Clinical significance of chromatin-spliceosome acute myeloid leukemia: a report from the Northern Italy Leukemia Group (NILG) randomized trial 02/06.

Secondary acute myeloid leukemia (sAML) after myelodysplastic or myeloproliferative disorders is a high-risk category currently identified by clinical history or specific morphological and cytogenetic abnormalities. However, in the absence of these features, uncertainties remain to identify the secondary nature of some cases otherwise defined as de novo AML. To test whether a chromatin-spliceosome (CS) mutational signature might better inform the definition of the de novo AML group, we analyzed a prospective cohort of 413 newly diagnosed AML patients enrolled into a randomized clinical trial (NILG AML 02/06) and provided with accurate cytogenetic and molecular characterization. Among clinically defined de novo AML, 17.6% carried CS mutations (CS-AML) and showed clinical characteristics closer to sAML (older age, lower white blood cell counts and higher rate of multilineage dysplasia). Outcomes in this group were adverse, more similar to those of sAML as compared to de novo AML (overall survival, 30% in CS-AML and 17% in sAML vs 61% in de novo AML, P<0.0001; disease free survival, 26% in CS-AML and 22% in sAML vs 54% of de novo AML, P<0.001) and independently confirmed by multivariable analysis. Allogeneic transplant in first complete remission improved survival in both sAML and CS-AML patients. In conclusion, these findings highlight the clinical significance of identifying CS-AML for improved prognostic prediction and potential therapeutic implications. (NILG AML 02/06: ClinicalTrials.gov Identifier: NCT00495287).

High-sensitivity analysis of clonal hematopoiesis reveals increased clonal complexity of potential-driver mutations in severe COVID-19 patients.

Whether Clonal Hematopoiesis (CH) represents a risk factor for severity of the COVID-19 disease remains a controversial issue. We report the first high- sensitivity analysis of CH in COVID-19 patients (threshold of detection at 0.5% vs 1 or 2% in previous studies). We analyzed 24 patients admitted to ICU for COVID-19 (COV-ICU) and 19 controls, including healthy subjects and asymptomatic SARS-CoV2-positive individuals. Despite the significantly higher numbers of CH mutations identified (80% mutations with <2% variant allele frequency, VAF), we did not find significant differences between COV-ICU patients and controls in the prevalence of CH or in the numbers, VAF or functional categories of the mutated genes, suggesting that CH is not overrepresented in patients with COVID-19. However, when considering potential drivers CH mutations (CH-PD), COV-ICU patients showed higher clonal complexity, in terms of both mutation numbers and VAF, and enrichment of variants reported in myeloid neoplasms. However, we did not score an impact of increased CH-PD on patient survival or clinical parameters associated with inflammation. These data suggest that COVID-19 influence the clonal composition of the peripheral blood and call for further investigations addressing the potential long-term clinical impact of CH on people experiencing severe COVID-19. We acknowledge that it will indispensable to perform further studies on larger patient cohorts in order to validate and generalize our conclusions. Moreover, we performed CH analysis at a single time point. It will be necessary to consider longitudinal approaches with long periods of follow-up in order to assess if the COVID-19 disease could have an impact on the evolution of CH and long-term consequences in patients that experienced severe COVID-19.

Barcode demultiplexing of nanopore sequencing raw signals by unsupervised machine learning.

Introduction: Oxford Nanopore Technologies (ONT) is a third generation sequencing approach that allows the analysis of individual, full-length nucleic acids. ONT records the alterations of an ionic current flowing across a nano-scaled pore while a DNA or RNA strand is threading through the pore. Basecalling methods are then leveraged to translate the recorded signal back to the nucleic acid sequence. However, basecall generally introduces errors that hinder the process of barcode demultiplexing, a pivotal task in single-cell RNA sequencing that allows for separating the sequenced transcripts on the basis of their cell of origin. Methods: To solve this issue, we present a novel framework, called UNPLEX, designed to tackle the barcode demultiplexing problem by operating directly on the recorded signals. UNPLEX combines two unsupervised machine learning methods: autoencoders and self-organizing maps (SOM). The autoencoders extract compact, latent representations of the recorded signals that are then clustered by the SOM. Results and Discussion: Our results, obtained on two datasets composed of in silico generated ONT-like signals, show that UNPLEX represents a promising starting point for the development of effective tools to cluster the signals corresponding to the same cell.

High-resolution Nanopore methylome-maps reveal random hyper-methylation at CpG-poor regions as driver of chemoresistance in leukemias.

Aberrant DNA methylation at CpG dinucleotides is a cancer hallmark that is associated with the emergence of resistance to anti cancer treatment, though molecular mechanisms and biological significance remain elusive. Genome scale methylation maps by currently used methods are based on chemical modification of DNA and are best suited for analyses of methylation at CpG rich regions (CpG islands). We report the first high coverage whole-genome map in cancer using the long read nanopore technology, which allows simultaneous DNA-sequence and -methylation analyses on native DNA. We analyzed clonal epigenomic/genomic evolution in Acute Myeloid Leukemias (AMLs) at diagnosis and relapse, after chemotherapy. Long read sequencing coupled to a novel computational method allowed definition of differential methylation at unprecedented resolution, and showed that the relapse methylome is characterized by hypermethylation at both CpG islands and sparse CpGs regions. Most differentially methylated genes, however, were not differentially expressed nor enriched for chemoresistance genes. A small fraction of under-expressed and hyper-methylated genes at sparse CpGs, in the gene body, was significantly enriched in transcription factors (TFs). Remarkably, these few TFs supported large gene-regulatory networks including 50% of all differentially expressed genes in the relapsed AMLs and highly-enriched in chemoresistance genes. Notably, hypermethylated regions at sparse CpGs were poorly conserved in the relapsed AMLs, under-represented at their genomic positions and showed higher methylation entropy, as compared to CpG islands. Analyses of available datasets confirmed TF binding to their target genes and conservation of the same gene-regulatory networks in large patient cohorts. Relapsed AMLs carried few patient specific structural variants and DNA mutations, apparently not involved in drug resistance. Thus, drug resistance in AMLs can be mainly ascribed to the selection of random epigenetic alterations at sparse CpGs of a few transcription factors, which then induce reprogramming of the relapsing phenotype, independently of clonal genomic evolution.

GASOLINE: detecting germline and somatic structural variants from long-reads data.

Long-read sequencing allows analyses of single nucleic-acid molecules and produces sequences in the order of tens to hundreds kilobases. Its application to whole-genome analyses allows identification of complex genomic structural-variants (SVs) with unprecedented resolution. SV identification, however, requires complex computational methods, based on either read-depth or intra- and inter-alignment signatures approaches, which are limited by size or type of SVs. Moreover, most currently available tools only detect germline variants, thus requiring separate computation of sample pairs for comparative analyses. To overcome these limits, we developed a novel tool (Germline And SOmatic structuraL varIants detectioN and gEnotyping; GASOLINE) that groups SV signatures using a sophisticated clustering procedure based on a modified reciprocal overlap criterion, and is designed to identify germline SVs, from single samples, and somatic SVs from paired test and control samples. GASOLINE is a collection of Perl, R and Fortran codes, it analyzes aligned data in BAM format and produces VCF files with statistically significant somatic SVs. Germline or somatic analysis of 30[Formula: see text] sequencing coverage experiments requires 4-5 h with 20 threads. GASOLINE outperformed currently available methods in the detection of both germline and somatic SVs in synthetic and real long-reads datasets. Notably, when applied on a pair of metastatic melanoma and matched-normal sample, GASOLINE identified five genuine somatic SVs that were missed using five different sequencing technologies and state-of-the art SV calling approaches. Thus, GASOLINE identifies germline and somatic SVs with unprecedented accuracy and resolution, outperforming currently available state-of-the-art WGS long-reads computational methods.

Corrigendum: Single-cell technologies to decipher the immune microenvironment in myeloid neoplasms: Perspectives and opportunities.

[This corrects the article DOI: 10.3389/fonc.2021.796477.].

Hypoplastic Myelodysplastic Syndromes: Just an Overlap Syndrome?

: Myelodysplasias with hypocellular bone marrow (hMDS) represent about 10-15% of MDS and are defined by reduced bone marrow cellularity (i.e., <25% or an inappropriately reduced cellularity for their age in young patients). Their diagnosis is still an object of debate and has not been clearly established in the recent WHO classification. Clinical and morphological overlaps with both normo/hypercellular MDS and aplastic anemia include cytopenias, the presence of marrow hypocellularity and dysplasia, and cytogenetic and molecular alterations. Activation of the immune system against the hematopoietic precursors, typical of aplastic anemia, is reckoned even in hMDS and may account for the response to immunosuppressive treatment. Finally, the hMDS outcome seems more favorable than that of normo/hypercellular MDS patients. In this review, we analyze the available literature on hMDS, focusing on clinical, immunological, and molecular features. We show that hMDS pathogenesis and clinical presentation are peculiar, albeit in-between aplastic anemia (AA) and normo/hypercellular MDS. Two different hMDS phenotypes may be encountered: one featured by inflammation and immune activation, with increased cytotoxic T cells, increased T and B regulatory cells, and better response to immunosuppression; and the other, resembling MDS, where T and B regulatory/suppressor cells prevail, leading to genetic clonal selection and an increased risk of leukemic evolution. The identification of the prevailing hMDS phenotype might assist treatment choice, inform prognosis, and suggest personalized monitoring.

Convalescent hyperimmune plasma for chemo-immunotherapy induced immunodeficiency in COVID-19 patients with hematological malignancies.

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak, patients with defective immunity after chemo-immunotherapy due to hematological disorders showed prolonged symptoms and worse prognosis of coronavirus disease-2019 (COVID-19) pneumonia, probably due to inadequate adaptive immune response and noneffective viral clearance. We describe a single-center series of hematological immunocompromised patients undergoing passive immunization with hyperimmune plasma for persistent COVID-19 symptoms. In all cases, such treatment was well tolerated and contributed to clinical and radiological improvement and recovery; viral clearance was also achieved in a patients' subset. Although requiring further investigation, these results suggest a specific role for hyperimmune plasma administration in hematological patients.

Single-Cell Technologies to Decipher the Immune Microenvironment in Myeloid Neoplasms: Perspectives and Opportunities.

Myeloid neoplasms (MN) are heterogeneous clonal disorders arising from the expansion of hematopoietic stem and progenitor cells. In parallel with genetic and epigenetic dynamics, the immune system plays a critical role in modulating tumorigenesis, evolution and therapeutic resistance at the various stages of disease progression. Single-cell technologies represent powerful tools to assess the cellular composition of the complex tumor ecosystem and its immune environment, to dissect interactions between neoplastic and non-neoplastic components, and to decipher their functional heterogeneity and plasticity. In addition, recent progress in multi-omics approaches provide an unprecedented opportunity to study multiple molecular layers (DNA, RNA, proteins) at the level of single-cell or single cellular clones during disease evolution or in response to therapy. Applying single-cell technologies to MN holds the promise to uncover novel cell subsets or phenotypic states and highlight the connections between clonal evolution and immune escape, which is crucial to fully understand disease progression and therapeutic resistance. This review provides a perspective on the various opportunities and challenges in the field, focusing on key questions in MN research and discussing their translational value, particularly for the development of more efficient immunotherapies.

High Throughput Molecular Characterization of Normal Karyotype Acute Myeloid Leukemia in the Context of the Prospective Trial 02/06 of the Northern Italy Leukemia Group (NILG).

By way of a Next-Generation Sequencing NGS high throughput approach, we defined the mutational profile in a cohort of 221 normal karyotype acute myeloid leukemia (NK-AML) enrolled into a prospective randomized clinical trial, designed to evaluate an intensified chemotherapy program for remission induction. NPM1, DNMT3A, and FLT3-ITD were the most frequently mutated genes while DNMT3A, FLT3, IDH1, PTPN11, and RAD21 mutations were more common in the NPM1 mutated patients (p < 0.05). IDH1 R132H mutation was strictly associated with NPM1 mutation and mutually exclusive with RUNX1 and ASXL1. In the whole cohort of NK-AML, no matter the induction chemotherapy used, by multivariate analysis, the achievement of complete remission was negatively affected by the SRSF2 mutation. Alterations of FLT3 (FLT3-ITD) and U2AF1 were associated with a worse overall and disease-free survival (p < 0.05). FLT3-ITD positive patients who proceeded to alloHSCT had a survival probability similar to FLT3-ITD negative patients and the transplant outcome was no different when comparing high and low-AR-FLT3-ITD subgroups in terms of both OS and DFS. In conclusion, a comprehensive molecular profile for NK-AML allows for the identification of genetic lesions associated to different clinical outcomes and the selection of the most appropriate and effective treatment strategies, including stem cell transplantation and targeted therapies.

Molecular Detection of Minimal Residual Disease before Allogeneic Stem Cell Transplantation Predicts a High Incidence of Early Relapse in Adult Patients with NPM1 Positive Acute Myeloid Leukemia.

We analyzed the impact of alloHSCT in a single center cohort of 89 newly diagnosed NPM1mut AML patients, consecutively treated according to the Northern Italy Leukemia Group protocol 02/06 [NCT00495287]. After two consolidation cycles, the detection of measurable residual disease (MRD) by RQ-PCR was strongly associated with an inferior three-year overall survival (OS, 45% versus 84%, p = 0.001) and disease-free survival (DFS, 44% versus 76%, p = 0.006). In MRD-negative patients, post-remissional consolidation with alloHSCT did not provide a significant additional benefit over a conventional chemotherapy in terms of overall survival [OS, 89% (95% CI 71-100%) versus 81% (95% CI 64-100%), p = 0.59] and disease-free survival [DFS, 80% (95% CI 59-100%) versus 75% (95% CI 56-99%), p = 0.87]. On the contrary, in patients with persistent MRD positivity, the three-year OS and DFS were improved in patients receiving an alloHSCT compared to those allocated to conventional chemotherapy (OS, 52% versus 31%, p = 0.45 and DFS, 50% versus 17%, p = 0.31, respectively). However, in this group of patients, the benefit of alloHSCT was still hampered by a high incidence of leukemia relapse during the first year after transplantation (43%, 95% CI 25-60%). Consolidative alloHSCT improves outcomes compared to standard chemotherapy in patients with persistent NPM1mut MRD positivity, but in these high-risk patients, the significant incidence of leukemia relapse must be tackled by post-transplant preemptive treatments.

Outcome assessment of the VADO approach in psychiatric rehabilitation: a partially randomised multicentric trial.

BACKGROUND: Recent studies on representative samples of psychiatric services have shown that low proportions of cases received effective rehabilitation interventions. The following are likely to be the most important causes: the scarcity of mental health workers trained in social and work skills strategies and the absence of a structured framework to formulate rehabilitation practices. The aim of this study was to assess if a specific structured planning and evaluation manual, called VADO (Valutazione delle Abilità e Definizione degli Obiettivi--in english: Skills Assessment and Definition of Goals), is more effective than routine interventions in reducing disability in patients with schizophrenia. METHOD: Each of 10 mental health services were invited to recruit 10 patients with a schizophrenic disorder. Altogether 98 patients were recruited. Of these, 62 patients were randomly allocated to the intervention/experimental or a control group. The remaining group of 36 patients was not randomised and it was considered as a parallel effectiveness study. Assessment measures at the beginning of the study and at the one-year follow-up included the FPS scale of social functioning and the BPRS 4.0. Between group (VADO vs. Routine) and time effects were examined with ANOVA, Chi-square or Fisher exact. Clinical "improvement" was defined as an increase of at least ten points on the FPS or a decrease of at least 20% on BPRS scores. RESULTS: 31 of the 62 randomized patients received the experimental interventions, while 31 followed the routine ones. At follow-up, the experimental group showed statistically and clinically greater improvements in psychopathology and social functioning. Better outcomes of both social functioning and symptom severity were observed in non randomised patients (parallel effectiveness study). CONCLUSION: The results suggest that setting personalised and measurable objectives, as recommended by the manual, can improve the outcome of rehabilitation of severe mental disorders. Better outcomes in the parallel effectiveness study could be attributed to the greater confidence and enthusiasm of staff in centres where the VADO approach originated.

Autoimmune diseases during treatment with immunomodulatory drugs in multiple myeloma: selective occurrence after lenalidomide.

Immunomodulatory drugs (IMiDs) may favor autoimmune disease (AD) occurrence. We conducted a retrospective study to evaluate AD occurrence among IMiD-treated patients with myeloma. Patients were grouped into three classes depending on the type of IMiD engaged. The first group included patients treated with thalidomide (Thal) (n = 474), the second group with lenalidomide (Len) (n = 140) and patients in the third group were first treated with Thal followed by Len (Thal-Len) (n = 94). Absolute risk of AD was 0.4% for patients treated with Thal, 4.3% for Len and 1.1% for Thal-Len. ADs manifested prevalently as autoimmune cytopenias (55%), although we observed one vasculitis, one optic neuritis, one Graves' disease and one polymyositis. ADs occurred preferentially in the first months of IMiD treatment. A previous autologous transplant was shown to be a significant risk factor. All ADs were managed with IMiD discontinuation and steroids, resolving in a few weeks, except for Graves' disease and polymyositis.