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Cytogenetics has long represented a critical component in the clinical evaluation of hematologic malignancies. Chromosome banding studies provide a simultaneous snapshot of genome-wide copy number and structural variation, which have been shown to drive tumorigenesis, define diseases, and guide treatment. Technological innovations in sequencing have ushered in our present-day clinical genomics era. With recent publications highlighting novel sequencing technologies as alternatives to conventional cytogenetic approaches, we, an international consortium of laboratory geneticists, pathologists, and oncologists, describe herein the advantages and limitations of both conventional chromosome banding and novel sequencing technologies and share our considerations on crucial next steps to implement these novel technologies in the global clinical setting for a more accurate cytogenetic evaluation, which may provide improved diagnosis and treatment management. Considering the clinical, logistic, technical, and financial implications, we provide points to consider for the global evolution of cytogenetic testing.

CD19-directed immunotherapies have revolutionized the treatment of advanced B-cell acute lymphoblastic leukemia (B-ALL). Despite initial impressive rates of complete remission (CR) many patients ultimately relapse. Patients with B-ALL successfully treated with CD19-directed T cells eventually relapse, which, coupled with the early onset of CD22 expression during B-cell development, suggests that preexisting CD34+CD22+CD19− (pre)-leukemic cells represent an “early progenitor origin-related” mechanism underlying phenotypic escape to CD19-directed immunotherapies. We demonstrate that CD22 expression precedes CD19 expression during B-cell development. CD34+CD19−CD22+ cells are found in diagnostic and relapsed bone marrow samples of ∼70% of patients with B-ALL, and their frequency increases twofold in patients with B-ALL in CR after CD19 CAR T-cell therapy. The median of CD34+CD19−CD22+ cells before treatment was threefold higher in patients in whom B-ALL relapsed after CD19-directed immunotherapy (median follow-up, 24 months). Fluorescence in situ hybridization analysis in flow-sorted cell populations and xenograft modeling revealed that CD34+CD19−CD22+ cells harbor the genetic abnormalities present at diagnosis and initiate leukemogenesis in vivo. Our data suggest that preleukemic CD34+CD19−CD22+ progenitors underlie phenotypic escape after CD19-directed immunotherapies and reinforce ongoing clinical studies aimed at CD19/CD22 dual targeting as a strategy for reducing CD19− relapses. The implementation of CD34/CD19/CD22 immunophenotyping in clinical laboratories for initial diagnosis and subsequent monitoring of patients with B-ALL during CD19-targeted therapy is encouraged., The work in P.M. and C.B.’s Laboratory was supported by the European Research Council (CoG-2014-646903, PoC-2018-811220) and the Spanish Ministry of Economy and Competitiveness (SAF2016-80481R, PID2019-108160RB-I00) (P.M.); the ISCIII (ISCIII/FEDER, PI17/01028 and PI20/00822), the Spanish Association against Cancer (AECC), and the FERO Foundation (C.B.). P.M. and J.M.R. acknowledge the support of ISCIII-RICORS within the Next Generation EU program (Plan de Recuperación, Transformación y Resiliencia). N.E. is supported by Cancer Research UK and a Children and Young People’s Cancer Innovation Award (DRCPGM\100058). A.R. is supported by a Wellcome Trust Clinical Research Career Development Fellowship (216632/Z/19/Z). A.O. was supported by ISCIII (PI19/011183). F.S. was supported by AGAUR/Generalitat de Catalunya (SGR288). The single-cell transcriptomic analysis was supported by MRC Discovery award MRCDA 0816-11 and the MRC WIMM Single Cell Facility and MRC-funded Oxford Consortium for Single-Cell Biology (MR/M00919X/1) provided assistance.

Haematopoietic malignancies are frequently characterized by karyotypic abnormalities. The development of targeted drugs has been pioneered with compounds against gene products of fusion genes caused by chromosomal translocations. While polysomies are equally frequent as translocations, for many of them we are lacking therapeutic approaches aimed at synthetic lethality. Here, we report two new cell lines, named MBU-7 and MBU-8, that differ in complete trisomy of chromosome18, a partial trisomy of chromosome 7 and a tetrasomy of the p-arm of chromosome 8, but otherwise share the same mutational pattern and complex karyotype. Both cell lines are divergent clones of U-937 cells and have the morphology and immunoprofile of monocytic cells. The distinct karyotypic differences between MBU-7 and MBU-8 are associated with a difference in the specific response to nucleoside analogues. Taken together, we propose the MBU-7 and MBU-8 cell lines described here as suitable in vitro models for screening and testing vulnerabilities that are associated with the disease-relevant polysomies of chromosome 7, 8 and 18.

Simple Summary Chromosomal abnormalities and somatic mutations are found in patients with myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) in around 50-80% of cases. The identification of these alterations is important for the accurate diagnosis and prognostic classification of these patients. Often, an apparently normal or failed karyotype might lead to an inadequate estimation of the prognostic risk, and several strategies should be combined to solve these cases. The aim of this study was to introduce a novel next-generation sequencing (NGS)-based strategy for the simultaneous detection of all the clinically relevant genetic alterations associated with these disorders. We validated this approach on a large cohort of patients by comparing our findings with those obtained with standard-of-care methods (i.e., karyotype and SNP-arrays). We show that our platform represents a significant improvement on current strategies in defining diagnosis and risk stratification of patients with MDS and myeloid-related disorders. Myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms are clonal disorders that share most of their cytogenetic and molecular alterations. Despite the increased knowledge of the prognostic importance of genetics in these malignancies, next-generation sequencing (NGS) has not been incorporated into clinical practice in a validated manner, and the conventional karyotype remains mandatory in the evaluation of suspected cases. However, non-informative cytogenetics might lead to an inadequate estimation of the prognostic risk. Here, we present a novel targeted NGS-based assay for the simultaneous detection of all the clinically relevant genetic alterations associated with these disorders. We validated this platform in a large cohort of patients by performing a one-to-one comparison with the lesions from karyotype and single-nucleotide polymorphism (SNP) arrays. Our strategy demonstrated an approximately 97% concordance with standard clinical assays, showing sensitivity at least equivalent to that of SNP arrays and higher than that of conventional cytogenetics. In addition, this NGS assay was able to identify both copy-neutral loss of heterozygosity events distributed genome-wide and copy number alterations, as well as somatic mutations within significant driver genes. In summary, we show a novel NGS platform that represents a significant improvement to current strategies in defining diagnosis and risk stratification of patients with MDS and myeloid-related disorders.

Simple Summary Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell malignancies characterized by ineffective differentiation of one or more bone marrow cell lineages. Only 50% of patients with de novo MDS will be found to have cytogenetic abnormalities, of which del(5q) is the most common. In 10% of MDS cases, del(5q) is found as a sole abnormality. In this work, a single cell approach was used to analyze intratumoral heterogeneity in four patients with MDS with isolated del(5q). We were able to observe that an ancestral event in one patient can appear as a secondary hit in another one, thus reflecting the high intratumoral heterogeneity in MDS with isolated del(5q) and the importance of patient-specific molecular characterization. Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological diseases. Among them, the most well characterized subtype is MDS with isolated chromosome 5q deletion (MDS del(5q)), which is the only one defined by a cytogenetic abnormality that makes these patients candidates to be treated with lenalidomide. During the last decade, single cell (SC) analysis has emerged as a powerful tool to decipher clonal architecture and to further understand cancer and other diseases at higher resolution level compared to bulk sequencing techniques. In this study, a SC approach was used to analyze intratumoral heterogeneity in four patients with MDS del(5q). Single CD34+CD117+CD45+CD19- bone marrow hematopoietic stem progenitor cells were isolated using the C1 system (Fluidigm) from diagnosis or before receiving any treatment and from available follow-up samples. Selected somatic alterations were further analyzed in SC by high-throughput qPCR (Biomark HD, Fluidigm) using specific TaqMan assays. A median of 175 cells per sample were analyzed. Inferred clonal architectures were relatively simple and either linear or branching. Similar to previous studies based on bulk sequencing to infer clonal architecture, we were able to observe that an ancestral event in one patient can appear as a secondary hit in another one, thus reflecting the high intratumoral heterogeneity in MDS del(5q) and the importance of patient-specific molecular characterization.

The potential prognostic value of conventional karyotyping in adult T-cell acute lymphoblastic leukemia (T-ALL) remains an open question. We hypothesized that a modified cytogenetic classification, based on the number and type of cytogenetic abnormalities, would allow the identification of high-risk adult T-ALL patients. Complex karyotype defined by the presence of >3 cytogenetic alterations identified T-ALL patients with poor prognosis in this study. Karyotypes with >3 abnormalities accounted for 16 % (22/139) of all evaluable karyotypes, corre-sponding to the largest poor prognosis cytogenetic subgroup of T-ALL identified so far. Patients carrying kar-yotypes with >3 cytogenetic alterations showed a significantly inferior response to therapy, and a poor outcome in terms of event-free survival (EFS), overall survival (OS) and cumulative incidence of relapse (CIR), inde-pendently of other baseline characteristics and the end-induction minimal residual disease (MRD) level. Addi-tional molecular analyses of patients carrying >3 cytogenetic alterations showed a unique molecular profile that could contribute to understand the underlying molecular mechanisms of resistance and to evaluate novel tar-geted therapies (e.g. IL7R directed) with potential impact on outcome of adult T-ALL patients.

The purpose of this article is to show the mediastinal dissection method used during the stage performed by a group of students from the Univrersity of palermo that, during the sumer of 2017, had the opportunity to spend a period of 4 weeks at the Department of Anatomy of The University of malta. The students were guided to practice dissection of some corpes to study various mediastinal organs. This experience permitted to the students to verify practically what they lernt in the books, and reperesented a unique opportunity for them to perform practice with cadavers, that is actually very difficult to do in italian universities.

BACKGROUND There are conflicting evidence on the association between atrial fibrillation (AF) pattern, such as persistent/permanent (Pers/Perm) and paroxysmal (PAF) AF and risk of ischemic events. We investigated if left atrial diameter (LAd) may affect the risk of cardiovascular outcomes according to AF pattern. METHODS Prospective multicenter observational including 1,252 non-valvular AF patients (533 PAF and 719 Pers/Perm AF). Study endpoints were cardiovascular events (CVEs), major adverse cardiac events (MACE) and CV death. LA anteroposterior diameter (LAd) was obtained by transthoracic echocardiography. RESULTS Pers/Perm AF patients had a higher proportion of LAd above median than PAF (≥44 mm, 59.5% vs 37.5% respectively, P

Clinical sequencing across a large prospective cohort of patients with myelodysplasic syndrome uncovers distinct associations between the mono- and biallelic states ofTP53and clinical presentation Tumor protein p53 (TP53) is the most frequently mutated gene in cancer(1,2). In patients with myelodysplastic syndromes (MDS),TP53mutations are associated with high-risk disease(3,4), rapid transformation to acute myeloid leukemia (AML)(5), resistance to conventional therapies(6-8)and dismal outcomes(9). Consistent with the tumor-suppressive role ofTP53, patients harbor both mono- and biallelic mutations(10). However, the biological and clinical implications ofTP53allelic state have not been fully investigated in MDS or any other cancer type. We analyzed 3,324 patients with MDS forTP53mutations and allelic imbalances and delineated two subsets of patients with distinct phenotypes and outcomes. One-third ofTP53-mutated patients had monoallelic mutations whereas two-thirds had multiple hits (multi-hit) consistent with biallelic targeting. Established associations with complex karyotype, few co-occurring mutations, high-risk presentation and poor outcomes were specific to multi-hit patients only.TP53multi-hit state predicted risk of death and leukemic transformation independently of the Revised International Prognostic Scoring System (IPSS-R)(11). Surprisingly, monoallelic patients did not differ fromTP53wild-type patients in outcomes and response to therapy. This study shows that consideration ofTP53allelic state is critical for diagnostic and prognostic precision in MDS as well as in future correlative studies of treatment response.

LoRa is a promising technology in the current Internet of Things market, which operates in un-licensed bands achieving long-range communications and with ultra power devices. In this work we capitalize on the idea introduced in [1], i.e. balance the Air-Time of the different modulation spreading factors (SF), and adapt it to operate in a typical metropolitan scenario comprising multiple gateways (GWs) interconnected to a same network server. Our proposed approach, named ADaptive Mitigation of the AIr-time pressure in lORa (AD MAIORA), relies on a suitable measure of the per-spreading-factor load at each GW - quantified by means of a so-called pressure table -, and on a relevant heuristic algorithm which attempts to balance such a per-SF-pressure. Especially in cases of very loaded scenarios, where a high number of nodes insist on the same GWs, the use of AD MAIORA shows significant performance gains, up to a factor of 5 improvements with respect to the legacy LoRaWAN's Adaptive Data Rate.

Minimal residual disease (MRD) assessment is an essential tool in contemporary acute lymphoblastic leukemia (ALL) protocols, being used for therapeutic decisions such as hematopoietic stem cell transplantation in high-risk patients. However, a significant proportion of adult ALL patients with negative MRD still relapse suggesting that other factors (ie, molecular alterations) must be considered in order to identify those patients with high risk of disease progression. We have identified partial IKZF1 gene deletions and CDKN2A/B deletions as markers of disease recurrence and poor survival in a series of uniformly treated adolescent and adult Philadelphia chromosome-negative B-cell progenitor ALL patients treated according to the Programa Espanol de Tratamientos en Hematologia protocols. Importantly, CDKN2A/B deletions showed independent significance of MRD at the end of induction, which points out the need for treatment intensification in these patients despite being MRD-negative after induction therapy.

Simple Summary Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are clonal myeloid neoplasms characterized, at the time of their presentation, by the simultaneous presence of both myelodysplastic and myeloproliferative features. In MDS/MPN, the karyotype is often normal but mutations in genes that are common across myeloid neoplasms can be detected in a high proportion of cases by targeted sequencing. In this review, we intend to summarize the main genetic findings across all MDS/MPN overlap syndromes and discuss their relevance in the management of patients. Abstract Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are myeloid neoplasms characterized by the presentation of overlapping features from both myelodysplastic syndromes and myeloproliferative neoplasms. Although the classification of MDS/MPN relies largely on clinical features and peripheral blood and bone marrow morphology, studies have demonstrated that a large proportion of patients (~90%) with this disease harbor somatic mutations in a group of genes that are common across myeloid neoplasms. These mutations play a role in the clinical heterogeneity of these diseases and their clinical evolution. Nevertheless, none of them is specific to MDS/MPN and current diagnostic criteria do not include molecular data. Even when such alterations can be helpful for differential diagnosis, they should not be used alone as proof of neoplasia because some of these mutations may also occur in healthy older people. Here, we intend to review the main genetic findings across all MDS/MPN overlap syndromes and discuss their relevance in the management of the patients.