Molecular Pathogenesis and Treatment Perspectives for Hypereosinophilia and Hypereosinophilic Syndromes.

Hypereosinophilia (HE) is a heterogeneous condition with a persistent elevated eosinophil count of >350/mm3, which is reported in various (inflammatory, allergic, infectious, or neoplastic) diseases with distinct pathophysiological pathways. HE may be associated with tissue or organ damage and, in this case, the disorder is classified as hypereosinophilic syndrome (HES). Different studies have allowed for the discovery of two major pathogenetic variants known as myeloid or lymphocytic HES. With the advent of molecular genetic analyses, such as T-cell receptor gene rearrangement assays and Next Generation Sequencing, it is possible to better characterize these syndromes and establish which patients will benefit from pharmacological targeted therapy. In this review, we highlight the molecular alterations that are involved in the pathogenesis of eosinophil disorders and revise possible therapeutic approaches, either implemented in clinical practice or currently under investigation in clinical trials.

Molecular characterization of V(D)J rearrangements in immature acute leukemias.

Early T-cell Precursor Acute Lymphoblastic Leukemia (ETP-ALL), T-Lymphoid/Myeloid Mixed Phenotype Acute Leukemia (T/M-MPAL), and Acute Myeloid Leukemia with minimal differentiation (AML-M0) are immature acute leukemias (AL) that present overlapping T-cell lymphoid and myeloid features at different degrees, with impact to disease classification. An interesting strategy to assess lymphoid lineage commitment and maturation is the analysis of V(D)J gene segment recombination, which can be applied to investigate leukemic cells in immature AL. Herein, we revisited 19 ETP-ALL, 8 T/M-MPAL, and 12 AML-M0 pediatric patients to characterize V(D)J rearrangement (V(D)J-r) profiles associated with other somatic alterations. V(D)J-r were identified in 74 %, 25 %, and 25 % of ETP-ALL, T/M-MPAL, and AML-M0, respectively. Forty-six percent of ETP-ALL harbored ≥ 3 V(D)J-r, while there was no more than one V(D)J-r per patient in AML-M0 and T/M-MPAL. TCRD was the most rearranged locus in ETPALL, but it was not rearranged in other AL. In ETP-ALL, N/KRAS mutations were associated with absence of V(D)J-r, while NF1 deletion was most frequent in patients with ≥ 3 V(D)J-r. Relapse and death occurred mainly in patients harboring one or no rearranged locus. Molecular characterization of V(D)J-r in our cohort indicates a distinct profile of ETP-ALL, compared to T/M-MPAL and AML-M0. Our findings also suggest that the clinical outcome of ETP-ALL patients may be affected by blast cell maturity, inferred from the number of rearranged TCR loci.

Evaluation of a worldwide EQA scheme for complex clonality analysis of clinical lymphoproliferative cases demonstrates a learning effect.

Clonality analysis of immunoglobulin (IG) or T-cell receptor (TR) gene rearrangements is routine practice to assist diagnosis of lymphoid malignancies. Participation in external quality assessment (EQA) aids laboratories in identifying systematic shortcomings. The aim of this study was to evaluate laboratories' improvement in IG/TR analysis and interpretation during five EQA rounds between 2014 and 2018. Each year, participants received a total of five cases for IG and five cases for TR testing. Paper-based cases were included for analysis of the final molecular conclusion that should be interpreted based on the integration of the individual PCR results. Wet cases were distributed for analysis of their routine protocol as well as evaluation of the final molecular conclusion. In total, 94.9% (506/533) of wet tests and 97.9% (829/847) of paper tests were correctly analyzed for IG, and 96.8% (507/524) wet tests and 93.2% (765/821) paper tests were correctly analyzed for TR. Analysis scores significantly improved when laboratories participated to more EQA rounds (p=0.001). Overall performance was significantly lower (p=0.008) for non-EuroClonality laboratories (95% for IG and 93% for TR) compared to EuroClonality laboratories (99% for IG and 97% for TR). The difference was not related to the EQA scheme year, anatomic origin of the sample, or final clinical diagnosis. This evaluation showed that repeated EQA participation helps to reduce performance differences between laboratories (EuroClonality versus non-EuroClonality) and between sample types (paper versus wet). The difficulties in interpreting oligoclonal cases highlighted the need for continued education by meetings and EQA schemes.

Usage of Droplet Digital PCR (ddPCR) Assays for T Cell Quantification in Cancer.

T cells fulfill a central role in cell-mediated immunity and can be found in the circulation and lymphoid organs upon maturation. For clinical applications, it can be important to quantify (infiltrated) T cells accurately in a variety of body fluids and tissues of benign, inflammatory, or malignant origin. For decades, flow cytometry and immunohistochemistry have been the accustomed methods to quantify T cells. Although these methods are widely used, they depend on the accessibility of T-cell epitopes and therefore require fresh, frozen, or fixated material of a certain quality. Whenever samples are low in quantity or quality, an accurate quantification can be impeded. By shifting the focus from epitopes to DNA, quantification of T cells remains achievable.Mature T cells differ genetically from other cell types as a result of T-cell receptor (TCR) gene rearrangements. This genetic dissimilarity can be exploited to quantify the T-cell fraction in DNA specimens. Conventionally, multiplex PCR and droplet digital PCR (ddPCR), combined with deep-sequencing techniques, can be applied to determine T-cell content. However, these approaches typically target the whole TCR repertoire, thereby supplying additional information about TCR use. Considering this, a simple T-cell quantification, unwantedly, turns into a complex, expensive, and time-consuming procedure. We have developed two generic single duplex ddPCR assays as alternative methods to quantify T cells in a relatively simple, cheap, and fast manner by targeting sequences located between the Dδ2 and Dδ3 genes (TRD locus) and Dß1 and Jß1.1 genes (TRB locus). These specific TCR loci become deleted systematically early during lymphoid differentiation and therefore will serve as biomarkers for the quantification of mature T cells. Here, we describe a simple and sensitive ddPCR-based method to quantify T cells relatively fast, accurately and independently of the cellular context.

Caracterización del perfil de reordenamientos génicos de inmunoglobulinas y receptores de linfocitos T en leucemia linfoblástica aguda pediátrica / Pattern of immunoglobulins and T lymphocyte receptor gene rearrangements in childhood acute lymphoblastic leukemia / Caracterização do perfil de rearranjos gênicos de imunoglobulinas e receptores de linfócitos T na leucemia linfoide aguda pediátrica

Resumen Durante la ontogenia linfocitaria se produce el reordenamiento de los segmentos génicos V-(D)-J que codifican para la región variable de las cadenas de inmunoglobulinas (Ig) y receptores de linfocitos T (TCR). Durante este proceso, los segmentos se reordenan al azar y ocurren deleciones e inserciones de nucleótidos en la región de unión entre ellos. Los objetivos del presente trabajo fueron describir las incidencias de los reordenamientos Ig/TCR y de los segmentos V-(D)-J involucrados, en niños con leucemia linfoblástica aguda (LLA). Para ello se estudiaron 769 pacientes pediátricos con LLA, diagnosticados entre 1999 y 2018 por los centros de la Sociedad Argentina de Hemato-Oncología Pediátrica. Se caracterizaron reordenamientos de Ig/TCR mediante PCR-multiplex y secuenciación para la búsqueda de recombinaciones génicas IGH, IGK, TCRB, TCRG y TCRD, en muestras de ADN obtenidas de médula ósea o sangre periférica al diagnóstico. El 95% (n=730) de los casos presentaron reordenamientos Ig/TCR. En el 68% de los casos se caracterizaron recombinaciones génicas IGH, en 43% IGK, en 25% TCRB, en 49% TCRG y en el 55% TCRD. Se caracterizó un total de 2506 reordenamientos de Ig/TCR que correspondían 1161 a inmunoglobulinas y 1345 a TCR. En la mayoría de los casos los reordenamientos de IGH fueron completos, IGK involucró a IGKde, TRCB se reordenó frecuentemente con el segmento Jb2, TCRG involucró preferentemente a Vg9 y los TCRD fueron principalmente reordenamientos incompletos. Este trabajo constituye el primer estudio realizado en la Argentina sobre la caracterización de reordenamientos Ig/TCR en un número muy significativo de pacientes con LLA pediátrica.

Abstract During lymphocyte ontogeny, the variable region of immunoglobulin (Ig) and T-cell receptor (TCR) is generated by rearrangements of the V-(D)-J gene segments. In this random process, nucleotide deletions and insertions occur between V-(D)-J segments. The aims of this work were to describe the incidence of Ig/TCR rearrangements, and the V-(D)-J segments involved in acute lymphoblastic leukemia (ALL) patients. With this purpose, 769 pediatric ALL patients belonging to Sociedad Argentina de Hemato-Oncología Pediátrica, diagnosed between 1999 and 2018, were studied. Ig/TCR rearrangements were characterized by multiplex PCR and sequencing to evaluate IGH, IGK, TCRB, TCRG and TCRD rearrangements in DNA samples obtained at diagnosis from bone marrow or peripheral blood. In total, 95% (n=730) of patients disclosed Ig/TCR rearrangements. IGH rearrangements were detected in 68% of cases; in 43% IGK, in 25% TCRB, in 49% TCRG and in 55% of cases, TCRD. A total of 2506 Ig/TCR rearrangements were characterized, being 1161 immunoglobulins and 1345 TCR. In most cases, IGH rearrangements were complete, IGK involved IGKde, TRCB was frequently rearranged with the Jb2 segment, TCRG preferentially involved Vg9, and TCRDs were mostly incomplete rearrangements. This work is the first study of Ig/TCR rearrangements characterization in a very significant number of childhood ALL carried out in Argentina.

Resumo Durante a ontogenia dos linfócitos, ocorre um rearranjo dos segmentos gênicos V-(D)-J que codificam para a região variável das cadeias de imunoglobulinas (Ig) e receptores de linfócitos T (TCR). Durante esse processo, os segmentos reorganizam-se aleatoriamente e exclusões e inserções de nucleotídeos ocorrem na região da união entre eles. Os objetivos do presente trabalho foram descrever as incidências dos rearranjos Ig/TCR e dos segmentos V-(D)-J envolvidos, em crianças com leucemia linfoide aguda (LLA). Para tanto, foram estudados 769 pacientes pediátricos com LLA, diagnosticados entre 1999 e 2018 pelos centros da Sociedade Argentina de Hemato-Oncologia Pediátrica. Rearranjos de Ig/TCR foram caracterizados através de PCR-multiplex e sequenciação para procurar recombinações gênicas IGH, IGK, TCRB, TCRG e TCRD em amostras de DNA obtidas da medula óssea ou sangue periférico no diagnóstico. Do total de pacientes estudados, 95% (n=730) apresentaram rearranjos de Ig/TCR. Os rearranjos gênicos IGH foram caracterizados em 68% dos casos, em 43% de IGK, em 25% de TCRB, em 49% de TCRG e em 55% de TCRD. Foi caracterizado um total de 2506 rearranjos de Ig/TCR, correspondendo 1161 a imunoglobulinas e 1345 a TCR. Na maioria dos casos, os rearranjos de IGH foram concluídos, o IGK envolveu o IGKde, o TRCB foi frequentemente rearranjado com o segmento Jb2, o TCRG preferencialmente envolveu o Vg9 e os TCRDs foram principalmente os rearranjos incompletos. Este trabalho constitui o primeiro estudo realizado na Argentina sobre a caracterização de rearranjos de Ig/TCR em um número muito significativo de pacientes com LLA pediátrica.