue was minced into 1mm3 pieces, then digested with 1 mg/mL collagenase P for 20-30 min at 37°C with gentle agitation. Digested tissue was rinsed three times with DMEM/1% BSA/10µM Y27632, then filtered through a 40µm mesh. Resulting cells were submitted to the University of Michigan Advanced Genomics Core for single cell sequencing using the 10x Genomics Platform. FASTQ files of all sequencing runs along with clinical annotation of donors is available through this dbGaP submission.

Study phs003229

Peripheral Blood Transcriptome Analysis of ALS Patients

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative disorders which overlap in their clinical presentation, pathology, and genetic origin. An over-representation of autoimmune disorders exists in both ALS and FTD, but this remains an unexplained epidemiologic observation. Expansions of a hexanucleotide repeat (GGGGCC) in the C9orf72 gene (GeneID:203228) are the most common cause of familial ALS and FTD (C9-ALS/FTD), leading to both repeat containing RNA and dipeptide accumulation coupled with decreased C9orf72 protein expression in brain and peripheral blood cells. Here we show that loss of C9orf72 in myeloid cells is sufficient to recapitulate the age dependent lymphoid hypertrophy and autoinflammation seen in complete C9orf72-/- mice. Dendritic cells isolated from C9orf72-/- mice showed marked early activation of the type I interferon response, and C9orf72-/- myeloid cells were primarily hyperresponsive to activators of stimulator of interferon gene (STING1 GeneID:340061), a key regulator of the innate immune response to cytosolic DNA. STING degradation through the autolysosomal pathway was diminished in C9orf72-/- mice myeloid cells, and blocking STING suppressed hyperactive type I interferon responses in C9orf72-/- myeloid cells and splenomegaly and inflammation in C9orf72-/- mice. Additionally, mice lacking one or both copies of C9orf72 were more susceptible to experimental autoimmune encephalitis, mirroring the susceptibility to autoimmune diseases seen in C9-ALS/FTD patients. Finally, we found that blood derived macrophages, peripheral blood monocytes, and brain tissue from C9-ALS/FTD patients all showed a hyperactive type I interferon signature compared to sporadic ALS/FTD subjects. Collectively, our results indicate that C9-ALS/FTD patients have an altered immunophenotype due to loss of C9orf72 regulation of STING mediated type I interferon activation.

Study phs002055

Resistance to Checkpoint Blockade Therapy Through Inactivation of Antigen Presentation

Although immune checkpoint blockade (CPB) leads to prolonged responses in 15-40% of patients with metastatic melanoma, treatment refractory disease and progression after initial response remain major causes of mortality. While predictors of response have been reported, the common mechanisms of both primary and acquired resistance are poorly understood. To identify mechanisms of resistance and examine the evolving landscape in response to CPB, we performed whole exome sequencing (WES), immunohistochemistry (IHC), and RNA-sequencing (RNAseq) of longitudinal tumor biopsies from 17 metastatic melanoma patients treated with various CPB therapies. We found no significant changes in both mutational and neoantigen loads over time between responders and nonresponders. However, we identified abnormalities in one gene, beta-2-microglobulin (B2M), an essential component of MHC Class I antigen presentation, that were present in samples during disease progression but not regression. In total, we identified B2M aberrations in 29.4% of patients, including multiple early frameshift mutations, loss of heterozygosity (LOH) overlapping B2M, and absence of tumor-specific B2M protein expression. Additional defects in the antigen presentation and IFNγ pathways were identified but were not restricted to progressing lesions in our cohort. In two independent cohorts of 105 and 38 melanoma patients treated with ipilimumab (anti-CTLA4) and pembrolizumab (anti-PD1) respectively, we found that B2M LOH was enriched 3-fold in nonresponders (~30%) vs. responders (~10%) and associated with poorer overall survival (log-rank p=0.01, p=0.006). Loss of both copies of B2M was found only in nonresponders. We also found evidence for association of LOH overlapping IFNGR1 with poorer overall survival exclusively in the anti-PD1 cohort. Thus, B2M loss is likely a common mechanism of primary and acquired resistance to therapies targeting CTLA4 or PD-1.

Study phs001427

Analysis of CD20 loss in patients treated with Mosunetuzumab

CD20 is an established therapeutic target in B-cell malignancies. The CD20xCD3 bispecific antibody mosunetuzumab has significant efficacy in B-cell non-Hodgkin lymphomas (NHL). Since target antigen loss is a recognized mechanism of resistance, we evaluated the contribution of CD20 expression to clinical response in patients with relapsed and/or refractory NHL in the phase 1/2 GO29781 trial (ClinicalTrials.gov: NCT02500407) investigating mosunetuzumab monotherapy. CD20 was studied using immunohistochemistry (IHC), RNA sequencing, and whole-exome sequencing performed centrally in biopsies collected prior to treatment, or as paired samples at pre-dose and on-treatment, or at-progression. Prior to treatment, most patients exhibited a high proportion of tumor cells expressing CD20; however, the proportion of CD20+ tumor cells in 14/293 (6%) patients was <10%. Analyses of paired biopsies from patients during treatment revealed that CD20 levels were maintained in 29/30 (97%), vs patients with samples at-progression, in which 11/32 (34%) showed CD20 loss. Reduced transcription or acquisition of truncating mutations explained most but not all cases of CD20 loss. In vitro modeling confirmed the effect of CD20 variants identified in clinical samples on reduction of CD20 expression and confirmed missense mutations in the extracellular domain that could block mosunetuzumab binding. This study expands knowledge about the occurrence of target antigen loss after anti-CD20 therapeutics to CD20-targeting bispecific antibodies and elucidates the underlying mechanisms for reduced CD20 expression at disease progression that may be generalizable to other anti-CD20 targeting agents. These results also establish the utility of readily-available IHC staining for CD20 as a tool to inform clinical decisions.

Study EGAS50000000151

RNAseq: Acquired non-permissive bone marrow microenvironment impairs hematopoietic stem cell proliferation and maintenance and B-cell development post-HSCT

Defects in B-cell reconstitution upon hematopoietic stem-cell (HSC) transplantation (HSCT) are a common observation, yet the mechanism remains unexplained. The bone marrow (BM) stroma, including mesenchymal stromal cells (MSCs), guides HSC maintenance and B-lymphopoiesis by secreting crucial cytokines. We report acquired, permanent, selective and complete B-cell deficiency in the context of full donor-chimerism in a patient with X-linked lymphoproliferative disease and aimed to identify the contribution of the BM-microenvironment in disrupted B-cell reconstitution post-HSCT. We studied longitudinal BM samples from the patient and his identical twin, both of whom underwent HSCT with the same donor with opposite outcomes in B-cell reconstitution. In the index patient BM, we observed progressive loss of proliferation of HSCs and a selective block at the pre-BI cell stage. In vitro modeling studies showed limited survival of patient-HSCs and a relative accumulation of pre-B cells. Patient-derived MSCs failed to support survival and proliferation of HSCs and B-cell development of healthy-HSCs which was correlated with reduced CXCL12 levels. Using bulk RNA-sequencing of MSCs and in vitro functional studies, we showed global changes in the patients’ MSCs and a progressive loss of CXCL12 expression. Indeed, survival of patients HSCs improved supplementing in vitro development culture with CXCL12, suggesting a contribution of defective CXCL12 signaling to the phenotype. In summary, our data show that an acquired defect in the BM-stromal microenvironment and exhaustion of HSCs and committed progenitors may cause a permanent non-permissive state for normal B-cell development.

Study EGAS50000001437

DNA methylation-based prognostic subtypes of chordoma tumors in tissue

Chordomas are rare malignant bone cancers of the skull-base and spine. Patient survival is variable and not reliably predicted using clinical factors or molecular features. This study identifies prognostic epigenetic chordoma subtypes that are detected noninvasively using plasma methylomes. Methylation profiles of 68 chordoma surgical samples were obtained between 1996 and 2018 across three international centers along with matched plasma methylomes where available. Consensus clustering identified two stable tissue clusters with a disease-specific survival difference that was independent of clinical factors in a multivariate Cox analysis (HR = 14.2, 95%CI: 2.1–94.8, P = 0.0063). Immune-related pathways with genes hypomethylated at promoters and increased immune cell abundance were observed in the poorperforming “Immune-infiltrated” subtype. Cell-to-cell interaction plus extracellular matrix pathway hypomethylation and higher tumor purity were observed in the better-performing “Cellular” subtype. The findings were validated in additional DNA methylation and RNA sequencing datasets as well as with immunohistochemical staining. Plasma methylomes distinguished chordomas from other clinical differential diagnoses by applying fifty chordoma-versusother binomial generalized linear models in random 20% testing sets (mean AUROC = 0.84, 95%CI: 0.52–1.00). Tissue-based and plasma-based methylation signals were highly correlated in both prognostic clusters. Additionally, leave-one-out models accurately classified all tumors into their correct cluster based on plasma methylation data. Here, we show the first identification of prognostic epigenetic chordoma subtypes and first use of plasma methylome based biomarkers to noninvasively diagnose and subtype chordomas. These results may transform patient management by allowing treatment aggressiveness to be balanced with patient risk according to prognosis.

Study EGAS00001006406

11932 results for "cancer rna-seq"

in 58.50 milliseconds.