SmMIP-tools: a computational toolset for processing and analysis of single-molecule molecular inversion probes-derived data.

MOTIVATION: Single-molecule molecular inversion probes (smMIPs) provide an exceptionally cost-effective and modular approach for routine or large-cohort next-generation sequencing. However, processing the derived raw data to generate highly accurate variants calls remains challenging. RESULTS: We introduce SmMIP-tools, a comprehensive computational method that promotes the detection of single nucleotide variants and short insertions and deletions from smMIP-based sequencing. Our approach delivered near-perfect performance when benchmarked against a set of known mutations in controlled experiments involving DNA dilutions and outperformed other commonly used computational methods for mutation detection. Comparison against clinically approved diagnostic testing of leukaemia patients demonstrated the ability to detect both previously reported variants and a set of pathogenic mutations that did not pass detection by clinical testing. Collectively, our results indicate that increased performance can be achieved when tailoring data processing and analysis to its related technology. The feasibility of using our method in research and clinical settings to benefit from low-cost smMIP technology is demonstrated. AVAILABILITY AND IMPLEMENTATION: The source code for SmMIP-tools, its manual and additional scripts aimed to foster large-scale data processing and analysis are all available on github (https://github.com/abelson-lab/smMIP-tools). Raw sequencing data generated in this study have been submitted to the European Genome-Phenome Archive (EGA; https://ega-archive.org) and can be accessed under accession number EGAS00001005359. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Tracing the origins of relapse in acute myeloid leukaemia to stem cells.

In acute myeloid leukaemia, long-term survival is poor as most patients relapse despite achieving remission. Historically, the failure of therapy has been thought to be due to mutations that produce drug resistance, possibly arising as a consequence of the mutagenic properties of chemotherapy drugs. However, other lines of evidence have pointed to the pre-existence of drug-resistant cells. For example, deep sequencing of paired diagnosis and relapse acute myeloid leukaemia samples has provided direct evidence that relapse in some cases is generated from minor genetic subclones present at diagnosis that survive chemotherapy, suggesting that resistant cells are generated by evolutionary processes before treatment and are selected by therapy. Nevertheless, the mechanisms of therapy failure and capacity for leukaemic regeneration remain obscure, as sequence analysis alone does not provide insight into the cell types that are fated to drive relapse. Although leukaemia stem cells have been linked to relapse owing to their dormancy and self-renewal properties, and leukaemia stem cell gene expression signatures are highly predictive of therapy failure, experimental studies have been primarily correlative and a role for leukaemia stem cells in acute myeloid leukaemia relapse has not been directly proved. Here, through combined genetic and functional analysis of purified subpopulations and xenografts from paired diagnosis/relapse samples, we identify therapy-resistant cells already present at diagnosis and two major patterns of relapse. In some cases, relapse originated from rare leukaemia stem cells with a haematopoietic stem/progenitor cell phenotype, while in other instances relapse developed from larger subclones of immunophenotypically committed leukaemia cells that retained strong stemness transcriptional signatures. The identification of distinct patterns of relapse should lead to improved methods for disease management and monitoring in acute myeloid leukaemia. Moreover, the shared functional and transcriptional stemness properties that underlie both cellular origins of relapse emphasize the importance of developing new therapeutic approaches that target stemness to prevent relapse.

Clonal hematopoiesis confers an increased mortality risk in orthotopic heart transplant recipients.

Novel risk stratification and non-invasive surveillance methods are needed in orthotopic heart transplant (OHT) to reduce morbidity and mortality post-transplant. Clonal hematopoiesis (CH) refers to the acquisition of specific gene mutations in hematopoietic stem cells linked to enhanced inflammation and worse cardiovascular outcomes. The purpose of this study was to investigate the association between CH and OHT. Blood samples were collected from 127 OHT recipients. Error-corrected sequencing was used to detect CH-associated mutations. We evaluated the association between CH and acute cellular rejection, CMV infection, cardiac allograft vasculopathy (CAV), malignancies, and survival. CH mutations were detected in 26 (20.5%) patients, mostly in DNMT3A, ASXL1, and TET2. Patients with CH showed a higher frequency of CAV grade 2 or 3 (0% vs. 18%, p < .001). Moreover, a higher mortality rate was observed in patients with CH (11 [42%] vs. 15 [15%], p = .008) with an adjusted hazard ratio of 2.9 (95% CI, 1.4-6.3; p = .003). CH was not associated with acute cellular rejection, CMV infection or malignancies. The prevalence of CH in OHT recipients is higher than previously reported for the general population of the same age group, with an associated higher prevalence of CAV and mortality.

Clinical significance of clonal hematopoiesis in the setting of autologous stem cell transplantation for lymphoma.

Autologous stem cell transplantation (ASCT) remains a key therapeutic strategy for treating patients with relapsed or refractory non-Hodgkin and Hodgkin lymphoma. Clonal hematopoiesis (CH) has been proposed as a major contributor not only to the development of therapy-related myeloid neoplasms but also to inferior overall survival (OS) in patients who had undergone ASCT. Herein, we aimed to investigate the prognostic implications of CH after ASCT in a cohort of 420 lymphoma patients using ultra-deep, highly sensitive error-correction sequencing. CH was identified in the stem cell product samples of 181 patients (43.1%) and was most common in those with T-cell lymphoma (72.2%). The presence of CH was associated with a longer time to neutrophil and platelet recovery. Moreover, patients with evidence of CH had inferior 5-year OS from the time of first relapse (39.4% vs. 45.8%, p = .043) and from the time of ASCT (51.8% vs. 59.3%, p = .018). The adverse prognostic impact of CH was not due to therapy-related myeloid neoplasms, the incidence of which was low in our cohort (10-year cumulative incidence of 3.3% vs. 3.0% in those with and without CH, p = .445). In terms of specific-gene mutations, adverse OS was mostly associated with PPM1D mutations (hazard ratio (HR) 1.74, 95% confidence interval (CI) 1.13-2.67, p = .011). In summary, we found that CH is associated with an increased risk of non-lymphoma-related death after ASCT, which suggests that lymphoma survivors with CH may need intensified surveillance strategies to prevent and treat late complications.

Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia.

In acute myeloid leukaemia (AML), the cell of origin, nature and biological consequences of initiating lesions, and order of subsequent mutations remain poorly understood, as AML is typically diagnosed without observation of a pre-leukaemic phase. Here, highly purified haematopoietic stem cells (HSCs), progenitor and mature cell fractions from the blood of AML patients were found to contain recurrent DNMT3A mutations (DNMT3A(mut)) at high allele frequency, but without coincident NPM1 mutations (NPM1c) present in AML blasts. DNMT3A(mut)-bearing HSCs showed a multilineage repopulation advantage over non-mutated HSCs in xenografts, establishing their identity as pre-leukaemic HSCs. Pre-leukaemic HSCs were found in remission samples, indicating that they survive chemotherapy. Therefore DNMT3A(mut) arises early in AML evolution, probably in HSCs, leading to a clonally expanded pool of pre-leukaemic HSCs from which AML evolves. Our findings provide a paradigm for the detection and treatment of pre-leukaemic clones before the acquisition of additional genetic lesions engenders greater therapeutic resistance.

Mutations in myeloid transcription factors and activated signaling genes predict chronic myeloid leukemia outcomes.

ABSTRACT: Advancements in genomics are transforming the clinical management of chronic myeloid leukemia (CML) toward precision medicine. The impact of somatic mutations on treatment outcomes is still under debate. We studied the association of somatic mutations in epigenetic modifier genes and activated signaling/myeloid transcription factors (AS/MTFs) with disease progression and treatment failure in patients with CML after tyrosine kinase inhibitor (TKI) therapy. A total of 394 CML samples were sequenced, including 254 samples collected at initial diagnosis and 140 samples taken during follow-up. Single-molecule molecular inversion probe (smMIP)-based next-generation sequencing (NGS) was conducted targeting recurrently mutated loci in 40 genes, with a limit of detection of 0.2%. Seventy mutations were detected in 57 diagnostic samples (22.4%), whereas 64 mutations were detected in 39 of the follow-up samples (27.9%). Carrying any mutation at initial diagnosis was associated with worse outcomes after TKI therapy, particularly in AS/MTF genes. Patients having these mutations at initial diagnosis and treated with imatinib showed higher risks of treatment failure (hazard ratio, 2.53; 95% confidence interval, 1.13-5.66; P = .0239). The adverse prognostic impact of the mutations was not clear for patients treated with second-generation TKIs. The multivariate analysis affirmed that mutations in AS/MTF genes independently serve as adverse prognostic factors for molecular response, failure-free survival, and progression risk. Additionally, there was an observable nonsignificant trend indicating a heightened risk of progression to advanced disease and worse overall survival. In conclusion, mutations in the AS/MTF genes using smMIP-based NGS can help identify patients with a potential risk of both treatment failure and progression and may help upfront TKI selection.

Clonal haematopoiesis is associated with higher mortality in patients with cardiogenic shock.

AIMS: Cardiogenic shock (CS) with variable systemic inflammation may be responsible for patient heterogeneity and the exceedingly high mortality rate. Cardiovascular events have been associated with clonal haematopoiesis (CH) where specific gene mutations in haematopoietic stem cells lead to clonal expansion and the development of inflammation. This study aims to assess the prevalence of CH and its association with survival in a population of CS patients in a quaternary centre. METHODS AND RESULTS: We compared the frequency of CH mutations among 341 CS patients and 345 ambulatory heart failure (HF) patients matched for age, sex, ejection fraction, and HF aetiology. The association of CH with survival and levels of circulating inflammatory cytokines was analysed. We detected 266 CH mutations in 149 of 686 (22%) patients. CS patients had a higher prevalence of CH-related mutations than HF patients (odds ratio 1.5; 95% confidence interval [CI] 1.0-2.1, p = 0.02) and was associated with decreased survival (30 days: hazard ratio [HR] 2.7; 95% CI 1.3-5.7, p = 0.006; 90 days: HR 2.2; 95% CI 1.3-3.9, p = 0.003; and 3 years: HR 1.7; 95% CI 1.1-2.8, p = 0.01). TET2 or ASXL1 mutations were associated with lower survival in CS patients at all time-points (p ≤ 0.03). CS patients with TET2 mutations had higher circulating levels of SCD40L, interferon-γ, interleukin-4, and tumour necrosis factor-α (p ≤ 0.04), while those with ASXL1 mutations had decreased levels of CCL7 (p = 0.03). CONCLUSIONS: Cardiogenic shock patients have high frequency of CH, notably mutations in TET2 and ASXL1. This was associated with reduced survival and dysregulation of circulating inflammatory cytokines in those CS patients with CH.

Recurrent deletions in clonal hematopoiesis are driven by microhomology-mediated end joining.

The mutational mechanisms underlying recurrent deletions in clonal hematopoiesis are not entirely clear. In the current study we inspect the genomic regions around recurrent deletions in myeloid malignancies, and identify microhomology-based signatures in CALR, ASXL1 and SRSF2 loci. We demonstrate that these deletions are the result of double stand break repair by a PARP1 dependent microhomology-mediated end joining (MMEJ) pathway. Importantly, we provide evidence that these recurrent deletions originate in pre-leukemic stem cells. While DNA polymerase theta (POLQ) is considered a key component in MMEJ repair, we provide evidence that pre-leukemic MMEJ (preL-MMEJ) deletions can be generated in POLQ knockout cells. In contrast, aphidicolin (an inhibitor of replicative polymerases and replication) treatment resulted in a significant reduction in preL-MMEJ. Altogether, our data indicate an association between POLQ independent MMEJ and clonal hematopoiesis and elucidate mutational mechanisms involved in the very first steps of leukemia evolution.

Mapping the cellular origin and early evolution of leukemia in Down syndrome.

Children with Down syndrome have a 150-fold increased risk of developing myeloid leukemia, but the mechanism of predisposition is unclear. Because Down syndrome leukemogenesis initiates during fetal development, we characterized the cellular and developmental context of preleukemic initiation and leukemic progression using gene editing in human disomic and trisomic fetal hematopoietic cells and xenotransplantation. GATA binding protein 1 (GATA1) mutations caused transient preleukemia when introduced into trisomy 21 long-term hematopoietic stem cells, where a subset of chromosome 21 microRNAs affected predisposition to preleukemia. By contrast, progression to leukemia was independent of trisomy 21 and originated in various stem and progenitor cells through additional mutations in cohesin genes. CD117+/KIT proto-oncogene (KIT) cells mediated the propagation of preleukemia and leukemia, and KIT inhibition targeted preleukemic stem cells.