Molecular Screening in Anaplastic Lymphoma Kinase-Positive Anaplastic Large Cell Lymphoma: Anaplastic Lymphoma Kinase Analysis, Next-Generation Sequencing Fusion Gene Detection, and T-Cell Receptor Immunoprofiling.

Anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (ALK+ ALCL) originates from the T-lineage and is marked by rearrangements of the ALK gene. More than 10 fusion partners with the ALK gene are known, with the most common being the t(2;5)(p23;q35) translocation resulting in the NPM1::ALK fusion. In 10% to 20% of the ALK+ ALCL cases, the ALK gene fuses with various other partners. Modern molecular techniques, especially next-generation sequencing (NGS), have eased the identification of ALK gene fusion partners and have allowed in-depth characterization of the T-cell receptor (TCR) repertoire. We devised a real-time quantitative reverse-transcription polymerase chain reaction to measure the expression of the translocated portion of the ALK gene. Fusion partners for the ALK gene were analyzed using rapid amplification of 5'cDNA ends (RACE) method or NGS. TCR immunoprofiling was performed by amplicon NGS. We studied 96 ALK+ ALCL patients. NPM1::ALK fusion gene was observed in 71 patients, ATIC::ALK in 9, and TPM3::ALK in 3. CLTC::ALK, MYH9::ALK, and RNF213::ALK fusions were identified in 2 patients each. We also discovered the TPM4::ALK and SATB1::ALK fusion genes, plus the following 2 previously unidentified ALK+ ALCL fusions: SQSTM1::ALK and CAPRIN1::ALK. High expression of the translocated ALK gene segment was observed in all 93 analyzed samples. TCR testing was conducted on 23 patients with available DNA. In 18 (78%) patients, we discerned at least one (ranging from 1 to 4) clonal TCR rearrangement. In 59% of the patients, clonal TCR beta junctions corresponded with sequences previously observed in both healthy donors and under various pathological conditions. Reverse-transcriptase quantitative detection of ALK expression is a fast and reliable method for both diagnosing and monitoring treatment response in ALK+ ALCL patients, irrespective of the ALK gene translocation. NGS reveals new ALK translocation partners. Both malignant and reactive TCR repertoires in ALK+ ALCL patients are unique and do not consistently occur among different patients.

B cell phenotype and serum levels of interferons, BAFF, and APRIL in multisystem inflammatory syndrome in children associated with COVID-19 (MIS-C).

BACKGROUND: Multisystem inflammatory syndrome in children associated with COVID-19 (MIS-C) is a late complication of pediatric COVID-19, which follows weeks after the original SARS-CoV-2 infection, regardless of its severity. It is characterized by hyperinflammation, neutrophilia, lymphopenia, and activation of T cells with elevated IFN-γ. Observing the production of autoantibodies and parallels with systemic autoimmune disorders, such as systemic lupus erythematodes (SLE), we explored B cell phenotype and serum levels of type I, II, and III interferons, as well as the cytokines BAFF and APRIL in a cohort of MIS-C patients and healthy children after COVID-19. RESULTS: We documented a significant elevation of IFN-γ, but not IFN-α and IFN-λ in MIS-C patients. BAFF was elevated in MIS-C patient sera and accompanied by decreased BAFFR expression on all B cell subtypes. The proportion of plasmablasts was significantly lower in patients compared to healthy post-COVID children. We noted the pre-IVIG presence of ENA Ro60 autoantibodies in 4/35 tested MIS-C patients. CONCLUSIONS: Our work shows the involvement of humoral immunity in MIS-C and hints at parallels with the pathophysiology of SLE, with autoreactive B cells driven towards autoantibody production by elevated BAFF.

NGS better discriminates true MRD positivity for the risk stratification of childhood ALL treated on an MRD-based protocol.

We compared minimal/measurable residual disease (MRD) levels evaluated by routinely used real-time quantitative polymerase chain reaction (qPCR) patient-specific assays and by next-generation sequencing (NGS) approach in 780 immunoglobulin (IG) and T-cell receptor (TR) markers in 432 children with B-cell precursor acute lymphoblastic leukemia treated on the AIEOP-BFM ALL 2009 protocol. Our aim was to compare the MRD-based risk stratification at the end of induction. The results were concordant in 639 of 780 (81.9%) of these markers; 37 of 780 (4.7%) markers were detected only by NGS. In 104 of 780 (13.3%) markers positive only by qPCR, a large fraction (23/104; 22.1%) was detected also by NGS, however, owing to the presence of identical IG/TR rearrangements in unrelated samples, we classified those as nonspecific/false-positive. Risk group stratification based on the MRD results by qPCR and NGS at the end of induction was concordant in 76% of the patients; 19% of the patients would be assigned to a lower risk group by NGS, largely owing to the elimination of false-positive qPCR results, and 5% of patients would be assigned to a higher risk group by NGS. NGS MRD is highly concordant with qPCR while providing more specific results and can be an alternative in the front line of MRD evaluation in forthcoming MRD-based protocols.

Clinical Outcomes, Immunogenicity, and Safety of BNT162b2 Vaccine in Primary Antibody Deficiency.

BACKGROUND: Common variable immunodeficiency (CVID) is characterized by an impaired postvaccination response, high susceptibility to respiratory tract infections, and a broad spectrum of noninfectious complications. Thus, patients with CVID may be at high risk for COVID-19, and vaccination's role in prevention is questionable. OBJECTIVE: We evaluated the clinical outcomes, safety, and dynamics of humoral and T-cell immune responses induced by the mRNA vaccine BNT162b2 in CVID. METHODS: This prospective observational cohort study focused on the clinical outcomes (proportion of infected patients and disease severity), safety (incidences of adverse events and changes in laboratory parameters), and dynamics of humoral (specific postvaccination and virus-neutralizing antibody assessment) and T-cell immune responses (anti-SARS-CoV-2-specific T-cell detection) in 21 patients with CVID after a two-dose administration of BNT162b2. The patients were observed for 6 months. RESULTS: Humoral response was observed in 52% of patients (11 of 21) at month 1 after vaccination but continuously decreased to 33.3% at month 6 (five of 15). Nevertheless, they had a remarkably lower anti-SARS-CoV-2 neutralizing antibody titer compared with healthy controls. The T-cell response was measurable in 46% of patients with CVID (six of 13) at month 1 and persisted over the study period. Mild infection occurred in three patients within the follow-up period (14.3%). The vaccine also exhibited a favorable safety profile. CONCLUSIONS: The BNT162b2 vaccine elicited a measurable antibody response in a high proportion of patients, but it was limited by low titer of virus-neutralizing antibodies and rapid waning of anti-receptor-binding domain SARS-CoV-2-specific antibodies. T-cell response was detected in one-third of patients and remained stable within the follow-up period. Vaccination has favorable safety and clinical-related outcomes in preventing severe COVID-19.