Outcome of adolescents and young adults with acute lymphoblastic leukemia in a single center in Brazil

ABSTRACT Introduction: Acute lymphoblastic leukemia (ALL) presents a poor prognosis in adults. The adoption of pediatric protocols has been changing this scenario, especially for adolescents and young adults (AYA). Objective and method: We aimed to evaluate a consecutive series of patients treated at the State Institute of Hematology of Rio de Janeiro between 2012 and 2020, focusing on the AYA subgroup. Result: The B-ALL was the most frequent subtype (81.6%) and AYA, the predominant age group (57.7%). The median overall survival (OS) was 9.4 months. High early mortality was observed and sepsis was the main cause of death. Better OS results were noted in AYA, in comparison to over 39y (13.3 × 6.2 months, respectively), the Berlin-Frankfurt-Münster (BFM) being the protocol of choice in this group. Conclusion: The use of the pediatric protocol seems to improve the OS of AYA, however, high rates of deaths from infection were observed, demonstrating the need for advances in the Brazilian public system clinical support.

The Intracellular and Secreted Sides of Osteopontin and Their Putative Physiopathological Roles.

Classically, osteopontin (OPN) has been described as a secreted glycophosprotein. Indeed, most data concerning its physiological and pathological roles are mainly related to the secreted OPN (sOPN). However, there are several instances in which intracellular OPN (iOPN) has been described, presenting some specific roles in distinct experimental models, such as in the immune system, cancer cells, and neurological disorders. We herein aimed to highlight and discuss some of these secreted and intracellular roles of OPN and their putative clinical and biological impacts. Moreover, by consolidating data from the OPN protein database, we also analyzed the occurrence of signal peptide (SP) sequences and putative subcellular localization, especially concerning currently known OPN splicing variants (OPN-SV). Comprehending the roles of OPN in its distinct cellular and tissue environments may provide data regarding the additional applications of this protein as biomarkers and targets for therapeutic purposes, besides further describing its pleiotropic roles.

Outcome of adolescents and young adults with acute lymphoblastic leukemia in a single center in Brazil.

INTRODUCTION: Acute lymphoblastic leukemia (ALL) presents a poor prognosis in adults. The adoption of pediatric protocols has been changing this scenario, especially for adolescents and young adults (AYA). OBJECTIVE AND METHOD: We aimed to evaluate a consecutive series of patients treated at the State Institute of Hematology of Rio de Janeiro between 2012 and 2020, focusing on the AYA subgroup. RESULT: The B-ALL was the most frequent subtype (81.6%) and AYA, the predominant age group (57.7%). The median overall survival (OS) was 9.4 months. High early mortality was observed and sepsis was the main cause of death. Better OS results were noted in AYA, in comparison to over 39y (13.3 × 6.2 months, respectively), the Berlin-Frankfurt-Münster (BFM) being the protocol of choice in this group. CONCLUSION: The use of the pediatric protocol seems to improve the OS of AYA, however, high rates of deaths from infection were observed, demonstrating the need for advances in the Brazilian public system clinical support.

Novel Diagnostic and Therapeutic Options for KMT2A-Rearranged Acute Leukemias.

The KMT2A (MLL) gene rearrangements (KMT2A-r) are associated with a diverse spectrum of acute leukemias. Although most KMT2A-r are restricted to nine partner genes, we have recently revealed that KMT2A-USP2 fusions are often missed during FISH screening of these genetic alterations. Therefore, complementary methods are important for appropriate detection of any KMT2A-r. Here we use a machine learning model to unravel the most appropriate markers for prediction of KMT2A-r in various types of acute leukemia. A Random Forest and LightGBM classifier was trained to predict KMT2A-r in patients with acute leukemia. Our results revealed a set of 20 genes capable of accurately estimating KMT2A-r. The SKIDA1 (AUC: 0.839; CI: 0.799-0.879) and LAMP5 (AUC: 0.746; CI: 0.685-0.806) overexpression were the better markers associated with KMT2A-r compared to CSPG4 (also named NG2; AUC: 0.722; CI: 0.659-0.784), regardless of the type of acute leukemia. Of importance, high expression levels of LAMP5 estimated the occurrence of all KMT2A-USP2 fusions. Also, we performed drug sensitivity analysis using IC50 data from 345 drugs available in the GDSC database to identify which ones could be used to treat KMT2A-r leukemia. We observed that KMT2A-r cell lines were more sensitive to 5-Fluorouracil (5FU), Gemcitabine (both antimetabolite chemotherapy drugs), WHI-P97 (JAK-3 inhibitor), Foretinib (MET/VEGFR inhibitor), SNX-2112 (Hsp90 inhibitor), AZD6482 (PI3Kß inhibitor), KU-60019 (ATM kinase inhibitor), and Pevonedistat (NEDD8-activating enzyme (NAE) inhibitor). Moreover, IC50 data from analyses of ex-vivo drug sensitivity to small-molecule inhibitors reveals that Foretinib is a promising drug option for AML patients carrying FLT3 activating mutations. Thus, we provide novel and accurate options for the diagnostic screening and therapy of KMT2A-r leukemia, regardless of leukemia subtype.

CRLF2 overexpression defines an immature-like subgroup which is rescued through restoration of the PRC2 function in T-cell precursor acute lymphoblastic leukemia.

CRLF2 overexpression has been described as a biomarker of poor prognosis in T-cell acute lymphoblastic leukemia (T-ALL). In the present study, we aimed to unravel the genomic profile underlying CRLF2 overexpression (CRLF2-high) by analyzing RNA-seq, WES, and SNP-array data from 264 T-ALL patients and five cell lines deposited on the TARGET initiative, Cancer Cell Line Encyclopedia and Gene Expression Omnibus. These data allowed us to delineate the genomic landscape of CRLF2-high in T-ALL, which was associated with PTEN, JAK3, PHF6, EZH2, and RUNX1 mutations. We also observed an enrichment of CRLF2-high in early T-precursor (ETP)-ALL (23.08% vs. 4.02%, P = 7.579e-06 ) and a very similar gene upregulation profile between these two entities. The inhibition of BET (iBET) proteins is a strategy previously demonstrated to reverse the gene upregulation pattern of ETP cells through restoration of polycomb repressive complex 2 (PRC2) activity. While CRLF2 expression was rescued by using this strategy in LOUCY (untreated vs. iBET P = 0.0095, DMSO vs. iBET P = 0.0286), a classical ETP-ALL cell line, PRC2 loss was not sufficient to promote CRLF2 upregulation in JURKAT, a more mature T-ALL cell line. Considering the role of IKZF1 in CRLF2 regulation and in recruitment of PCR2, we evaluated IKZF1 status according to CRLF2-expression subgroups. We identified that IKZF1 transcripts with intron retention were upregulated in the CRLF2-high subgroup. Here, we delineated the gene expression profile of CRLF2-high T-ALL samples and unraveled the crucial role of PRC2 in CRLF2 regulation in ETP-ALL.

IKZF1 deletions associate with CRLF2 overexpression leading to a poor prognosis in B-cell precursor acute lymphoblastic leukaemia.

Cytokine Receptor-Like Factor 2 (CRLF2) overexpression occurs in 5-15% of B-cell precursor acute lymphoblastic leukaemia (B-ALL). In ∼50% of these cases, the mechanisms underlying this dysregulation are unknown. IKAROS Family Zinc Finger 1 (IKZF1) is a possible candidate to play a role in this dysregulation since it binds to the CRLF2 promoter region and suppresses its expression. We hypothesised that IKZF1 loss of function, caused by deletions or its short isoforms expression, could be associated with CRLF2 overexpression in B-ALL. A total of 131 paediatric and adult patients and 7 B-ALL cell lines were analysed to investigate the presence of IKZF1 deletions and its splicing isoforms expression levels, the presence of CRLF2 rearrangements or mutations, CRLF2 expression and JAK2 mutations. Overall survival analyses were performed according to the CRLF2 and IKZF1 subgroups. Our analyses showed that 25.2% of patients exhibited CRLF2 overexpression (CRLF2-high). CRLF2-high was associated with the presence of IKZF1 deletions (IKZF1del, p = 0.001), particularly with those resulting in dominant-negative isoforms (p = 0.006). Moreover, CRLF2 expression was higher in paediatric samples with high loads of the short isoform IK4 (p = 0.011). It was also associated with the occurrence of the IKZF1 plus subgroup (p = 0.004). Furthermore, patients with CRLF2-high/IKZF1del had a poorer prognosis in the RELLA05 protocol (p = 0.067, 36.1 months, 95%CI 0.0-85.9) and adult cohort (p = 0.094, 29.7 months, 95%CI 11.8-47.5). In this study, we show that IKZF1 status is associated with CRLF2-high and dismal outcomes in B-ALL patients regardless of age.

Implementation of a pharmacogenomic program in a Brazilian public institution.

This narrative review describes implementation, current status and perspectives of a pharmacogenomic (PGx) program at the Brazilian National Cancer Institute (INCA), targeting the cancer chemotherapeutic drugs - fluoropyrimidines, irinotecan and thiopurines. This initiative, designed as a research project, was supported by a grant from the Brazilian Ministry of Health. A dedicated task force developed standard operational procedures from recruitment of patients to creating PGx reports with dosing recommendations, which were successfully applied to test 100 gastrointestinal cancer INCA outpatients and 162 acute lymphoblastic leukemia pediatric patients from INCA and seven other hospitals. The program has been subsequently expanded to include gastrointestinal cancer patients from three additional cancer treatment centers. We anticipate implementation of routine pre-emptive PGx testing at INCA but acknowledge challenges associated with this transition, such as continuous financing support, availability of trained personnel, adoption of the PGx-informed prescription by the clinical staff and, ultimately, evidence of cost-effectiveness.

CD9 predicts ETV6-RUNX1 in childhood B-cell precursor acute lymphoblastic leukemia

ABSTRACT Introduction: The ETV6-RUNX1 is a fusion gene associated with a good outcome in B-cell precursor lymphoblastic leukemia. Objective: This study aimed to re-evaluate the CD9 cellular expression by flow cytometry (FC) as a possible tool to predict the presence of ETV6-RUNX1. Method: Childhood B-cell precursor lymphoblastic leukemia cases were included (n = 186). The percentage of CD9-labeled cells and the median fluorescence intensity ratio were used for correlation with the molecular tests. Receiver Operating Characteristic curves were performed to determine the likelihood of the CD9 expression predicting ETV6-RUNX1. Results: The ETV6-RUNX1 was found in 44/186 (23.6%) cases. Data analysis revealed that the best cutoff for CD9 percentage was 64%, with an accuracy of 0.84, whereas the best cutoff for CD9 median fluorescence intensity ratio was 12.52, with an accuracy of 0.80. A strong association was observed between the level of CD9 expression and the presence of ETV6-RUNX1. Conclusion: These data confirm that the CD9 expression could be used for risk stratification in clinical practice.

FLT3 overexpression in acute leukaemias: New insights into the search for molecular mechanisms.

FLT3 overexpression is a recurrent event in various acute leukaemia subtypes. This transcriptional deregulation is important to define the prognostic risk for many patients. Of note, the molecular mechanisms leading to this gene upregulation are unknown for a substantial number of cases. In this Mini-Review, we highlight the role of FLT3 overexpression in acute leukaemia and discuss emerging mechanisms accounting for this upregulation. The benefits of using targeted therapy are also addressed in the overexpression context, posing other therapeutic possibilities based on state-of-the-art knowledge that could be considered for future research.

CD9 predicts ETV6-RUNX1 in childhood B-cell precursor acute lymphoblastic leukemia.

INTRODUCTION: The ETV6-RUNX1 is a fusion gene associated with a good outcome in B-cell precursor lymphoblastic leukemia. OBJECTIVE: This study aimed to re-evaluate the CD9 cellular expression by flow cytometry (FC) as a possible tool to predict the presence of ETV6-RUNX1. METHOD: Childhood B-cell precursor lymphoblastic leukemia cases were included (n=186). The percentage of CD9-labeled cells and the median fluorescence intensity ratio were used for correlation with the molecular tests. Receiver Operating Characteristic curves were performed to determine the likelihood of the CD9 expression predicting ETV6-RUNX1. RESULTS: The ETV6-RUNX1 was found in 44/186 (23.6%) cases. Data analysis revealed that the best cutoff for CD9 percentage was 64%, with an accuracy of 0.84, whereas the best cutoff for CD9 median fluorescence intensity ratio was 12.52, with an accuracy of 0.80. A strong association was observed between the level of CD9 expression and the presence of ETV6-RUNX1. CONCLUSION: These data confirm that the CD9 expression could be used for risk stratification in clinical practice.

IKZF1 Deletions with COBL Breakpoints Are Not Driven by RAG-Mediated Recombination Events in Acute Lymphoblastic Leukemia.

IKZF1 deletion (ΔIKZF1) is an important predictor of relapse in both childhood and adult B-cell precursor acute lymphoblastic leukemia (B-ALL). Previously, we revealed that COBL is a hotspot for breakpoints in leukemia and could promote IKZF1 deletions. Through an international collaboration, we provide a detailed genetic and clinical picture of B-ALL with COBL rearrangements (COBL-r). Patients with B-ALL and IKZF1 deletion (n = 133) were included. IKZF1 ∆1-8 were associated with large alterations within chromosome 7: monosomy 7 (18%), isochromosome 7q (10%), 7p loss (19%), and interstitial deletions (53%). The latter included COBL-r, which were found in 12% of the IKZF1 ∆1-8 cohort. Patients with COBL-r are mostly classified as intermediate cytogenetic risk and frequently harbor ETV6, PAX5, CDKN2A/B deletions. Overall, 56% of breakpoints were located within COBL intron 5. Cryptic recombination signal sequence motifs were broadly distributed within the sequence of COBL, and no enrichment for the breakpoint cluster region was found. In summary, a diverse spectrum of alterations characterizes ΔIKZF1 and they also include deletion breakpoints within COBL. We confirmed that COBL is a hotspot associated with ΔIKZF1, but these rearrangements are not driven by RAG-mediated recombination.

Validation of the United Kingdom copy-number alteration classifier in 3239 children with B-cell precursor ALL.

Genetic abnormalities provide vital diagnostic and prognostic information in pediatric acute lymphoblastic leukemia (ALL) and are increasingly used to assign patients to risk groups. We recently proposed a novel classifier based on the copy-number alteration (CNA) profile of the 8 most commonly deleted genes in B-cell precursor ALL. This classifier defined 3 CNA subgroups in consecutive UK trials and was able to discriminate patients with intermediate-risk cytogenetics. In this study, we sought to validate the United Kingdom ALL (UKALL)-CNA classifier and reevaluate the interaction with cytogenetic risk groups using individual patient data from 3239 cases collected from 12 groups within the International BFM Study Group. The classifier was validated and defined 3 risk groups with distinct event-free survival (EFS) rates: good (88%), intermediate (76%), and poor (68%) (P < .001). There was no evidence of heterogeneity, even within trials that used minimal residual disease to guide therapy. By integrating CNA and cytogenetic data, we replicated our original key observation that patients with intermediate-risk cytogenetics can be stratified into 2 prognostic subgroups. Group A had an EFS rate of 86% (similar to patients with good-risk cytogenetics), while group B patients had a significantly inferior rate (73%, P < .001). Finally, we revised the overall genetic classification by defining 4 risk groups with distinct EFS rates: very good (91%), good (81%), intermediate (73%), and poor (54%), P < .001. In conclusion, the UKALL-CNA classifier is a robust prognostic tool that can be deployed in different trial settings and used to refine established cytogenetic risk groups.

IKZF1 deletion and co-occurrence with other aberrations in a child with chronic myeloid leukemia progressing to acute lymphoblastic leukemia.

Chronic myeloid leukemia (CML) is a rare disease in children. Different from that in adults, childhood CML involves transformative events occurring over a short time period. CML transformation to lymphoid blast phase (BP) is associated with copy number abnormalities, characteristic of BCR-ABL1 positive acute lymphoblastic leukemia, but not of CML in the chronic phase. Here, we present an unusual case of CML progressing to BP in a 1.6-year-old child, harboring IKZF1, PAX5, CDKN2A, and ETV6 deletions at diagnosis. It remains to be addressed whether distinct mechanisms might account for CML pathogenesis in early childhood.