Table1_Comprehensive detection of CRLF2 alterations in acute lymphoblastic leukemia: a rapid and accurate novel approach.xlsx

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.</p

DataSheet1_Comprehensive detection of CRLF2 alterations in acute lymphoblastic leukemia: a rapid and accurate novel approach.docx

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.</p

Image2_Comprehensive detection of CRLF2 alterations in acute lymphoblastic leukemia: a rapid and accurate novel approach.PNG

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.</p

Image1_Comprehensive detection of CRLF2 alterations in acute lymphoblastic leukemia: a rapid and accurate novel approach.PNG

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.</p

Single-Nucleotide Polymorphism Array-Based Karyotyping of Acute Promyelocytic Leukemia

<div><p>Acute promyelocytic leukemia (APL) is characterized by the t(15;17)(q22;q21), but additional chromosomal abnormalities (ACA) and other rearrangements can contribute in the development of the whole leukemic phenotype. We hypothesized that some ACA not detected by conventional techniques may be informative of the onset of APL. We performed the high-resolution SNP array (SNP-A) 6.0 (Affymetrix) in 48 patients diagnosed with APL on matched diagnosis and remission sample. Forty-six abnormalities were found as an acquired event in 23 patients (48%): 22 duplications, 23 deletions and 1 Copy-Neutral Loss of Heterozygocity (CN-LOH), being a duplication of 8(q24) (23%) and a deletion of 7(q33-qter) (6%) the most frequent copy-number abnormalities (CNA). Four patients (8%) showed CNAs adjacent to the breakpoints of the translocation. We compared our results with other APL series and found that, except for dup(8q24) and del(7q33-qter), ACA were infrequent (≤3%) but most of them recurrent (70%). Interestingly, having CNA or <i>FLT3</i> mutation were mutually exclusive events. Neither the number of CNA, nor any specific CNA was associated significantly with prognosis. This study has delineated recurrent abnormalities in addition to t(15;17) that may act as secondary events and could explain leukemogenesis in up to 40% of APL cases with no ACA by conventional cytogenetics.</p></div

Schematic representation of CNA adjacent to the translocation breakpoints found in our series.

<p>On the left panel, the Smooth Signal of chromosome 15 and 17 from case APL_32 are represented. Results from diagnosis sample are shown in blue and from complete remission sample, in green. On the right, chromosomes 15 and 17 are depicted with G-banding and arrows pointing the location of the <i>PML</i> and <i>RARA</i> genes. Areas shaded in blue show regions duplicated and in red, deleted. Panel (A) corresponds to isochromosome der(17)t(15;17); Panel (B) shows a small duplication of the <i>PML</i> gene, and in panel (C) both the <i>PML</i> and the <i>RARA</i> genes are duplicated. These two cases had a cryptic t(15;17) by CC, that was revealed by FISH. In panel (D) two small deletions are found distally to the translocation breakpoints.</p

Mutated genes with a higher frequency in our cohort than expected in the 1000 genomes repository.

<p>Mutated genes with a higher frequency in our cohort than expected in the 1000 genomes repository.</p

Distribution of selected mutations along the different affected genes and their related functional categories.

<p>A) Number of mutated samples by gene according to the described mutation filtering protocol (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148346#pone.0148346.s002" target="_blank">S2 Fig</a>). Only 33 recurrent genes were included. B) Number of mutated samples by gene category (bottom). The corresponding number of genes included in each functional category (top) is also represented in order to avoid any size bias. C) The functional categories are distributed depending on the observed mutated samples and the known number of belonging genes. Both transcription factor and ubiquitination categories shown and excess of mutated samples. D) The number of mutated samples per category in the haloplex cohort were compared against a reference population of healthy samples (1000 genomes). Here, while metabolism and signalling genes categories appear poorly mutated, ubiquitination appears remarkably more mutated than the reference population.</p

Network of significant gene co-occurrences.

<p>Genes are represented by nodes and their sizes defined from the number of significant co-occurrences they are implied. Edges represent co-occurrences between pairs of genes. Every edge is labelled with the number of samples that carries the mutated pair of genes as follows: higher than expected co-occurrences are coloured in green, while lower than expected (only one) in red. Edge width is proportional to the statistical p-value of chi-square test. Those genes co-occurring only at one single patient are painted in white. Seven co-occurrence subnetworks arise from the significant co-occurrence network, where remarkably a single component connected the half of represented genes. In contrast, 3 pairs are simultaneously mutated only in 2 different individuals, and 3 significant co-occurrence subnetworks, only in 1 patient.</p

Comparison of type, size and number of SNP-A abnormalities in reported APL series.

<p>*one Uniparental Tetrasomy has been included both in duplication and CN-LOH group.</p><p>CNA: Copy-Number Abnormality. CN-LOH: Copy-Neutral Loss of Heterozygocity.</p