Subclones with variants of uncertain clinical significance might contribute to ineffective hemopoiesis and leukemia predisposition.

BACKGROUND: Splicing modifications, genomic instability, and hypomethylation are central mechanisms promoting myelodysplasia and acute myeloid leukemia (AML). In this real-life retrospective study, to elucidate pathophysiology of clonal hemopoiesis in hematological malignancies, we investigated clinical significance of mutations in leukemia-related genes of known pathogenetic significance and of variants of uncertain clinical significance (VUS) in a cohort of patients with MDS and AML. METHODS: A total of 59 consecutive subjects diagnosed with MDS, 48 with AML, and 17 with clonal cytopenia with unknown significance were screened for somatic mutations in AML-related genes by next-generation sequencing. RESULTS: We showed that TET2, SETBP1, ASXL1, EZH2, RUNX1, SRSF2, DNMT3A, and IDH1/2 were commonly mutated. MDS patients also showed a high genetic complexity, especially for SETBP1. Moreover, the presence of SETBP1 wild-type or two or more simultaneous VUS variants identified a subgroup of AML and MDS patients with better outcome, while the presence of single SETBP1 VUS variant was related to a worse prognosis, regardless TET2 mutational status. CONCLUSIONS: In conclusions, we linked both pathogenic and VUS variants in AML-related genes to clonal hematopoiesis; therefore, we proposed to consider those variants as prognostic markers in leukemia and myelodysplasia. However, further studies in larger prospective cohorts are required to validate our results.

c-Kit M541L variant is related to ineffective hemopoiesis predisposing to clonal evolution in 3D in vitro biomimetic co-culture model of bone marrow niche.

Hematopoietic stem cell (HSC) maintenance in vitro is challenging because stem cell survival relies on cell-to-cell contacts and paracrine signals from bone marrow (BM) microenvironment. Indeed, HSCs easily differentiate in conventional culture systems, and in vitro study of stem cell biology, leukemogenesis, and evolutionary trajectories is limited. 3D-culture systems can mimic tissue architecture and microenvironment thus preserving HSC phenotype. In this study, we developed a calcium alginate hydrogel-based 3D co-culture system of BM mononuclear cells (BMMCs) and BM-derived mesenchymal stem cells (BM-MSCs) to study hemopoiesis in health and disease, such as biological roles of c-Kit M541L somatic mutation of unknown significance. BMMCs and peripheral blood stem cells were obtained from an acute myeloid leukemia patient who experienced graft failure and his haploidentical donor, and from a healthy donor. Cells embedded in alginate scaffolds were cultured for up to 21 days, and flow cytometry immunophenotyping was performed at baseline and every seven days. Our results showed suitability of our 3D culture system in preserving HSC vitality and phenotype throughout the culture period, and also in maintaining composition and vitality of total BMMCs. Moreover, 3D in vitro culture results suggested that M541L c-Kit somatic mutation could be a loss-of-function alteration by reducing HSC maintenance ability thus quickly promoting differentiation, as documented by in vivo graft failure and in vitro absence of long-term culture stability. In conclusions, our 3D BM-like biomimetic culture system allowed long-term stemness maintenance, making it a valid and effective tool for in vitro study of physiological and pathological hemopoiesis.

MiR-27a downregulates 14-3-3θ, RUNX1, AF4, and MLL-AF4, crucial drivers of blast transformation in t(4;11) leukemia cells.

The chromosomal translocation t(4;11)(q21;q23), a hallmark of an aggressive form of acute lymphoblastic leukemia (ALL), encodes mixed-lineage leukemia (MLL)-AF4 oncogenic chimera that triggers aberrant transcription of genes involved in lymphocyte differentiation, including HOXA9 and MEIS1. The scaffold protein 14-3-3θ, which promotes the binding of MLL-AF4 to the HOXA9 promoter, is a target of MiR-27a, a tumor suppressor in different human leukemia cell types. We herein study the role of MiR-27a in the pathogenesis of t(4;11) ALL. Reverse transcription quantitative PCR (qPCR) reveals that MiR-27a and 14-3-3θ expression is inversely correlated in t(4;11) ALL cell lines; interestingly, MiR-27a relative expression is significantly lower in patients affected by t(4;11) ALL than in patients affected by the less severe t(12;21) leukemia. In t(4;11) leukemia cells, ectopic expression of MiR-27a decreases protein level of 14-3-3θ and of the key transcription factor RUNX1. We show for the first time that MiR-27a also targets AF4 and MLL-AF4; in agreement, MiR-27a overexpression strongly reduces AF4 and MLL-AF4 protein levels in RS4;11 cells. Consequent to AF4 and MLL-AF4 downregulation, MiR-27a overexpression negatively affects transcription of HOXA9 and MEIS1 in different t(4;11) leukemia cell lines. In agreement, we show through chromatin immunoprecipitation experiments that MiR-27a overexpression impairs the binding of MLL-AF4 to the HOXA9 promoter. Lastly, we found that MiR-27a overexpression decreases viability, proliferation, and clonogenicity of t(4;11) cells, whereas it enhances their apoptotic rate. Overall, our study identifies the first microRNAthat strikes in one hit four crucial drivers of blast transformation in t(4;11) leukemia. Therefore, MiR-27a emerges as a new promising therapeutic target for this aggressive and poorly curable form of leukemia.

Impact of BCR::ABL1 transcript type on RT-qPCR amplification performance and molecular response to therapy.

Several studies have reported that chronic myeloid leukaemia (CML) patients expressing e14a2 BCR::ABL1 have a faster molecular response to therapy compared to patients expressing e13a2. To explore the reason for this difference we undertook a detailed technical comparison of the commonly used Europe Against Cancer (EAC) BCR::ABL1 reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assay in European Treatment and Outcome Study (EUTOS) reference laboratories (n = 10). We found the amplification ratio of the e13a2 amplicon was 38% greater than e14a2 (p = 0.015), and the amplification efficiency was 2% greater (P = 0.17). This subtle difference led to measurable transcript-type dependent variation in estimates of residual disease which could be corrected by (i) taking the qPCR amplification efficiency into account, (ii) using alternative RT-qPCR approaches or (iii) droplet digital PCR (ddPCR), a technique which is relatively insensitive to differences in amplification kinetics. In CML patients, higher levels of BCR::ABL1/GUSB were identified at diagnosis for patients expressing e13a2 (n = 67) compared to e14a2 (n = 78) when analysed by RT-qPCR (P = 0.0005) but not ddPCR (P = 0.5). These data indicate that widely used RT-qPCR assays result in subtly different estimates of disease depending on BCR::ABL1 transcript type; these differences are small but may need to be considered for optimal patient management.

Standardization of molecular monitoring of CML: results and recommendations from the European treatment and outcome study.

Standardized monitoring of BCR::ABL1 mRNA levels is essential for the management of chronic myeloid leukemia (CML) patients. From 2016 to 2021 the European Treatment and Outcome Study for CML (EUTOS) explored the use of secondary, lyophilized cell-based BCR::ABL1 reference panels traceable to the World Health Organization primary reference material to standardize and validate local laboratory tests. Panels were used to assign and validate conversion factors (CFs) to the International Scale and assess the ability of laboratories to assess deep molecular response (DMR). The study also explored aspects of internal quality control. The percentage of EUTOS reference laboratories (n = 50) with CFs validated as optimal or satisfactory increased from 67.5% to 97.6% and 36.4% to 91.7% for ABL1 and GUSB, respectively, during the study period and 98% of laboratories were able to detect MR4.5 in most samples. Laboratories with unvalidated CFs had a higher coefficient of variation for BCR::ABL1IS and some laboratories had a limit of blank greater than zero which could affect the accurate reporting of DMR. Our study indicates that secondary reference panels can be used effectively to obtain and validate CFs in a manner equivalent to sample exchange and can also be used to monitor additional aspects of quality assurance.

Loss of Detection of sgN Precedes Viral Abridged Replication in COVID-19-Affected Patients-A Target for SARS-CoV-2 Propagation.

The development of prophylactic agents against the SARS-CoV-2 virus is a public health priority in the search for new surrogate markers of active virus replication. Early detection markers are needed to follow disease progression and foresee patient negativization. Subgenomic RNA transcripts (with a focus on sgN) were evaluated in oro/nasopharyngeal swabs from COVID-19-affected patients with an analysis of 315 positive samples using qPCR technology. Cut-off Cq values for sgN (Cq < 33.15) and sgE (Cq < 34.06) showed correlations to high viral loads. The specific loss of sgN in home-isolated and hospitalized COVID-19-positive patients indicated negativization of patient condition, 3-7 days from the first swab, respectively. A new detection kit for sgN, gene E, gene ORF1ab, and gene RNAse P was developed recently. In addition, in vitro studies have shown that 2'-O-methyl antisense RNA (related to the sgN sequence) can impair SARS-CoV-2 N protein synthesis, viral replication, and syncytia formation in human cells (i.e., HEK-293T cells overexpressing ACE2) upon infection with VOC Alpha (B.1.1.7)-SARS-CoV-2 variant, defining the use that this procedure might have for future therapeutic actions against SARS-CoV-2.

Identification of SARS-CoV-2 Proteins from Nasopharyngeal Swabs Probed by Multiple Reaction Monitoring Tandem Mass Spectrometry.

Numerous reverse transcription polymerase chain reaction (RT-PCR) tests have emerged over the past year as the gold standard for detecting millions of cases of SARS-CoV-2 reported daily worldwide. However, problems with critical shortages of key reagents such as PCR primers and RNA extraction kits and unpredictable test reliability related to high viral replication cycles have triggered the need for alternative methodologies to PCR to detect specific COVID-19 proteins. Several authors have developed methods based on liquid chromatography with tandem mass spectrometry (LC-MS/MS) to confirm the potential of the technique to detect two major proteins, the spike and the nucleoprotein, of COVID-19. In the present work, an S-Trap mini spin column digestion protocol was used for sample preparation prodromal to LC-MS/MS analysis in multiple reactions monitoring ion mode (MRM) to obtain a comprehensive method capable of detecting different viral proteins. The developed method was applied to n. 81 oro/nasopharyngeal swabs submitted in parallel to quantitative reverse transcription PCR (RT-qPCR) assays to detect RdRP, the S and N genes specific for COVID-19, and the E gene for all Sarbecoviruses, including SARS-CoV-2 (with cycle negativity threshold set to 40). A total of 23 peptides representative of the six specific viral proteins were detected in the monitoring of 128 transitions found to have good ionic currents extracted in clinical samples that reacted differently to the PCR assay. The best instrumental response came from the FLPFQFGR sequence of spike [558-566] peptide used to test the analytical performance of the method that has good sensitivity with a low false-negative rate. Transition monitoring using a targeted MS approach has the great potential to detect the fragmentation reactions of any peptide molecularly defined by a specific amino acid sequence, offering the extensibility of the approach to any viral sequence including derived variants and thus providing insights into the development of new types of clinical diagnostics.

Droplet Digital PCR for BCR-ABL1 Monitoring in Diagnostic Routine: Ready to Start?

BCR-ABL1 mRNA levels represent the key molecular marker for the evaluation of minimal residual disease (MRD) in chronic myeloid leukemia (CML) patients and real-time quantitative PCR (RT-qPCR) is currently the standard method to monitor it. In the era of tyrosine kinase inhibitors (TKIs) discontinuation, droplet digital PCR (ddPCR) has emerged to provide a more precise detection of MRD. To hypothesize the use of ddPCR in clinical practice, we designed a multicentric study to evaluate the potential value of ddPCR in the diagnostic routine. Thirty-seven RNA samples from CML patients and five from healthy donors were analyzed using both ddPCR QXDxTMBCR-ABL %IS Kit and LabNet-approved RT-qPCR methodologies in three different Italian laboratories. Our results show that ddPCR has a good agreement with RT-qPCR, but it is more precise to quantify BCR-ABL1 transcript levels. Furthermore, we did not find differences between duplicate or quadruplicate analysis in terms of BCR-ABL1% IS values. Droplet digital PCR could be confidently introduced into the diagnostic routine as a complement to the RT-qPCR.

Long-chain polyphosphates impair SARS-CoV-2 infection and replication.

Inorganic polyphosphates (polyPs) are linear polymers composed of repeated phosphate (PO4 3-) units linked together by multiple high-energy phosphoanhydride bonds. In addition to being a source of energy, polyPs have cytoprotective and antiviral activities. Here, we investigated the antiviral activities of long-chain polyPs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In molecular docking analyses, polyPs interacted with several conserved amino acid residues in angiotensin-converting enzyme 2 (ACE2), the host receptor that facilitates virus entry, and in viral RNA-dependent RNA polymerase (RdRp). ELISA and limited proteolysis assays using nano- LC-MS/MS mapped polyP120 binding to ACE2, and site-directed mutagenesis confirmed interactions between ACE2 and SARS-CoV-2 RdRp and identified the specific amino acid residues involved. PolyP120 enhanced the proteasomal degradation of both ACE2 and RdRp, thus impairing replication of the British B.1.1.7 SARS-CoV-2 variant. We thus tested polyPs for functional interactions with the virus in SARS-CoV-2-infected Vero E6 and Caco2 cells and in primary human nasal epithelial cells. Delivery of a nebulized form of polyP120 reduced the amounts of viral positive-sense genomic and subgenomic RNAs, of RNA transcripts encoding proinflammatory cytokines, and of viral structural proteins, thereby presenting SARS-CoV-2 infection in cells in vitro.

WT1 Expression Levels Combined with Flow Cytometry Blast Counts for Risk Stratification of Acute Myeloid Leukemia and Myelodysplastic Syndromes.

Wilm's tumor 1 (WT1), a zinc-finger transcription factor and an epigenetic modifier, is frequently overexpressed in several hematologic disorders and solid tumors, and it has been proposed as diagnostic and prognostic marker of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, the exact role of WT1 in leukemogenesis and disease progression remains unclear. In this real-world evidence retrospective study, we investigated prognostic role of WT1-mRNA expression levels in AML and MDS patients and correlations with complete blood counts, flow cytometry counts, and molecular features. A total of 71 patients (AML, n = 46; and MDS, n = 25) were included in this study, and WT1 levels were assessed at diagnosis, during treatment and follow-up. We showed that WT1 expression levels were inversely correlated with normal hemopoiesis in both AML and MDS, and positively associated with blast counts. Flow cytometry was more sensitive and specific in distinguishing normal myeloid cells from neoplastic counterpart even just using linear parameters and CD45 expression. Moreover, we showed that a simple integrated approach combining blast counts by flow cytometry, FLT3 mutational status, and WT1 expression levels might be a useful tool for a better prognostic definition in both AML and MDS patients.

SARS-CoV-2 Subgenomic N (sgN) Transcripts in Oro-Nasopharyngeal Swabs Correlate with the Highest Viral Load, as Evaluated by Five Different Molecular Methods.

The COVID-19 pandemic has forced diagnostic laboratories to focus on the early diagnostics of SARS-CoV-2. The positivity of a molecular test cannot respond to the question regarding the viral capability to replicate, spread, and give different clinical effects. Despite the fact that some targets are covered by commercially-available assays, the identification of new biomarkers is desired in order to improve the quality of the information given by these assays. Therefore, since the subgenomic transcripts (sgN and sgE) are considered markers of viral activity, we evaluated these subgenomic transcripts in relation to the genomic amplification obtained using five different commercial CE-IVD tools. Methods: Five CE-IVD kits were compared in terms of their capability to detect both synthetic SARS-CoV-2 viral constructs (spiked in TMB or PBS medium) and targets (N, E, RdRp and Orf1ab genes) in twenty COVID-19-positive patients' swabs. The sgN and sgE were assayed by real-time RT-qPCR and digital PCR. Results: None of the diagnostic kits missed the viral target genes when they were applied to targets spiked in TMB or PBS (at dilutions ranging from 100 pg to 0.1 pg). Nevertheless, once they were applied to RNA extracted from the patients' swabs, the superimposability ranged from 50% to 100%, regardless of the extraction procedure. The sgN RNA transcript was detected only in samples with a higher viral load (Ct ≤ 22.5), while sgE was within all of the Ct ranges. Conclusions: The five kits show variable performances depending on the assay layout. It is worthy of note that the detection of the sgN transcript is associated with a higher viral load, thus representing a new marker of early and more severe infection.

Novel Multiplex Droplet Digital PCR Assays to Monitor Minimal Residual Disease in Chronic Myeloid Leukemia Patients Showing Atypical BCR-ABL1 Transcripts.

BCR-ABL1 fusion transcript is the minimal residual disease marker in chronic myeloid leukemia; 2% of patients show unusual breakpoints generating atypical transcripts, not quantifiable by standardized real-time PCR (RT-PCR). Response monitoring is performed by non-quantitative NESTED PCR, useless for evaluating patients' molecular remission, excluding them from treatment-free-remission protocols. Droplet digital PCR (ddPCR) is highly sensitive technology, allowing an absolute quantification independent of standard curves. Based on this, we have developed assays able to evaluate the molecular response in atypical patients. We designed new ddPCR-based molecular assays able to quantify atypical BCR-ABL1 transcripts, with a detection limit of 0.001%, validated in a cohort of 65 RNA from 11 patients. Fifty samples were identified congruently by ddPCR and NESTED PCR (40 positives and 10 negatives for atypical BCR-ABL1 transcript), while 11 positive samples were detected only by ddPCR. Our results highlight ddPCR usefulness, primarily when the BCR-ABL1/ABL1 level is less than 1.5% and NESTED PCR results are often inaccurate. Furthermore, we identified 3 patients who maintained a deep molecular response for at least one year, who could be considered good candidates for treatment-free remission approaches. Here, we describe a new promising molecular approach, highly sensitive, to monitor atypical BCR-ABL1 patients, paving the foundation to include them in treatment-free remission protocols.

The Q-LAMP Method Represents a Valid and Rapid Alternative for the Detection of the BCR-ABL1 Rearrangement in Philadelphia-Positive Leukemias.

Molecular detection of the BCR-ABL1 fusion transcripts is necessary for the genetic confirmation of a chronic myeloid leukemia diagnosis and for the risk classification of acute lymphoblastic leukemia. BCR-ABL1 mRNAs are usually identified using a conventional RT-PCR technique according to the BIOMED-1 method. In this study, we evaluated 122 BCR-ABL1-positive samples with the Q-LAMP assay to establish if this technology may represent a valid alternative to the qualitative BIOMED-1 PCR technique usually employed for the detection and the discrimination of the common BCR-ABL1 transcripts (p190 and p210 isoforms). We found a 100% concordance rate between the two methods. Specifically, the p190- and p210-positive samples were amplified by Q-LAMP with a median threshold time (Tt) of 26.70 min (range: 24.45-31.80 min) and 20.26 min (range: 15.25-34.57 min), respectively. A median time of 19.63 was observed in samples displaying both (e13a2/e14a2) p210 isoforms. Moreover, the Q-LAMP assay allowed recognition of the BCR-ABL1 e13a2 and e14a2 isoforms (median Tts 18.48 for e13a2 vs. 26.08 min for e14a2; p < 0.001). Finally, 20 samples harboring rare BCR-ABL1 isoforms (e1a3, e13a3, e14a3, and e19a2) were correctly identified by the Q-LAMP assay. We conclude that the Q-LAMP assay may represent a faster and valid alternative to the qualitative BIOMED-1 RT-PCR for the diagnosis at BCR-ABL1-positive leukemias, especially when samples are analyzed in centers with restricted resources and/or limited technical expertise.

Monitoring Chronic Myeloid Leukemia: How Molecular Tools May Drive Therapeutic Approaches.

More than 15 years ago, imatinib entered into the clinical practice as a "magic bullet"; from that point on, the prognosis of patients affected by chronic myeloid leukemia (CML) became comparable to that of aged-matched healthy subjects. The aims of treatment with tyrosine kinase inhibitors (TKIs) are for complete hematological response after 3 months of treatment, complete cytogenetic response after 6 months, and a reduction of the molecular disease of at least 3 logs after 12 months. Patients who do not reach their goal can switch to another TKI. Thus, the molecular monitoring of response is the main consideration of management of CML patients. Moreover, cases in deep and persistent molecular response can tempt the physician to interrupt treatment, and this "dream" is possible due to the quantitative PCR. After great international effort, today the BCR-ABL1 expression obtained in each laboratory is standardized and expressed as "international scale." This aim has been reached after the establishment of the EUTOS program (in Europe) and the LabNet network (in Italy), the platforms where biologists meet clinicians. In the field of quantitative PCR, the digital PCR is now a new and promising, sensitive and accurate tool. Some authors reported that digital PCR is able to better classify patients in precise "molecular classes," which could lead to a better identification of those cases that will benefit from the interruption of therapy. In addition, digital PCR can be used to identify a point mutation in the ABL1 domain, mutations that are often responsible for the TKI resistance. In the field of resistance, a prominent role is played by the NGS that enables identification of any mutation in ABL1 domain, even at sub-clonal levels. This manuscript reviews how the molecular tools can lead the management of CML patients, focusing on the more recent technical advances.

Laboratory medicine: health evaluation in elite athletes.

The need to evaluate the health status of an athlete represents a crucial aim in preventive and protective sports science in order to identify the best diagnostic strategy to improve performance and reduce risks related to physical exercise. In the present review we aim to define the main biochemical and haematological markers that vary significantly during and after sports training to identify risk factors, at competitive and professional levels and to highlight the set up of a specific parameter's panel for elite athletes. Moreover, we also intend to consider additional biomarkers, still under investigation, which could further contribute to laboratory sports medicine and provide reliable data that can be used by athlete's competent staff in order to establish personal attitudes and prevent sports injuries.

Molecular analysis of cluster headache.

OBJECTIVES: Cluster headache (CH) is characterized by severe, recurrent, unilateral attacks of extreme intensity and brief duration. Variants in a myriad of genes were studied in sporadic CH patients, often with conflicting results. METHODS: We studied gene mutations in some candidate genes, hypocretin receptor 2, Clock, and alcohol dehydrogenase 4 (ADH4), in 54 unrelated sporadic CH patients and in 200 controls in 8 kindreds/families that included more affected and nonaffected cases. Furthermore, we performed the whole-genome scanning by comparative genomic hybridization, searching for rearrangements associated with DNA gain or loss in a subset of sporadic and familial CH and control participants. RESULTS: The analysis of candidate genes revealed that only allele and genotype frequency of the 2 ADH4 mutations resulted significantly between sporadic CH and controls; the same mutations were homozygous in CH patients from 2 families. The comparative genomic hybridization analysis revealed 2 novel rearrangements that involved the intron regions of thyrotropin-releasing hormone-degrading enzyme and neurexin 3 (NRXN3) genes, respectively. The first arrangement was present either in CH or in controls, whereas the second one was specifically found in some sporadic and familial CH cases. CONCLUSIONS: Our data (although obtained on a small number of cases) confirm the genetic heterogeneity of CH, suggesting that mutations in the ADH4 gene and a novel rearrangement involving NRXN3 gene might be related to CH in a subset of cases.

Real-time quantitative polymerase chain reaction detection of minimal residual disease by standardized WT1 assay to enhance risk stratification in acute myeloid leukemia: a European LeukemiaNet study.

PURPOSE: Risk stratification in acute myeloid leukemia (AML) is currently based on pretreatment characteristics. It remains to be established whether relapse risk can be better predicted through assessment of minimal residual disease (MRD). One proposed marker is the Wilms tumor gene WT1, which is overexpressed in most patients with AML, thus providing a putative target for immunotherapy, although in the absence of a standardized assay, its utility for MRD monitoring remains controversial. PATIENTS AND METHODS: Nine published and in-house real-time quantitative polymerase chain reaction WT1 assays were systematically evaluated within the European LeukemiaNet; the best-performing assay was applied to diagnostic AML samples (n = 620), follow-up samples from 129 patients treated with intensive combination chemotherapy, and 204 normal peripheral blood (PB) and bone marrow (BM) controls. RESULTS: Considering relative levels of expression detected in normal PB and BM, WT1 was sufficiently overexpressed to discriminate > or = 2-log reduction in transcripts in 46% and 13% of AML patients, according to the respective follow-up sample source. In this informative group, greater WT1 transcript reduction after induction predicted reduced relapse risk (hazard ratio, 0.54 per log reduction; 95% CI, 0.36 to 0.83; P = .004) that remained significant when adjusted for age, WBC count, and cytogenetics. Failure to reduce WT1 transcripts below the threshold limits defined in normal controls by the end of consolidation also predicted increased relapse risk (P = .004). CONCLUSION: Application of a standardized WT1 assay provides independent prognostic information in AML, lending support to incorporation of early assessment of MRD to develop more robust risk scores, to enhance risk stratification, and to identify patients who may benefit from allogeneic transplantation.