Design and Validation of a Custom Next-Generation Sequencing Panel in Pediatric Acute Lymphoblastic Leukemia.

The molecular landscape of acute lymphoblastic leukemia (ALL) is highly heterogeneous, and genetic lesions are clinically relevant for diagnosis, risk stratification, and treatment guidance. Next-generation sequencing (NGS) has become an essential tool for clinical laboratories, where disease-targeted panels are able to capture the most relevant alterations in a cost-effective and fast way. However, comprehensive ALL panels assessing all relevant alterations are scarce. Here, we design and validate an NGS panel including single-nucleotide variants (SNVs), insertion-deletions (indels), copy number variations (CNVs), fusions, and gene expression (ALLseq). ALLseq sequencing metrics were acceptable for clinical use and showed 100% sensitivity and specificity for virtually all types of alterations. The limit of detection was established at a 2% variant allele frequency for SNVs and indels, and at a 0.5 copy number ratio for CNVs. Overall, ALLseq is able to provide clinically relevant information to more than 83% of pediatric patients, making it an attractive tool for the molecular characterization of ALL in clinical settings.

RNA Sequencing Analysis for the Identification of a PCM1/PDGFRB Fusion Gene Responsive to Imatinib.

The platelet-derived growth factor receptor ß (PDGFRB) gene translocations lead to a spectrum of chronic myeloid neoplasms, frequently associated with eosinophilia. Clinical heterogeneity is associated with a molecular one. Here, we report a novel case of a patient harboring a t(5;8)(q33;p22) translocation, resulting in the PCM1/PDGFRB fusion. Conventional cytogenetics and RNA sequencing were performed to identify the chromosomes and the genes involved in the rearrangement, respectively. This study shows that the combination of different strategies is pivotal to fine-tune the diagnosis and the clinical management of the patient. After 1 year of treatment with imatinib, the patient achieves hematological and molecular remission. We present an attractive strategy to identify novel and/or cryptic fusions, which will be relevant for clinicians dealing with the diagnosis of the patients with myelodysplastic syndrome/myeloproliferative diseases with atypical manifestations.

Comprehensive detection of CRLF2 alterations in acute lymphoblastic leukemia: a rapid and accurate novel approach.

Introduction: Acute lymphoblastic leukemia (ALL) is a prevalent childhood cancer with high cure rate, but poses a significant medical challenge in adults and relapsed patients. Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype, with approximately half of cases characterized by CRLF2 overexpression and frequent concomitant IKZF1 deletions. Methods: To address the need for efficient, rapid, and cost-effective detection of CRLF2 alterations, we developed a novel RT-qPCR technique combining SYBR Green and highresolution melting analysis on a single plate. Results: The method successfully identified CRLF2 expression, P2RY8::CRLF2 fusions, and CRLF2 and JAK2 variants, achieving a 100% sensitivity and specificity. Application of this method across 61 samples revealed that 24.59% exhibited CRLF2 overexpression, predominantly driven by IGH::CRLF2 (73.33%). High Resolution Melting analysis unveiled concurrent CRLF2 or JAK2 variants in 8.19% of samples, as well as a dynamic nature of CRLF2 alterations during disease progression. Discussion: Overall, this approach provides an accurate identification of CRLF2 alterations, enabling improved diagnostic and facilitating therapeutic decision-making.

Methotrexate & rheumatoid arthritis associated atherosclerosis: A narrative review of multidisciplinary approach for risk modification by the international board of experts.

Rheumatoid arthritis (RA) is an idiopathic, autoimmune connective tissue disorder that primarily affects the synovial joints, causing symmetric, erosive-deforming polyarthritis. It is also associated with extra-articular manifestations, particularly cardiovascular (CV) diseases (CVD). CV risk modification in RA remains unsolved despite recent advances in the management of RA. RA is an independent risk factor for atherosclerosis. RA and atherosclerosis share similar pathophysiological features (such as the pro-inflammatory cascade activation including interleukin-6) and risk factors (such as microflora dysbacteriosis and smoking). Patients with RA experience an exacerbation of atherogenesis, with atheromas destabilization, endothelial dysfunction, vasculitis, and hypercytokinemia. Consequently, the inflammatory response associated with RA is the basis for CVD development. The treat-to-target strategy not only improved RA control but also had a favorable effect on the morpho-functional state of the CV system in patients living with RA. Thus, disease-modifying antirheumatic drugs (DMARDs) - in particular methotrexate - may have a beneficial effect on the prevention of CV events in RA. It must be mentioned that RA is a serious multi-system disease, not only because of a window period during which the course of RA can be reversed, but also due to early damage to the heart and blood vessels. For this reason, a thorough cardiological assessment must be performed for all patients with RA, regardless of sex, age, disease stage, and disease activity score.

Cytogenetic Assessment and Risk Stratification in Myelofibrosis with Optical Genome Mapping.

Cytogenetic assessment in myelofibrosis is essential for risk stratification and patient management. However, an informative karyotype is unavailable in a significant proportion of patients. Optical genome mapping (OGM) is a promising technique that allows for a high-resolution assessment of chromosomal aberrations (structural variants, copy number variants, and loss of heterozygosity) in a single workflow. In this study, peripheral blood samples from a series of 21 myelofibrosis patients were analyzed via OGM. We assessed the clinical impact of the application of OGM for disease risk stratification using the DIPSS-plus, GIPSS, and MIPSS70+v2 prognostic scores compared with the standard-of-care approach. OGM, in combination with NGS, allowed for risk classification in all cases, compared to only 52% when conventional techniques were used. Cases with unsuccessful karyotypes (n = 10) using conventional techniques were fully characterized using OGM. In total, 19 additional cryptic aberrations were identified in 9 out of 21 patients (43%). No alterations were found via OGM in 4/21 patients with previously normal karyotypes. OGM upgraded the risk category for three patients with available karyotypes. This is the first study using OGM in myelofibrosis. Our data support that OGM is a valuable tool that can greatly contribute to improve disease risk stratification in myelofibrosis patients.

A Single-Run Next-Generation Sequencing (NGS) Assay for the Simultaneous Detection of Both Gene Mutations and Large Chromosomal Abnormalities in Patients with Myelodysplastic Syndromes (MDS) and Related Myeloid Neoplasms.

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.

Evaluation of immune responses to seasonal influenza vaccination in healthy volunteers and in patients after stem cell transplantation.

BACKGROUND: Influenza causes significant morbidity and mortality in immunocompromised stem cell transplantation (SCT) recipients. Measurement of cellular and humoral immunological responses might increase our understanding of how to estimate a protective response to influenza vaccination. METHODS: Eighteen healthy subjects and 14 SCT patients tested before and 4 weeks after influenza vaccination were included in the study. Peripheral blood lymphocytes were stimulated with influenza peptides and Enzyme-Linked ImmunoSpot assays to measure the production of intracellular interferon-gamma, interleukin-4 and interleukin-13. Prelabeled major histocompatibility complex class I pentamers were used for the detection of influenza-specific CD8+ T-cells. B-cell Enzyme-Linked ImmunoSpot and hemagglutination inhibition assays were performed to enumerate influenza-specific antibody-secreting cells and titer of neutralizing antibodies. RESULTS: Influenza vaccination elicited strong cell-mediated immune responses in the healthy controls (P< or =0.003 for all four peptides) and SCT patients (P< or =0.008). The percentage of CD8+ specific cells increased significantly after vaccination both in volunteers (P=0.005) and in patients (P< or =0.003). The number of influenza-specific antibody-secreting cells increased after vaccination both in volunteers (P=0.009) and in patients (P=0.01). Twenty-nine percent of SCT patients demonstrated protective antibody levels to influenza A H1/N1 serotype. CONCLUSIONS: Seasonal vaccination against influenza boosts the cellular immune response both in SCT patients and healthy controls. The protective effect is lower in the patients in general and especially on those, vaccinated early after SCT.

Novel complex t(V;9;22) rearrangements in three cases with chronic myeloid leukemia and a rare translocation in a case with classical Philadelphia chromosome.

The fusion gene BCR/ABL arises in connection with a complex translocation event in 2-10% of cases with chronic myeloid leukemia (CML). Due to causative treatment with Imatinib most cases with variant rearrangements show no specific prognostic significance, though the events of therapy resistance remain to be studied. Herein we report on three CML cases with complex chromosomal aberrations not observed before, involving chromosomal regions such as 1p32, 2q11 and 6q12. Additionally we report on one case with the rare translocation t(3;8)(p22;q22) along with the classic Philadelphia (Ph) chromosome. In two cases, two different breakpoints on chromosome 22 were found. Moreover, in one of them two breakpoints on chromosome 9 were observed. The following chromosomal studies, during therapy by Imatinib, have revealed different cytogenetic responses.

Risk factors for the development of cytomegalovirus disease after allogeneic stem cell transplantation.

BACKGROUND AND OBJECTIVES: Cytomegalovirus (CMV) disease remains an important complication of allogeneic stem cell transplantation (SCT). We studied viral load kinetics and correlated the viral load and other transplant factors with the development of CMV disease. DESIGN AND METHODS: We studied 162 consecutive patients who were CMV seropositive or had CMV seropositive donors. Quantification of CMV DNA was performed by real-time polymerase chain reaction. RESULTS: CMV DNA detected was detected in 105 of the 162 patients. The mean peak viral loads were similar at first and subsequent reactivations. The serologic status of the donors and recipients prior to SCT significantly influenced the viral load. The cumulative incidence of CMV disease was 1.8% at 100 days and 6.3% at 365 days after SCT. The peak viral load were higher in patients who developed CMV disease than in patients without CMV disease (log10 3.5; SE +/- 0.26/200,000 cells vs. log10 2.7; SE +/- 0.09/200,000 cells; p=0.02). However, in multivariate analysis, only acute graft-versus-host disease (GVHD) grade II-IV and a graft from a CMV-negative donor to a CMV-positive patient were significant risk factors for CMV disease. In patients who required more than one course of pre-emptive therapy, acute GVHD and the rate of decrease in viral load during first pre-emptive therapy were significant risk factors for subsequent development of CMV disease. INTERPRETATION AND CONCLUSIONS: A decrease in viral load during pre-emptive therapy is an important factor for later development of CMV disease.

Respiratory syncytial virus infection in recipients of allogeneic stem-cell transplantation: a retrospective study of the incidence, clinical features, and outcome.

BACKGROUND: Respiratory syncytial virus (RSV) is a common cause of serious respiratory infections in hematopoietic stem-cell transplant (HSCT) recipients. We aimed to determine the frequency, risk factors, and outcome of RSV infection in allo-HSCT recipients. DESIGN AND METHODS: Data were collected from 275 allo-HSCT recipients and identified 32 patients (11.6%, 26 adults and 6 children) RSV infection. A control group was selected matched for age, conditioning intensity and regimens, year of transplant. RESULTS: Eighteen patients had upper and 14 had lower respiratory tract infection (LRTI). The duration of viral shedding was long (20 days; 7-84). Twenty-eight patients received ribavirin (6 received intravenously, 11 orally, 9 both, 1 oral +aerosolized, and 1 received all three forms). The median duration of therapy was 22.5 days (7-54 days). Three (1.1% of entire cohort, 9.4% of infected patients; 21.4% of patients with LRTI) patients died from the RSV (attributable mortality), whereas two patients died from other subsequent infections 38 days to 5 months after diagnosis of RSV. Seven patients died from progression of underlying disease. Long-term respiratory function was assessed in 14 patients. Two patients died from respiratory failure. Three of 14 patients developed marked respiratory dysfunction after SCT. Three of 16 patients in the RSV group had normal respiratory function compared with 18 of 26 in the control group (P<0.01). CONCLUSION: RSV infection results in a low overall attributable mortality after allo-HSCT, but progression of the infection to LRTI is associated with increased risk for death. Late respiratory dysfunction is more common among patients, experienced RSV infection compared with controls.