XRCC4 rs28360071 intronic variant is associated with increased risk for infant acute lymphoblastic leukemia with KMT2A rearrangements.

Early age acute leukemia (EAL) shows a high frequency of KMT2A-rearrangements (KMT2A-r). Previous investigations highlighted double-strand breaks arising from maternal exposure to xenobiotics during pregnancy as a risk factor for EAL and KMT2A-r. In this case-control study, we investigated the relationship between EAL and genetic variants of the nonhomologous end-joining (XRCC6 rs5751129, XRCC4 rs6869366 and rs28360071), since they might affect DNA repair capacity, leading to KMT2A-r and leukemogenesis. Samples from 577 individuals (acute lymphoblastic leukemia-ALL, n=164; acute myeloid leukemia-AML, n=113; controls, n=300) were genotyped. No significant association was found for rs5751129 and rs6869366, whereas rs28360071 was associated with an increased risk for ALL with KMT2A-r (IIxID: OR - Odds ratio 2.23, CI 1.17-4.25, p=0.014). Bone marrow samples from ALL patients showed a higher expression of XRCC4 compared to AML patients (p=0.025). Human Splicing Finder 3.1 predicted that the deleted allele of rs28360071 is potentially associated with the activation of a 5' cryptic splice site in intron 3 of XRCC4. The sequencing of cDNA did not show any differences on the splicing process for the rs28360071 genotypes. Our results suggest that the deleted allele for rs28360071 increases the risk for ALL with KMT2A-r, but not by modifying the XRCC4 expression levels or its structure.

IKZF1 and BTG1 silencing reduces glucocorticoid response in B-cell precursor acute leukemia cell line.

INTRODUCTION: Secondary genetic alterations, which contribute to the dysregulation of cell cycle progression and lymphoid specialization, are frequently observed in B-cell precursor acute lymphoblastic leukemia (B-ALL). As IKZF1 and BTG1 deletions are associated with a worse outcome in B-ALL, this study aimed to address whether they synergistically promote glucocorticoid resistance. METHODS: Small interfering RNA was used to downregulate either IKZF1, or BTG1, or both genes in the 207 B-ALL cell line. Cell viability was investigated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and trypan blue exclusion assays. The expression levels of IKZF1, BTG1 and glucocorticoid-responsive genes (DUSP1, SGK1, FBXW7 and NR3C1) were evaluated by real time quantitative real time polymerase chain reaction (PCR). RESULTS: Isolated silencing of BTG1, IKZF1, or both genes in combination under dexamethasone treatment increased cell viability by 24%, 40% and 84%, respectively. Although BTG1 silencing did not alter the expression of glucocorticoid-responsive genes, IKZF1 knockdown decreased the transcript levels of DUSP1 (2.6-fold), SGK1 (1.8-fold), FBXW7 (2.2-fold) and NR3C1 (1.7-fold). The expression of glucocorticoid-responsive genes reached even lower levels (reducing 2.4-4 fold) when IKZF1 and BTG1 silencing occurred in combination. CONCLUSIONS: IKZF1 silencing impairs the transcription of glucocorticoid-responsive genes; this effect is enhanced by concomitant loss of BTG1. These results demonstrate the molecular mechanism by which the combination of both genetic deletions might contribute to higher relapse rates in B-ALL.

Description of lymphocyte and cytokine profiles in individuals with acute myeloid leukemia associated with FLT3-ITD and NPM1 mutation status.

The pathogenesis of acute myeloid leukemia (AML) involves mutations in genes such as FLT3 and NPM1, which are also associated with the prognosis of the disease. The immune system influences disease progression, but the mechanisms underlying the interaction between the immune system and AML are not clear. In this study, the profiles of lymphocytes and cytokines were described in individuals with AML stratified by molecular changes associated with prognosis. The participants included in this study were newly diagnosed AML patients (n = 43) who were about to undergo chemotherapy. Subtypes of lymphocytes in peripheral blood, including B cells, T cells, and natural killer cells, and serum concentrations of cytokines, including Th1, Th2, and Th17, were studied by flow cytometry assays (BD FACSCanto II). The correlations between lymphocyte subsets, cytokines, and genetic/prognostic risk stratification (based on the FLT3 and NPM1 genes) were analyzed. The differences in B lymphocytes (%), T lymphocytes (%), plasmablasts (%), leukocytes (cells/µl), and tumor necrosis factor (pg/ml) were determined between groups with FLT3-ITD+ and FLT3-ITD- mutations. The presence of mutations in NPM1 and FLT3-ITD and age suggested changes in the lymphocyte and cytokine profile in individuals with AML.

Characterisation of cells markers associated with IKZF1plus in BCP-ALL.

The presence of IKZF1 deletions has been associated with an increased relapse rate in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). There is a particular subset of IKZF1del cases called IKZF1plus (defined by the co-occurrence of IKZF1del and deletions in CDKN2A/B, PAX5, or the PAR1 region, in the absence of ERG deletions), which is also associated with worse prognosis, but some recent studies have not found major differences between the IKZF1del and IKZF1plus groups. Therefore, the IKZF1plus group still needs further comprehension and our study aims to characterise the molecular heterogeneity and identify molecular markers exclusively associated with IKZF1plus. Two independent series of cases (TARGET, n = 125 and GenLAb, n = 60) were evaluated by segregating patients into 3 groups: IKZF1plus, IKZF1del, and IKZF1wild. Differential expression analyses showed that the membrane protein-coding genes most associated with the IKZF1plus group were: KCNA5, GREB1, EPOR, SDK1, and PTPRB. Notably, KCNA5 and GREB1 differential expression levels were validated in the GenLAb validation series. Regarding copy number alterations, we observed a high frequency of VPREB1 deletions in the IKZF1plus group, as well as additional exclusive deletions in the CD200 and BTLA genes. Recent research suggests that the importance of the IKZF1plus profile varies depending on the genetic subgroup. In this scenario, we found associations between IKZF1plus and certain genes in BCP-ALL, being KCNA5 and GREB1 the most promising biomarkers for predicting IKZF1plus. A deeper understanding of these genetic profiles will allow a better risk assessment and offer precise rationale for therapeutic strategies in BCP-ALL.

Demographics, clinical findings and diagnoses of cranial thoracic myelopathies (T1-T6 vertebrae) in cats.

OBJECTIVES: The aim of the study was to describe the patient demographics, clinicopathological features and presumptive or final diagnoses in cats with myelopathies between the T1 and T6 vertebrae. METHODS: This retrospective multicentre case study enrolled cases between 2015 and 2022 that were diagnosed with myelopathies between the T1 and T6 vertebrae as the primary cause for the presenting clinical signs. RESULTS: A total of 21 cases matched the inclusion criteria, 13 males (11 castrated and 2 entire) and 8 spayed females (median age 93 months; range 5-192). Most of the cases presented with a chronic and progressive history (76% and 86%, respectively), with a median duration of 29 days (range 1-2880). At the time of presentation, 90% of the cases were localised to the T3-L3 spinal cord segments based on neurological examination. The most common underlying pathology was neoplasia (42.9%), followed by inflammatory (24%), anomalous (19%), degenerative (9.5%) and vascular (4.8%) disorders. The most common location was T3-T4 (29%), followed by T2-T3 and T5-T6 (19% each). The cutaneous trunci reflex was normal in 86% of the cases and most of the cases (71%) did not show spinal discomfort upon admission. CONCLUSIONS AND RELEVANCE: Neoplasia was the most common cause of cranial thoracic myelopathy in this study. The lack of pathognomonic clinical signs for this specific region highlights the importance of assessing the entire thoracolumbar region up to and including at least the T1 vertebra when investigating cases with signs consistent with a T3-L3 myelopathy.

Thoracic to lumbar vertebral column length and length ratios in miniature dachshunds with and without thoracolumbar intervertebral disc extrusion.

BACKGROUND: The chondrodystrophic body type predisposes miniature dachshunds to thoracolumbar intervertebral disc extrusion (IVDE). However, the relationship between thoracolumbar IVDE and the relative lengths of the thoracic and lumbar vertebral columns has not yet been evaluated. METHODS: This prospective multicentre study included 151 miniature dachshunds with (n = 47) and without (n = 104) thoracolumbar IVDE. All dogs had their thoracic and lumbar vertebral columns measured with a tape measure. Detailed descriptions were provided to facilitate consistent measurement. A thoracic to lumbar vertebral column ratio was calculated. Thoracolumbar IVDE was confirmed by magnetic resonance imaging or computed tomography. RESULTS: The thoracic to lumbar vertebral column length ratio and absolute thoracic vertebral column length were significantly smaller in miniature dachshunds with IVDE than in those without IVDE (p < 0.0001 for both). There were no significant differences in lumbar vertebral column length, age, sex or neuter status between the two groups. LIMITATIONS: The dogs without IVDE did not undergo a neurological examination and the thoracic and lumbar vertebral column measurements were not validated. CONCLUSIONS: The relative lengths of the thoracic and lumbar vertebral column segments could contribute to the development of thoracolumbar IVDE in miniature dachshunds. Further studies are needed to evaluate ideal thoracic to lumbar vertebral column length ratios in miniature dachshunds.

What Is Your Diagnosis?

In collaboration with the American College of Veterinary Radiology.

Cranial thoracic myelopathies (T1-T6 vertebrae): Retrospective evaluation of the signalment, clinical presentation, and, presumptive or final diagnoses in 84 dogs.

The aim of the study was to describe the signalment, clinical presentation and presumptive or final diagnoses of dogs with cranial thoracic spinal cord lesions identified on advanced imaging. Retrospective evaluation of the databases of three veterinary specialty centres, between 2009 and 2021, was performed to identify dogs with a lesion affecting the cranial thoracic vertebral column (T1-T6 vertebrae) as the primary cause for presenting signs of myelopathy and/or spinal pain. Eighty-four dogs were included in the study, with the majority (n = 76) presenting with a progressive history of over 4-weeks' duration. On neurologic examination, most dogs were ambulatory (n = 64), and the most common neuroanatomic localisation was the T3-L3 spinal cord segments (n = 63). Twelve dogs (14%) showed a short-strided thoracic limb gait on clinical examination. The most common diagnosis was neoplasia (n = 33), followed by anomalies (n = 22, including vertebral body malformations in 14 dogs) and degenerative disorders (n = 16, with intervertebral disc protrusion diagnosed in 9 dogs). The most common vertebrae affected were T3 and T5. Most dogs with degenerative conditions showed asymmetric clinical signs, and the majority of dogs with neoplasia showed signs of spinal hyperaesthesia on examination. The findings of this study describe the clinical signs and presumptive or final diagnoses associated with lesions affecting the cranial thoracic spinal cord. When combined with the signalment and clinical history, this information can assist in both the recognition of and problem-based approach to these cases.

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.

Therapy-related acute myeloid leukemia with KMT2A-SNX9 gene fusion associated with a hyperdiploid karyotype after hemophagocytic lymphohistiocytosis.

Therapy-related acute myeloid leukemia (t-AML) following treatment with topoisomerase-II inhibitors has been increasingly reported. These compounds (e.g. etoposide) promote DNA damage and are associated with KMT2A rearrangements. They are widely used as first-line treatment in hemophagocytic lymphohistiocytosis (HLH). Here we describe a newborn who developed t-AML after HLH treatment. We provide detailed clinical, cytogenetic, and molecular characteristics of this patient, including the identification of a novel gene fusion - KMT2A-SNX9 - in t-AML. Considering the dismal outcome of this case, we discuss the side-effects of etoposide administration during HLH treatment in infants.

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.

Molecular approaches identify a cryptic MECOM rearrangement in a child with a rapidly progressive myeloid neoplasm.

Myeloid neoplasms are a heterogeneous group of hematologic disorders with divergent patterns of cell differentiation and proliferation, as well as divergent clinical courses. Rare recurrent genetic abnormalities related to this group of cancers are associated with poor outcomes. One such abnormality is the MECOM gene rearrangement that typically occurs in cases with chromosome 7 abnormalities. MECOM encodes a transcription factor that plays an essential role in cell proliferation and maintenance and also in epigenetic regulation. Aberrant expression of this gene is associated with reduced survival. Hence, its detailed characterization provides biological and clinical information relevant to the management of pediatric myeloid neoplasms. In this work, we describe a rare karyotype harboring three copies of MECOM with overexpression of the gene in a child with a very aggressive myeloid neoplasm. Cytogenetic studies defined the karyotype as 46,XX,der(7)t(3;7)(q26.2;q21.2). Array comparative genomic hybridization (aCGH) revealed a gain of 26.04 Mb in the 3q26.2-3qter region and a loss of 66.6 Mb in the 7q21.2-7qter region. RT-qPCR analysis detected elevated expression of the MECOM and CDK6 genes (458.5-fold and 35.2-fold, respectively). Overall, we show the importance of performing detailed molecular cytogenetic analysis of MECOM to enable appropriate management of high-risk pediatric myeloid neoplasms.

IKZF1 Gene in Childhood B-cell Precursor Acute Lymphoblastic Leukemia: Interplay between Genetic Susceptibility and Somatic Abnormalities.

SNPs in IKZF1 are associated with inherited susceptibility to B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Besides, somatic copy number abnormalities (CNA) in genes related to lymphopoiesis (e.g., IKZF1, CDKN2A/B, BTG1) impact patient's outcome. Therefore, this study aimed to investigate an association between germline susceptibility and CNAs in BCP-ALL. The IKZF1 SNPs (rs11978267 and rs4132601) were genotyped in 276 cases and 467 controls. Bone marrow samples were used to determine the presence of somatic abnormalities. The IKZF1 transcript levels were quantified and associated with the SNPs and CNAs. Categorical variables were compared by χ2 test. ORs were estimated with unconditional logistic regression with 95% confidence interval (CI). The variant allele of IKZF1 rs4132601 conferred increased risk of BCP-ALL (OR, 2.09; 95% CI, 1.16-3.74). Individuals with either rs11978267 or rs4132601 had an increased risk for harboring IKZF1 deletion (OR, 2.80; 95% CI, 1.25-6.23 and OR, 2.88; 95% CI, 1.24-6.69, respectively). Increased risks were observed for individuals harboring both IKZF1 and BTG1 deletions (OR, 4.90; 95% CI, 1.65-14.55, rs11978267 and OR, 5.80; 95% CI, 1.94-17.41, rs4132601). Germline genetic variation increases the risk for childhood ALL in general, but also acts as a susceptibility factor bound for risk of specific somatic alterations. These findings provide new insight into the development of childhood ALL regarding causal variants and the biological basis of the risk association, offering the opportunity for future tailored research. Cancer Prev Res; 10(12); 738-44. ©2017 AACR.

Effect of pre-anesthetic fasting on gastric emptying and plasma glucose in healthy dogs of different age groups / Efeito do jejum pré-anestésico sobre o esvaziamento gástrico e a glicemia plasmática em cães hígidos de diferentes faixas etárias

Gastric emptying and plasma glucose were evaluated in young and adult dogs, fed with dry and wet food, submitted to different periods of pre-anesthetic fasting (6, 8, and 12 hours). Forty healthy dogs were selected, which were segmented into four groups according to the age group and type of diet. It was evaluated the gastric emptying by ultrasound and serum glycemia. Only 17.5% presented complete gastric emptying, and no significant differences were found between the 6 and 8-hour fasting evaluations, or between the age groups and the diets, considering significance level p<0.05. Mean plasma glucose values from the groups indicated normal glycemia at all times of evaluation. A significant difference was found between the means of glycemia in young and adult dogs, with the 8-hour fasting with wet diet (p=0.03) and with 12 hours with dry diet (p=0.04). Healthy young and adult dogs, in physiological equilibrium, maintain average values of plasma glucose despite prolonged periods of pre-anesthetic fasting, which may be necessary, since 8-hour fasting for solid food is not enough to provide complete gastric emptying.(AU)

Avaliou-se o esvaziamento gástrico e a glicemia plasmática em cães jovens e adultos, alimentados com ração seca e úmida, submetidos a diferentes períodos de jejum pré-anestésico (6, 8 e 12 horas). Foram selecionados 40 cães hígidos, os quais foram segmentados em 4 grupos de acordo com a faixa etária e o tipo de dieta administrada. Foi avaliado o esvaziamento gástrico por ultrassonografia e a glicemia sérica. Apenas 17,5% apresentaram completo esvaziamento gástrico, não sendo encontradas diferenças significativas entre as avaliações com 6 e 8 horas de jejum, ou entre as faixas etárias e dietas, considerando nível de significância p<0,05. Os valores médios da glicose plasmática dos grupos indicaram normoglicemia em todos os momentos de avaliação. Foi encontrada diferença significativa entre as médias da glicemia dos cães jovens e adultos, no período de 8 horas de jejum com dieta úmida (p=0,03) e com 12 horas nos animais com dieta seca (p=0,04). Conclui-se que cães hígidos jovens e adultos, em equilíbrio fisiológico, mantêm valores normais de glicemia plasmática apesar de períodos prolongados de jejum pré-anestésico, os quais podem ser necessários, tendo em vista que 8 horas de jejum alimentar de sólidos não é suficiente para proporcionar completo esvaziamento gástrico.(AU)