Landscape of driver mutations and their clinical effects on Down syndrome-related myeloid neoplasms.

ABSTRACT: Transient abnormal myelopoiesis (TAM) is a common complication in newborns with Down syndrome (DS). It commonly progresses to myeloid leukemia (ML-DS) after spontaneous regression. In contrast to the favorable prognosis of primary ML-DS, patients with refractory/relapsed ML-DS have poor outcomes. However, the molecular basis for refractoriness and relapse and the full spectrum of driver mutations in ML-DS remain largely unknown. We conducted a genomic profiling study of 143 TAM, 204 ML-DS, and 34 non-DS acute megakaryoblastic leukemia cases, including 39 ML-DS cases analyzed by exome sequencing. Sixteen novel mutational targets were identified in ML-DS samples. Of these, inactivations of IRX1 (16.2%) and ZBTB7A (13.2%) were commonly implicated in the upregulation of the MYC pathway and were potential targets for ML-DS treatment with bromodomain-containing protein 4 inhibitors. Partial tandem duplications of RUNX1 on chromosome 21 were also found, specifically in ML-DS samples (13.7%), presenting its essential role in DS leukemia progression. Finally, in 177 patients with ML-DS treated following the same ML-DS protocol (the Japanese Pediatric Leukemia and Lymphoma Study Group acute myeloid leukemia -D05/D11), CDKN2A, TP53, ZBTB7A, and JAK2 alterations were associated with a poor prognosis. Patients with CDKN2A deletions (n = 7) or TP53 mutations (n = 4) had substantially lower 3-year event-free survival (28.6% vs 90.5%; P < .001; 25.0% vs 89.5%; P < .001) than those without these mutations. These findings considerably change the mutational landscape of ML-DS, provide new insights into the mechanisms of progression from TAM to ML-DS, and help identify new therapeutic targets and strategies for ML-DS.

The impact of obesity-induced inflammation on clonal hematopoiesis.

PURPOSE OF REVIEW: This review meticulously delves into existing literature and recent findings to elucidate the intricate link between obesity and clonal hematopoiesis of indeterminate potential (CHIP) associated clonal hematopoiesis. It aims to enhance our comprehension of this multifaceted association, offering insights into potential avenues for future research and therapeutic interventions. RECENT FINDINGS: Recent insights reveal that mutations in CHIP-associated genes are not limited to symptomatic patients but are also present in asymptomatic individuals. This section focuses on the impact of obesity-induced inflammation and fatty bone marrow (FBM) on the development of CHIP-associated diseases. Common comorbidities such as obesity, diabetes, and infection, fostering pro-inflammatory environments, play a pivotal role in the acceleration of these pathologies. Our research underscores a notable association between CHIP and an increased waist-to-hip ratio (WHR), emphasizing the link between obesity and myeloid leukemia. Recent studies highlight a strong correlation between obesity and myeloid leukemias in both children and adults, with increased risks and poorer survival outcomes in overweight individuals. SUMMARY: We discuss recent insights into how CHIP-associated pathologies respond to obesity-induced inflammation, offering implications for future studies in the intricate field of clonal hematopoiesis.

Promoter-centred chromatin interactions associated with EVI1 expression in EVI1+3q- myeloid leukaemia cells.

EVI1 expression is associated with poor prognosis in myeloid leukaemia, which can result from Chr.3q alterations that juxtapose enhancers to induce EVI1 expression via long-range chromatin interactions. More often, however, EVI1 expression occurs unrelated to 3q alterations, and it remained unclear if, in these cases, EVI1 expression is similarly caused by aberrant enhancer activation. Here, we report that, in EVI1+3q- myeloid leukaemia cells, the EVI1 promoter interacts via long-range chromatin interactions with promoters of distally located, active genes, rather than with enhancer elements. Unlike in 3q+ cells, EVI1 expression and long-range interactions appear to not depend on CTCF/cohesin, though EVI1+3q- cells utilise an EVI1 promoter-proximal site to enhance its expression that is also involved in CTCF-mediated looping in 3q+ cells. Long-range interactions in 3q- cells connect EVI1 to promoters of multiple genes, whose transcription correlates with EVI1 in EVI1+3q- cell lines, suggesting a shared mechanism of transcriptional regulation. In line with this, CRISPR interference-induced silencing of two of these sites minimally, but consistently reduced EVI1 expression. Together, we provide novel evidence of features associated with EVI1 expression in 3q- leukaemia and consolidate the view that EVI1 in 3q- leukaemia is largely promoter-driven, potentially involving long-distance promoter clustering.

The transcriptome of acute dehydration in myeloid leukemic cells.

Human myeloid leukemia cells (HL-60/S4) exposed to hyperosmotic stress with sucrose undergo dehydration and cell shrinkage. Interphase chromatin and mitotic chromosomes congeal, exhibiting altered phase separation (demixing) of chromatin proteins. To investigate changes in the transcriptome, we exposed HL-60/S4 cells to hyperosmotic sucrose stress (~600 milliOsmolar) for 30 and 60 minutes. We employed RNA-Seq of polyA mRNA to identify genes with increased or decreased transcript levels relative to untreated control cells (i.e., differential gene expression). These genes were examined for over-representation of Gene Ontology (GO) terms.  In stressed cells, multiple GO terms associated with transcription, translation, mitochondrial function and proteosome activity, as well as "replication-dependent histones", were over-represented among genes with increased transcript levels; whereas, genes with decreased transcript levels were over-represented with transcription repressors. The transcriptome profiles of hyperosmotically-stressed cells suggest acquisition of cellular rebuilding, a futile homeostatic response, as these cells are ultimately doomed to a dehydrated death.

Association of HLA-E single nucleotide polymorphisms with human myeloid leukemia.

Single nucleotide polymorphisms (SNPs) of HLA-E are related to the occurrence of many diseases, but their functions remain unclear. In this study, the function of SNPs at HLA-E rs76971248 and rs1264457 on the myeloid leukemia cells was analyzed by a progressive procedure, included genotyping, mRNA transcription, regulatory element, protein expression, and anti-tumor effect. The frequencies of rs76971248 G and rs1264457 G were found higher in myeloid leukemia patients than those in healthy blood donors (p < 0.05). For myeloid leukemia, rs76971248 T was protective, while rs1264457 G was susceptible. We also found that rs76971248 affected HLA-E mRNA transcription and membrane HLA-E (mHLA-E) expression in K562 cells through differently binding to transcription factor HOXA5 (p < 0.0001), while rs1264457 affected mHLA-E expression by changing mRNA transcription and an encoding amino acid (p < 0.01). In contrast, the expression of soluble HLA-E (sHLA-E) was not influenced by both rs1264457 and rs76971248. The higher HLA-E expression was detected among myeloid leukemia patients, and the K562 cells with higher HLA-E molecules played a significant inhibitory effect on the killing activity of NK-92MI cells (p < 0.05). In conclusion, the higher HLA-E expression of myeloid leukemia cells is promoted by rs76971248 G and rs1264457 G, which helps escape from NK-92MI cells' killing.

miR-1202 acts as anti-oncomiR in myeloid leukaemia by down-modulating GATA-1S expression.

Transient abnormal myelopoiesis (TAM) is a Down syndrome-related pre-leukaemic condition characterized by somatic mutations in the haematopoietic transcription factor GATA-1 that result in exclusive production of its shorter isoform (GATA-1S). Given the common hallmark of altered miRNA expression profiles in haematological malignancies and the pro-leukaemic role of GATA-1S, we aimed to search for miRNAs potentially able to modulate the expression of GATA-1 isoforms. Starting from an in silico prediction of miRNA binding sites in the GATA-1 transcript, miR-1202 came into our sight as potential regulator of GATA-1 expression. Expression studies in K562 cells revealed that miR-1202 directly targets GATA-1, negatively regulates its expression, impairs GATA-1S production, reduces cell proliferation, and increases apoptosis sensitivity. Furthermore, data from TAM and myeloid leukaemia patients provided substantial support to our study by showing that miR-1202 down-modulation is accompanied by increased GATA-1 levels, with more marked effects on GATA-1S. These findings indicate that miR-1202 acts as an anti-oncomiR in myeloid cells and may impact leukaemogenesis at least in part by down-modulating GATA-1S levels.

Nucleic acid therapeutics as differentiation agents for myeloid leukemias.

Differentiation therapy has proven to be a success story for patients with acute promyelocytic leukemia. However, the remaining subtypes of acute myeloid leukemia (AML) are treated with cytotoxic chemotherapies that have limited efficacy and a high likelihood of resistance. As differentiation arrest is a hallmark of AML, there is increased interest in developing differentiation-inducing agents to enhance disease-free survival. Here, we provide a comprehensive review of current reports and future avenues of nucleic acid therapeutics for AML, focusing on the use of targeted nucleic acid drugs to promote differentiation. Specifically, we compare and discuss the precision of small interfering RNA, small activating RNA, antisense oligonucleotides, and aptamers to modulate gene expression patterns that drive leukemic cell differentiation. We delve into preclinical and clinical studies that demonstrate the efficacy of nucleic acid-based differentiation therapies to induce leukemic cell maturation and reduce disease burden. By directly influencing the expression of key genes involved in myeloid maturation, nucleic acid therapeutics hold the potential to induce the differentiation of leukemic cells towards a more mature and less aggressive phenotype. Furthermore, we discuss the most critical challenges associated with developing nucleic acid therapeutics for myeloid malignancies. By introducing the progress in the field and identifying future opportunities, we aim to highlight the power of nucleic acid therapeutics in reshaping the landscape of myeloid leukemia treatment.

Pre-analytical parameters associated with unsuccessful karyotyping in myeloid neoplasm: a study of 421 samples

Cytogenetics is essential in myeloid neoplasms (MN) and pre-analytical variables are important for karyotyping. We assessed the relationship between pre-analytical variables (time from collection to sample processing, material type, sample cellularity, and diagnosis) and failures of karyotyping. Bone marrow (BM, n=352) and peripheral blood (PB, n=69) samples were analyzed from acute myeloid leukemia (n=113), myelodysplastic syndromes (n=73), myelodysplastic syndromes/myeloproliferative neoplasms (n=17), myeloproliferative neoplasms (n=137), and other with conclusive diagnosis (n=6), and reactive disorders/no conclusive diagnosis (n=75). The rate of unsuccessful karyotyping was 18.5% and was associated with the use of PB and a low number of nucleated cells (≤7×103/µL) in the sample. High and low cellularity in BM and high and low cellularity in PB samples showed no metaphases in 3.9, 39.7, 41.9, and 84.6% of cases, respectively. Collecting a good BM sample is the key for the success of karyotyping in MN and avoids the use of expensive molecular techniques.

Acute leukemia in early childhood

Acute leukemia in early childhood is biologically and clinically distinct. The particular characteristics of this malignancy diagnosed during the first months of life have provided remarkable insights into the etiology of the disease. The pro-B, CD10 negative immunophenotype is typically found in infant acute leukemia, and the most common genetic alterations are the rearrangements of the MLL gene. In addition, the TEL/AML1 fusion gene is most frequently found in children older than 24 months. A molecular study on a Brazilian cohort (age range 0-23 months) has detected TEL/AML1+ve (N = 9), E2A/PBX1+ve (N = 4), PML/RARA+ve (N = 4), and AML1/ETO+ve (N = 2) cases. Undoubtedly, the great majority of genetic events occurring in these patients arise prenatally. The environmental exposure to damaging agents that give rise to genetic changes prenatally may be accurately determined in infants since the window of exposure is limited and known. Several studies have shown maternal exposures that may give rise to leukemogenic changes. The Brazilian Collaborative Study Group of Infant Acute Leukemia has found that mothers exposed to dipyrone, pesticides and hormones had an increased chance to give birth to babies with infant acute leukemia [OR = 1.48 (95 percentCI = 1.05-2.07), OR = 2.27 (95 percentCI = 1.56-3.31) and OR = 9.08 (95 percentCI = 2.95-27.96)], respectively. This review aims to summarize recent clues that have facilitated the elucidation of the biology of early childhood leukemias, with emphasis on infant acute leukemia in the Brazilian population.

Asociación HLA clase I y leucemia en pacientes mestizos del estado Zulia, Venezuela

Resumen. La asociación entre los Alelos Leucocitarios Humanos (HLA) clase I (A, B, C) y las Leucemias Linfoides Agudas y Mieloides, se determinó mediante la técnica de Reacción en cadena de la polimerasa con iniciadores de secuencia específica (PCR-SSP) en un grupo de 60 pacientes y 30 controles sanos. Los valores obtenidos se expresaron como frecuencias alélicas y haplotipicas. Se utilizaron como métodos estadísticos la prueba de Chi-cuadrado corregida, test de Fisher, riesgo relativo y la fracción etiológica. Se observó una asociación positiva significativa de los alelos HLA-B*39 (RR = 16,184, p = 0,0237) y HLA C*03 (RR =5,0; p = 0,0127) con las Leucemias Mieloides (aguda y crónica). Así mismo se encontraron asociaciones positivas de los haplotipos de 2 loci: HLA-A*02-C*03 (RR = 6,0; p = 0,0153), A*24-C*03 (RR = 16,184; p = 0,0237), B*40-C*03 (RR = 10,706; p = 0,0021) y un haplotipo de 3 loci: HLA-A*02-B*40-C*03 (RR = 8,11; p = 0,0102) con las Leucemias Mieloides. No se evidenció ningún tipo de asociación con la Leucemia Linfoide Aguda. No se observaron asociaciones negativas con ningún tipo de Leucemia.

Abstract. The Human Leukocyte Allele (HLA) Class I (A, B, C) and Acute Lymphoid Leukemia and Myeloid Leukemia association, was determined by polymerase chain reaction - sequence specific primers (PCR-SSP) in 60 patients and 30 healthy controls. The results were reported as allelic frequencies and haplotype. The Chi-square corrected, Fisher’s Test, Relative risk and etiologic fraction were calculated. A significant positive association was showed between HLA-B*39 (RR = 16.184; p = 0.0237) and HLA C*03 (RR =5.0; p = 0.0127) alleles and Myeloid Leukaemia. Positive associations between haplotypes 2 loci: HLA-A*02-C*03 (RR = 6.0; p = 0.0153), A*24-C*03 (RR = 16.184; p = 0.0237), B*40-C*03 (RR = 10.706; p = 0.0021) and haplotype 3 loci: HLA-A*02-B*40-C*03 (RR = 8.11; p = 0.0102) and Myeloid Leukemia were found. No association was evident in Acute Lymphoid Leukemia. No negative association with Leukemias were observed.

Estudio citogenético en médula ósea de 26 casos de leucemia mieloide crónica en pacientes del Hospital San Juan de Dios / Cytogenetic study in Bone Marrow of twenty six cases of Leukemia Myeloid Chronic in patients of the Hospital San Juan de Dios

Se presentan los resultados citogenéticos obtenidos en médula ósea de veintiseis pacientes con leucemia mieloide crónica. Veintiun pacientes fueron tratados, con el medicamento quimioterapéutico convencional, tres con alfa interferón y dos recibieron transplante de médula ósea. Se inicia la importancia de las técnicas citogenéticas en este tipo de paciente y en la valoración de resultados de estos tratamientos.

Estudio de los haplotipos HLA en leucemias / Study of HLA haplotypes in leukemias

Se determinaron las combinaciones alélicas de los genes HLA A y B en 144 pacientes con leucemias; 89 con leucemia linfoide aguda (LLA), 28 con leucemia mieloide aguda (LMA) y 27 con leucemia mieloide crónica (LMC), estraficados fenotípicamente en blancos, negros y mestizos, para evaluar la posible asociación entre los haplotipos HLA y esas enfermedades. Se utilizaron como controles 276 personas aparentemente estratificados también por el color de la piel. Los haplotipos A2-B12, A9-B12 y A2-B35 fueron los más frecuentes en el grupo total de enfermos y en los individuos blancos con LLA. La combinación de las especificidades A2 y B5 mostró un desequilibrio de asociación en el conjunto de enfermos y en los sujetos blancos con LMA

Ph1-negative chronic myelogenous leukemia with clonal chromosome abnormalities

Säo relatados os achados clínicos, hematológicos e citogenéticos de dois pacientes com síndromes mieloproliferativas Ph1-negativas. Um dos pacientes, um menino de 5 anos de idade com leucemia mielóide crônica "juvenil", exibia um cariótipo anormal 45,XO restrito a todas as células da medula óssea, enquanto outros tecidos somáticos tinham padräo normal 46,XY. A segunda paciente era portadora de uma forma atípica de leucemia mielóide crônica, acompanhada de eritrocitose e linfoadenopatia, mostrava um cariótipo 46,XX t(8;13) (p23;q14) em todas as células de medula óssea, baço e gânglios linfáticos, ao passo que eram normais os linfócitos do sangue periférico. Estes achados comprovam a natureza clonal da leucemia mielóide crônica Ph1-negativa, que compreenderia um grupo heterogêneo de doenças näo relacionadas com as formas típicas Ph1-positivas

Alteraçöes citogenética como fatores prognósticos em leucemias mielóides agudas / Cytogenetic abnormalities as prognostic factors in acute myeloide leukemia

O estudo das alteraçoes cromossômica nas leucemias mielóides agudas (LMA) vem-se tornando importante no diagnóstico e na caracterizaçao de subtipos, pois associam-se a características clínicas, morfológicas e imunológicas definidas à resposta a tratamento e à sobrevida. OBJETIVO. O presente trabalho objetiva avaliar a importância relativa das alteraçoes citogenética em portadores de LMA. MATERIAL. Foram estudados, ao diagnóstico, 13 pacientes com LMA e com idade mediana igual a 38 anos. O estudo citogenético foi realizado em material medular.RESULTADOS. Os subtipos FAB M1 e M2 foram o mais freqüentes (61,6 por cento). A análise citogenética mostrou cariótipo anormal em 61,5 por cento dos casos e, dentre estes, apenas 15,3 por cento tinham alteraçoes indicadoras de bom prognóstico [t(l5;17) e t(8;21)]. Na data de avaliaçao do estudo havia três pacientes vivos, dois em remissao completa contínua e um em segunda remissao. A sobrevida mediana global foi de 7 meses. Os pacientes foram divididos em dois grupos: um intitulado "bom prognóstico", que englobou cinco indivíduos com cariótipo normal e dois com as translocaçoes t(l5;17) e t(8;21), e outro, "mau prognóstico", com oito pacientes com alteraçoes cromossômicas desfavoráveis. O grupo "bom prognóstico" teve sobrevida mediana de nove meses, enquanto outro, de 6,2 meses, mas sem diferença estatisticamente significante (p= 0,180084), provavelmente devido ao pequeno número de casos em cada grupo. Entretanto, ao se analisar os casos em separado nota-se que os pacientes com translocaçoes (8;21) e (15;17), tidas como de bom prognóstico, tiveram sobrevidas mais longas. CONCLUSAO. Concluímos que o trabalho evidenciou sobrevida desigual entre os dois grupos, ressaltando a importância da análise citogenética que permite distinguir o paciente que terá evoluçao favorável.

Detecçäo de mRNAs quiméricos BCR/ABL em pacientes portadores de leucemia mielóide crônica, leucemia linfóide aguda/mielóide aguda pela reaçäo de polimerizaçäo em cadeia (PCR) / Detection of BCR/ABL chimeric mRNAs in patients with chronic myeloid leukemia, acute lymphocytic leukemia/ acute myeloid by polimerase chain reaction (PCR)

A leucemia mielóide crônica (LMC) é uma doença caracterizada pela proliferaçao e acumulaçao de células mielóides e seus precursores, induzida pela transformaçao neoplásica de uma célula hematopoética pluripotente, a stem-cell. Esse clone possui uma anormalidade citogenética que se caracteriza pela translocaçao recíproca entre os cromossomos 9 e 22 [t(9;22) (q34;q11)], resultando na formaçao do cromossomo Philadelphia (Ph1). O resultado dessa translocaçao é a justaposiçao do gene BCR com o protooncogene ABL, originando um gene híbrido que codifica uma proteína anormal com atividade tirosina quinase exacerbada. A reaçao de polimerizaçao em cadeia (PCR) baseada na amplificaçao do cDNA híbrido BCR/ABL tem sido considerada um meio altamente sensível e específico para detecçao dessa patologia, em nível molecular. O RNA total foi extraído pelo método de isocianato de guanidina, de leucócitos de sangue periférico e (ou) medula óssea. A primeira fita (cDNA) foi sintetizada utilizando primer complementar à regiao 3' do mRNA quimérico BCR/ABL, transcriptase reversa (Super Script II - Gibco-BRL) segundo sugestao do fabricante. A reaçao de PCR foi realizada em duas etapas: a primeira utilizando primers complementares à regiao BCR e ABL, num total de 25 ciclos com temperatura de 94 graus Celsius, 49 graus Celsius e 72 graus Celsius para desnaturaçao, anelamento e extensao, respectivamente; na segunda etapa foram utilizados primers internos aos primeiros (Nested-PCR) num total de 35 ciclos com temperatura de anelamento de 60 graus Celsius (primers sintetizados pela Genomic - Engenharia Molecular, SP). O controle de todo o procedimento técnico foi realizado pela amplificaçao de uma regiao do gene ABL. A presença de bandas características da LMC foram visualizadas após eletroforese em gel de poliacrilamida 6 por cento e coloraçao por brometo de etídio. O método foi considerado extremamente sensível e rápido para a detecçao da t(9;22) quando comparada com a citogenética convencional.

Seguimiento citogenético e hibridización in situ (FISH) en la enfermedad mínima residual / [Cytogenetic and in situ hybridization (FISH) follow-up in minimal residual disease]

Cytogenetic studies are an important tool for diagnosis, prognosis and minimal residual disease (MRD) follow up specially in oncohematology. Conventional cytogenetics, also known as G banding technique, brings complete information for the spectrum of aneuploidies and structural changes present in malignant cells with a sensitivity of 1-5

depending on the number of metaphases analyzed. FISH technique with a higher sensitivity, 10(-2)-10(-4), attempts to make up for one of the principal limitations of G banding which is the absence of metaphases, thus employing different probes and allowing the study of interphase nucleus. Our patients were monitored with both methodologies during the course of the disease looking for MRD. FISH is the best methodology for quantifying the abnormal clone until we can apply quantitative PCR routinely. It will also be important for the follow up of rearrangements for which we do not yet have molecular determinations available. Our findings are coincident with those of other research groups in the need of evaluating higher series of patients with different pathologies in order to arrive at conclusions allowing us to predict relapse with any certainty.

Simplified method for the analysis of cellular karyotype and phenotype in leukemias

The objective of this study was to develop a simplified method for the simultaneous analysis of cellular karyotype and phenotype which would permit the identification of cell origin. We studied 6 patients with AML, 3 with CML (one of which was in blastic transformation) and one ALL. We used a method in which the suspension of bone marrow cells was incubated in TC 199 medium with colchicine and with hypotonic solution formed from glycerol, NaCl, KCl, CaCl2, MgCl2 and sucrose. The slides were prepared from this cell suspension by cytospin and stained for peroxidase, PAS, esterases and iron. The karyotype was studied by direct method and culture. It was possible to relate the cytogenetic marker with cytochemistry characteristics in the same cell in 3 cases, showing the feasibility of cytochemistry techniques in cytogenetical preparations. The best preparations were found through peroxidase. The presence of iron granules allowed identification of erythroblastic lineage in the combined staining. Mitosis with a marker chromosome of leukemic clone in an AML cell with negative peroxidase probably showed a proliferation of more primitive precursor not sufficiently differentiated to show markers

Fisiopatologia e tratamento da leucemia mielóide crônica: novos conceitos e perspectivas / Physiopathology and treatment of chronic myeloid leukemia: new concepts and perspectives

Descobertas recentas sobre a atividade do gene quimérico BCR/ABL têm auxiliado na elucidaçäo de diversos mecanismos envolvidos na gênese e progressäo da leucemia mielóide crônica (LMC). Apesar de a LMC ser ainda, uma doença incurável para os pacientes que näo podem submeter-se a um transplante alogênico de medula óssea, a sobrevida geral tem aumentado progressivamente, devido especialmente a medidas capazes de prolongar a fase crônica. A técnica de reaçäo da polimerase em cadeia (PCR) para a detecçäo do gene quimérico BCR/ABL tem sido um teste bastante valioso para a identificaçäo de casos Philadelphia negativos que apresentam o rearranjo genético ao nível molecular e para a detecçäo de doença residual mínima, especialmente em indivíduos transplantados. Novas formas de tratamento devem traduzir-se em maior sobrevida nos próximos anos quando utilizadas em estágios precoces da doença: transplante autólogo de células-tronco com células mobilizadas e coletadas após quimioterapia em altas doses, o uso de interferon e a terapia gênica. O interferon já é a droga de escolha para o tratamento da maioria dos pacientes...