Duplicación interna en tándem del gen de la tirosina quinasa 3

Introducción: La reacción en cadena de la polimerasa requiere de varios procedimientos y controles para garantizar la eficacia del estudio. Se debe realizar un control de calidad a la reverso transcripción con el fin de determinar la efectividad del proceso y pasar a la siguiente etapa. Se comprueba con la amplificación de un gen normal, que se interpreta mediante electroforesis. Por dificultades en la importación de reactivos, se propuso sustituir este paso por la identificación del gen de la tirosina quinasa 3, que en la leucemia mieloide aguda puede afectarse por una mutación del tipo duplicación interna en tándem. Objetivo: Evaluar si el biomarcador duplicación interna en tándem del gen de la tirosina quinasa 3 es útil como control del proceso de reverso transcripción dentro de la técnica de reacción en cadena de la polimerasa para los estudios moleculares. Métodos: Se analizaron 235 muestras de aspirado medular de pacientes hematológicos, en el laboratorio de biología molecular del Instituto de Hematología e Inmunología entre junio de 2017 y junio de 2019. Resultados: El 85,53 % presentó solo una banda en la electroforesis capilar, correspondiente al alelo salvaje, y en el resto se observaron la banda normal y la correspondiente al alelo mutado. De esta forma, se comprobó la presencia de ADN complementario en todas las muestras. Conclusiones: La sustitución del control basado en un gen normal, por el biomarcador duplicación interna en tándem del gen de la tirosina quinasa 3 dentro de la técnica de reacción en cadena de la polimerasa, se realiza como parte del protocolo de laboratorio para el estudio de pacientes con enfermedades oncohematológicas.

Introduction: The polymerase chain reaction requires several procedures and controls to ensure the quality of the study. Quality control must be performed on the reverse transcript in order to determine the effectiveness of the process and move to the next step. It is checked with the amplification of a normal gene, which is interpreted by electrophoresis. Due to difficulties in importing reagents, it was proposed to replace this step with the identification of the tyrosine kinase 3 gene, which in acute myeloid leukemia can be affected by an internal tandem duplication type mutation. Objective: To evaluate whether the biomarker internal tandem duplication of the tyrosine kinase 3 gene is useful as a control of the reverse transcription process within the polymerase chain reaction technique for molecular studies. Methods: 235 bone marrow aspirate samples from hematological patients were analyzed in the molecular biology laboratory of the Institute of Hematology and Immunology between June 2017 and June 2019. Results: 85.53% presented only one band in capillary electrophoresis, corresponding to the wild-type allele, and in the rest, the normal band and the band corresponding to the mutated allele were observed. Thus, the presence of cDNA was verified in all samples. Conclusions: The substitution of the control based on a normal gene, by the biomarker internal tandem duplication of the tyrosine kinase 3 gene, within the polymerase chain reaction technique, is possible and, moreover, useful as part of the laboratory protocol for the study of patients with oncohematological diseases.

FLT3L-dependent dendritic cells control tumor immunity by modulating Treg and NK cell homeostasis.

FLT3-L-dependent classical dendritic cells (cDCs) recruit anti-tumor and tumor-protecting lymphocytes. We evaluate cancer growth in mice with low, normal, or high levels of cDCs. Paradoxically, both low or high numbers of cDCs improve survival in mice with melanoma. In low cDC context, tumors are restrained by the adaptive immune system through influx of effector T cells and depletion of Tregs and NK cells. High cDC numbers favor the innate anti-tumor response, with massive recruitment of activated NK cells, despite high Treg infiltration. Anti CTLA-4 but not anti PD-1 therapy synergizes with FLT3-L therapy in the cDCHi but not in the cDCLo context. A combination of cDC boost and Treg depletion dramatically improves survival of tumor-bearing mice. Transcriptomic data confirm the paradoxical effect of cDC levels on survival in several human tumor types. cDCHi-TregLo state in such patients predicts best survival. Modulating cDC numbers via FLT3 signaling may have therapeutic potential in human cancer.

Incidence and clinical significance of FLT3 and nucleophosmin mutation in childhood acute myeloid leukemia in Chile

Abstract Introduction Acute myeloid leukemia (AML) is a heterogeneous disease and approximately one-third of its carriers do not have evident genetic abnormalities. The mutation of specific molecular markers, such as fms-like tyrosine kinase 3 (FTL3) internal tandem duplication (ITD), FLT3 tyrosine kinase domain (TKD) and nucleophosmin (NPM1), are associated with an adverse and favorable prognosis, respectively. Objective The objective was to determine the prevalence of FLT3/ITD and NPM1 in Chilean patients and their association with clinical data and prognosis. Method and Results Two hundred and thirty-two children were studied between 2011 and 2017, the median being 8.6 years (ranging from 1 to 18 months). Acute promyelocytic leukemia (APL) was diagnosed in 29%. The FLT3/ITD-mutated in non-promyelocytic AML was at 10% (14/133) and the FLT3/TKD, at 3.7% (2/54). In APL, it was at 25.4% (16/63). In non-promyelocytic AML, the FLT3/ITD-mutated was associated with a high leucocyte count, the median being 28.5 x mm3 (n= 14) versus 19.4 x mm3 (n= 119), (p= 0.25), in non-mutated cases. In APL, the median was 33.6 x mm3 (n= 15) versus 2.8 x mm3 (n= 47), (p < 0.001). The five-year overall survival (OS) in non-promyelocytic AML with non-mutated and mutated FLT3/ITD were 62.7% and 21.4%, respectively, (p < 0.001); the 5-year event-free survival (EFS) were 79.5% and 50%, respectively, (p < 0.01). The five-year OS in APL with non-mutated and mutated FLT3/ITD was 84.7% and 62.5%, respectively, (p= 0.05); the 5-year EFS was 84.7% and 68.8%, respectively, (p= 0.122). The NPM1 mutation was observed in 3.2% (5/155), all non-promyelocytic AML with the normal karyotype. Conclusion The FLT3/ITD mutation was observed more frequently in APL and associated with a higher white cell count at diagnosis. However, the most important finding was that the FLT3/ITD mutation was associated with a shorter survival in non-promyelocytic AML.

Incidence and clinical significance of FLT3 and nucleophosmin mutation in childhood acute myeloid leukemia in Chile.

INTRODUCTION: Acute myeloid leukemia (AML) is a heterogeneous disease and approximately one-third of its carriers do not have evident genetic abnormalities. The mutation of specific molecular markers, such as fms-like tyrosine kinase 3 (FTL3) internal tandem duplication (ITD), FLT3 tyrosine kinase domain (TKD) and nucleophosmin (NPM1), are associated with an adverse and favorable prognosis, respectively. OBJECTIVE: The objective was to determine the prevalence of FLT3/ITD and NPM1 in Chilean patients and their association with clinical data and prognosis. METHOD AND RESULTS: Two hundred and thirty-two children were studied between 2011 and 2017, the median being 8.6 years (ranging from 1 to 18 months). Acute promyelocytic leukemia (APL) was diagnosed in 29%. The FLT3/ITD-mutated in non-promyelocytic AML was at 10% (14/133) and the FLT3/TKD, at 3.7% (2/54). In APL, it was at 25.4% (16/63). In non-promyelocytic AML, the FLT3/ITD-mutated was associated with a high leucocyte count, the median being 28.5 x mm3 (n = 14) versus 19.4 x mm3 (n = 119), (p = 0.25), in non-mutated cases. In APL, the median was 33.6 x mm3 (n = 15) versus 2.8 x mm3 (n = 47), (p < 0.001). The five-year overall survival (OS) in non-promyelocytic AML with non-mutated and mutated FLT3/ITD were 62.7% and 21.4%, respectively, (p < 0.001); the 5-year event-free survival (EFS) were 79.5% and 50%, respectively, (p < 0.01). The five-year OS in APL with non-mutated and mutated FLT3/ITD was 84.7% and 62.5%, respectively, (p = 0.05); the 5-year EFS was 84.7% and 68.8%, respectively, (p = 0.122). The NPM1 mutation was observed in 3.2% (5/155), all non-promyelocytic AML with the normal karyotype. CONCLUSION: The FLT3/ITD mutation was observed more frequently in APL and associated with a higher white cell count at diagnosis. However, the most important finding was that the FLT3/ITD mutation was associated with a shorter survival in non-promyelocytic AML.

Biomarcador FLT3-ITD: perspectivas y retos / FLT3-ITD biomarker: perspectives and challenges

Introducción: Las mutaciones del gen que codifica para el factor de la tirosina quinasa 3 (FLT3) son de especial importancia en la leucemia mieloide aguda debido a que sirven de guía para la confirmación del diagnóstico, la estimación del pronóstico y la toma de decisiones terapéuticas. Entre las alteraciones más importantes está la duplicación interna en tándem (FLT3-ITD). Objetivo: Exponer los aspectos más relevantes respecto al biomarcador FLT3-ITD en el contexto de la leucemia mieloide aguda. Métodos: Se realizó una búsqueda de artículos científicos actualizados en los idiomas inglés y español, en PubMed, EMBASE, Google Scholar y SciELO. Se seleccionaron artículos publicados en los últimos cinco años. Se revisaron los aspectos más relevantes sobre el biomarcador en el contexto de la leucemia mieloide aguda, su base biológica, el impacto del tamaño de los fragmentos y la carga alélica en la estimación del pronóstico de los pacientes, las nuevas estrategias terapéuticas y los retos en cuanto a los métodos de laboratorio para su diagnóstico. Análisis y síntesis de la información: Más allá de la positividad o no de dicho biomarcador, el tamaño de la duplicación interna en tándem, así como la carga alélica -determinada por la razón alelo mutado/alelo salvaje-, podrían tener un gran impacto en el pronóstico. Sin embargo, persisten diferencias en los criterios para establecer los algoritmos de predicción del riesgo, el punto de corte a utilizar como referencia y el protocolo de laboratorio específico para un estudio más detallado del biomarcador. Conclusiones: La comunidad científica necesita seguir trabajando en el esclarecimiento de la utilidad práctica de estos parámetros, validándolos en series amplias y diversas epidemiológicamente. Se debe determinar el punto de corte exacto para comparar la razón y estandarizar los métodos de laboratorio más adecuados y factibles para su estudio(AU)

ABSTRACT Introduction: Mutations in the tyrosine kinase 3 gene (FLT3) are of special importance in acute myeloid leukemia because they serve as a guide to confirm the diagnosis, estimate the prognosis, and make therapeutic decisions in the patient. Internal tandem duplication (FLT3-ITD) is the most important alteration of this gene. Objective: To present the most relevant aspects regarding the FLT3-ITD biomarker in the context of acute myeloid leukemia. Methods: a search was carried out for updated scientific articles, in English and Spanish, in PubMed, EMBASE, Google Scholar and SciELO. Articles published in the last five years were selected. The most relevant aspects of the biomarker in the context of acute myeloid leukemia, its biological basis, the impact of the size of the fragments and the allelic load in the estimation of the prognosis of the patients, the new therapeutic strategies and the challenges in regarding the laboratory methods for its diagnosis. Information analysis and synthesis: Beyond the biomarker positivity or not, the size of the ITD, as well as the allelic ratio determined by the mutated allele / wild-type allele, could have a great impact on the prognosis of patients. However, differences persist in the criteria for establishing risk prediction algorithms, the cut-off point to be used as a reference, and the specific laboratory protocol for a more detailed study of the biomarker. Conclusions: The scientific community needs to continue working to clarify the practical utility of these parameters, validating them in broad and epidemiologically diverse series. The exact cut-off point should be determined as a reference to compare the relationship and standardize the most suitable and feasible laboratory methods for its study(AU)

Primary splenic histiocytic sarcoma associated with hemophagocytic lymphohistiocytosis: A case report and review of literature of next-generation sequencing involving FLT3, NOTCH2, and KMT2A mutations.

BACKGROUND: Histiocytic sarcoma is a very rare monocyte/macrophage-derived hematopoietic system tumor with a poor prognosis whose diagnosis is pathologically challenging due to its extreme rarity and histological overlap with various mimicking entities in which histiocytes also predominate. CASE: We report the case of a 33-year-old male patient with hemophagocytic lymphohistiocytosis, purpuric syndrome, and significant splenomegaly. The patient underwent splenectomy; subsequent macroscopic examination revealed a spleen weighing 2065 grams with hyperemic red pulp and multiple infarcts at the periphery. The histological and immunohistochemical study established a diagnosis of primary splenic histiocytic sarcoma with frequent hemophagocytosis. Next-generation sequencing demonstrated mutations in FLT3, NOTCH2, and KMT2A, microsatellite stability, and a tumor mutational burden of 2 mut/Mb. The patient's condition deteriorated clinically from the appearance of the first symptoms and he died 6 months later from multi-organ failure. CONCLUSION: Primary splenic histiocytic sarcoma is one of the rarest tumors of the hematopoietic system. We report the first case with mutations in FLT3, NOTCH2, and KMT2A, and associated hemophagocytic lymphohistiocytosis.

Update on drug transporter proteins in acute myeloid leukemia: Pathological implication and clinical setting.

Acute myeloid leukemia (AML) is one of the most common hematological neoplasia causing death worldwide. The long-term overall survival is unsatisfactory due to many factors including older age, genetic heterogeneity and molecular characteristics comprising additional mutations, and resistance to chemotherapeutic drugs. The expression of ABCB1/P-glycoprotein, ABCC1/MRP1, ABCG2/BCRP and LRP transporter proteins is considered the major reason for multidrug resistance (MDR) in AML, however conflicting data have been reported. Here, we review the main issues about drug transporter proteins in AML clinical scenario, and highlight the clinicopathological significance of MDR phenotype associated with ABCB1 polymorphisms and FLT3 mutation.

The Role of FLT3-ITD and CCAAT-Enhancer-Binding Protein α Mutations on Prognosis of Acute Lymphoblastic Leukaemia in Turkish Patients

ABSTRACT Background: Acute lymphoblastic leukaemia (ALL) is the most common malignancy in childhood. Although some prognostic factors have been defined to date, the estimation of prognosis is currently not perfect. Previous studies had shown an association of FLT3 with poor prognosis and CCAAT-enhancer-binding protein α (CEBPA) mutation with the development of acute myeloid leukaemia (AML). Here, we aimed to evaluate the prognostic value of FLT3-ITD and CEBPA mutations in ALL. Methods: Sixty-one patients with ALL were included in the study. The patients were divided into three risk groups according to BFM risk classification. All of the patients were examined for FLT3-ITD mutations and 45 of them for CEBPA mutations. Mutation positive and negative patients were compared in terms of their risk groups, translocations and cell lineage. The clinical courses of the patients were appraised. Results: FLT3-ITD mutation was detected in 3 of the 61 patients, and CEBPA mutations were detected in 11 of the 45 patients. The incidence of established prognostic indicators including BFM risk classification, t(9; 22); BCR-ABL, t(1; 19); E2A-PBX1, t(12; 21); TEL-AML1, t(4; 11); MLL-AF4 were similar between FLT3-ITD and CEBPA positive and negative patients. A patient with an FLT3-ITD mutation was very susceptible to pancytopenia after maintenance treatment and two other patients with FLT3-ITD mutations were more prone to febrile neutropenia. Conclusion: Our results suggested that CEBPA or FLT3-ITD mutations might not be related to ALL prognosis in the sampled Turkish patients. However, FLT3-ITD mutation might have an influence on the response of bone marrow to chemotherapy.

Sunitinib-Containing Carborane Pharmacophore with the Ability to Inhibit Tyrosine Kinases Receptors FLT3, KIT and PDGFR-ß, Exhibits Powerful In Vivo Anti-Glioblastoma Activity.

Malignant gliomas are the most common malignant and aggressive primary brain tumors in adults, the prognosis being-especially for glioblastomas-extremely poor. There are no effective treatments yet. However, tyrosine kinase receptor (TKR) inhibitors and boron neutron capture therapy (BNCT), together, have been proposed as future therapeutic strategies. In this sense in our ongoing project of developing new anti-glioblastoma drugs, we identified a sunitinib-carborane hybrid agent, 1, with both in vitro selective cytotoxicity and excellent BNCT-behavior. Consequently, we studied the ability of compound 1 to inhibit TKRs, its promotion of cellular death processes, and its effects on the cell cycle. Moreover, we analyzed some relevant drug-like properties of 1, i.e., mutagenicity and ability to cross the blood-brain barrier. These results encouraged us to perform an in vivo anti-glioblastoma proof of concept assay. It turned out to be a selective FLT3, KIT, and PDGFR-ß inhibitor and increased the apoptotic glioma-cell numbers and arrested sub-G1-phase cell cycle. Its in vivo activity in immunosuppressed mice bearing U87 MG human glioblastoma evidenced excellent anti-tumor behavior.

Profiling FLT3 Mutations in Mexican Acute Myeloid Leukemia Pediatric Patients: Impact on Overall Survival.

Background: Acute myeloid leukemia (AML) is the second most frequent leukemia in childhood. The FLT3 gene participates in hematopoietic stem cell proliferation. FLT3 mutations are recurrent in AML and influence prognosis. In Mexican pediatric AML patients, FLT3 mutational profile, and their clinical impact have not been evaluated. Aim of the study: This study aimed to identify the profile of FLT3 mutations in pediatric patients with de novo AML and to assess their possible influence on overall survival (OS) and other clinical features. Methods: Massive parallel target sequencing of FLT3 was performed in 80 patients. Results: FLT3 mutations [internal tandem duplication (ITD) or tyrosine kinase domain (TKD)] were identified in 24% of them. OS was significantly lower in FLT3 POS cases than in FLT3 NEG (p = 0.03). The average OS for FLT3 POS was 1.2 vs. 2.2 years in FLT3 NEG. There were no significant differences in the children's sex, age, percentage of blasts in bone marrow aspirate, or white blood cell count in peripheral blood at diagnosis between both groups. No differences were identified stratifying by the mutational load (high > 0.4) or type of mutation. The negative effect of FLT3 mutations was also observed in patients with acute promyelocytic leukemia (APL). Conclusions: FLT3 mutational profile is described in Mexican pediatric AML patients for the first time. Mutated FLT3 negatively impacts the outcome of AML patients, even considering the APL group. The clinical benefit from treatment with tyrosine kinase inhibitors in the FLT3 POS pediatric patients needs to be assessed in clinical trials. FLT3 testing may contribute to better risk stratification in our pediatric AML patients.

Identificación de alteraciones moleculares en pacientes Venezolanos con diagnóstico de leucemia linfoblástica aguda / Identification of molecular alterations in Venezuelan patients with acute lymphoblastic leukemia diagnosis

La Leucemia Linfoblástica Aguda (LLA) es la neoplasia más frecuente en edad pediátrica. En los últimos años, entre el 15 y 20% de los pacientes fracasan en el tratamiento. Conocimientos en citogenética y biología molecular repercuten de manera importante en la determinación del pronóstico y del esquema de tratamiento adecuado. En Venezuela existe un conocimiento limitado en cuanto a la genética molecular de esta alteración onco-hematológica. El objetivo del trabajo fue evaluar las alteraciones genéticas más frecuentes en pacientes venezolanos con diagnóstico clínico de leucemia linfoblástica aguda. Se realizó un estudio transversal, descriptivo y prospectivo de 2006 a 2014, en el que se evaluaron las translocaciones ETV6/RUNX1, MLL/AF4, TCF3/PBX1, BCR/ABL1, así como las mutaciones en los genes PAX5 y FLT3 mediante el uso de diferentes tipos de PCR. Ciento treinta pacientes con diagnóstico clínico de leucemia linfocítica aguda fueron incluidos en el estudio. Se identificaron alteraciones moleculares en 56 pacientes (43,1%), en los que observamos la presencia de una o varias alteraciones en conjunción en un mismo paciente. Las alteraciones identificadas fueron t(12;21) (11,5%), t(4;11) (8,5%), t(1;19) (10%), t(9;22) (20,8%), ITD-FLT3 (14,8%), mutación P80S (4,2%) y S77del (4,2%) en el gen PAX5. La prevalencia de BCR/ ABL, es una de las más altas que ha sido descrita hasta ahora en casos de LLA donde la mayor parte de la población está conformada por pacientes pediátricos. Estos resultados representan el primer estudio molecular de la LLA en Venezuela, sentando las bases para el diagnóstico y seguimiento de la enfermedad en su población.

Acute Lymphoblastic Leukemia (ALL) is the most common neoplasm in pediatric age. In recent years, between 15 and 20% of patients failed in their treatments. Knowledge on cytogenetics and molecular biology has an important impact on the determination of the prognosis and the appropriate treatment scheme. In Venezuela there is limited knowledge regarding the molecular genetics of this onco-hematological alteration. The aim of this work was to evaluate the most frequent genetic alterations in Venezuelan patients with a clinical diagnosis of acute lymphoblastic leukemia. A cross-sectional, descriptive and prospective study was carried out from 2006 to 2014, in which the translocations ETV6/RUNX1, MLL/AF4, TCF3/PBX1, BCR/ABL1, as well as mutations in the PAX5 and FLT3 genes were evaluated through the use of different types of PCR. One hundred and thirty patients with a clinical diagnosis of acute lymphocytic leukemia were included in the study. Molecular alterations were identified in 56 patients (43.1%), in which we observed the presence of one or several alterations in conjunction in the same patient. The alterations identified were t(12; 21) (11.5%), t(4; 11) (8.5%), t(1; 19) (10%), t(9; 22) (20.8%), ITD-FLT3 (14.8%), P80S mutation (4.2%) and S77del (4.2%) in the PAX5 gene. The prevalence of BCR/ABL is one of the highest described so far in cases of ALL where most of the population is made up of pediatric patients. These results represent the first molecular study of ALL in Venezuela, laying the foundations for the diagnosis and monitoring of the disease in its population.

Theoretical Studies Aimed at Finding FLT3 Inhibitors and a Promising Compound and Molecular Pattern with Dual Aurora B/FLT3 Activity.

FLT3 and dual Aurora B/FLT3 inhibitors have shown relevance in the search for promising new anticancer compounds, mainly for acute myeloid leukemia (AML). This study was designed to investigate the interactions between human FLT3 in the kinase domain with several indolin-2-one derivatives, structurally similar to Sunitinib. Molegro Virtual Docker (MVD) software was utilized in docking analyses. The predicted model of the training group, considering nineteen amino acid residues, performed in Chemoface, achieved an R2 of 0.82, suggesting that the binding conformations of the ligands with FLT3 are reasonable, and the data can be used to predict the interaction energy of other FLT3 inhibitors with similar molecular patterns. The MolDock Score for energy for compound 1 showed more stable interaction energy (-233.25 kcal mol-1) than the other inhibitors studied, while Sunitinib presented as one of the least stable (-160.94 kcal mol-1). Compounds IAF70, IAF72, IAF75, IAF80, IAF84, and IAF88 can be highlighted as promising derivatives for synthesis and biological evaluation against FLT3. Furthermore, IAF79 can be considered to be a promising dual Aurora B/FLT3 inhibitor, and its molecular pattern can be exploited synthetically to search for new indolin-2-one derivatives that may become drugs used in the treatment of cancers, including AML.

The Impact of Flt3 Gene Mutations in Acute Promyelocytic Leukemia: A Meta-Analysis.

The association of FLT3 mutations with white blood cell (WBC) counts at diagnosis and early death was studied in patients with acute promyelocytic leukemia (APL). Publications indexed in databases of biomedical literature were analyzed. Potential publication bias was evaluated by analyzing the standard error in funnel plots using the estimated relative risk (RR). Mixed-effect models were used to obtain the consolidated RR. All analyses were conducted using the R statistical software package. We used 24 publications in the final meta-analysis. Of 1005 males and 1376 females included in these 24 publications, 645 had FLT3-ITD (internal tandem duplication) mutations. Information on FLT3-D835 mutations was available in 10 publications for 175 patients. Concurrent occurrence of the two mutations was rare. WBC count at diagnosis was ≥10 × 109/L in 351 patients. For patients with the FLT3-ITD mutation, RR was 0.59 for overall survival (OS) and 1.62 for death during induction. For those with FLT3-D835 mutations, the RR was 0.50 for OS and 1.77 for death during induction. RR for WBC count ≥10 × 109/L was 3.29 and 1.48 for patients with FLT3-ITD and FLT3-D835, respectively. APL patients with FLT3-ITD or FLT3-D835 are more likely to present with elevated WBC counts and poorer prognosis than those without these mutations.

Genetic alterations of triple negative breast cancer (TNBC) in women from Northeastern Mexico.

Triple negative breast cancer (TNBC) is a subtype of breast cancer of heterogeneous nature that is negative for estrogen receptor (ER), progesterone receptor (PR) and growth factor human epidermal 2 (HER2) following immunohistochemical analysis. TNBC is frequently characterized by relapse and reduced survival. To date, there is no targeted therapy for this type of cancer. Chemotherapy, radiotherapy, and surgery remain as the standard treatments options. The lack of a target therapy and the heterogeneity of TNBC highlight the need to seek new therapeutic options. In this study, fresh tissue samples of TNBC were analyzed with a panel of 48 driver genes (212 amplicons) that are likely to be therapeutic targets. We found intron variants, missense, stop gained and splicing variants in TP53, PIK3CA and FLT3 genes. Interestingly, all the analyzed samples had at least two variants in the TP53 gene, one being a drug response variant, rs1042522, found in 94% of our samples. We also found seven additional variants not previously reported in the TP53 gene, to the best of our knowledge, with probable deleterious characteristics of the tumor suppressor gene. We found four genetic variants in the PIK3CA gene, including two missense variants. The rs2491231 variant in the FLT3 gene was identified in 84% (16/19) of the samples, which not yet reported for TNBC, to the best of our knowledge. In conclusion, genetic variants in TP53 were found in all TNBC tumors, with rs1042522 being the most frequent (94% of TNBC biopsies), which had not been previously reported in TNBC. Also, we found two missense variants in the PIK3CA gene. These results justify the validation of these genetic variants in a large cohort, as well as the extensive study of their impact on the prognosis and therapy management of TBNC.

Treatment of Acute Myeloid Leukemia with the FLT3 Gene Mutation.

In acute myeloid leukemia (AML), mutations of the Fms-like tyrosine kinase 3 receptor (FLT3) and its overexpression are related with hyperleukocytosis, higher risk of relapse, and decrease of both disease-free survival and overall survival. It has been suggested that this phenomenon confers proliferative and survival advantages to the malignant blast cells. As a consequence, it is an attractive therapeutic target. As the best treatment strategy for mutated FLT3 AML remains to be defined, the addition of FLT3 inhibitor drugs to chemotherapy or to the bone marrow transplant approach has become a growing strategy. With encouraging results, this combination seems to be an attractive option. Relevant data regarding the current treatment trends on mutated FLT3 AML is reviewed here.

Analysis of the presence of FLT3 gene mutation and association with prognostic factors in adult and pediatric acute leukemia patients

ABSTRACT When the FLT3 gene is mutated, it originates a modified receptor with structural changes, which give survival advantage and malignant hematopoietic cell proliferation. Thus, the presence of mutations in this gene is considered an unfavorable prognostic factor. A total of 85 consecutive samples of newly diagnosed untreated patients with AL were included in the study after they provided their informed consent. FLT3 gene mutations were detected by PCR. For the pediatric group, a positive correlation was observed between WBC count and the presence of FLT3-ITD in patients with AML and ALL. Furthermore, children with AML who had the FLT3-ITD mutation showed a tendency to express CD34 in blast cells. In the adult group, the AML patients with FLT3-ITD who expressed CD34 in blast cells had a tendency to worse progression. The present data indicate no association between the prognostic factors evaluated and FLT3 gene mutations in adult with AL. Yet, the presence of FLT3-ITD mutation was significantly related with WBC count in the pediatric group. These findings demonstrate that FLT3 gene mutations can be considered as independent poor prognostic factors.

Estudio del ratio de FLT3-ITD como factor pronóstico en leucemias agudas mieloides: primeros casos estudiados en Uruguay

Introducción: en los últimos años ha existido un avance significativo en el conocimiento biológico de la leucemia aguda mieloide (LAM) que se ha traducido en que el tratamiento de los pacientes afectados se realice guiado por el perfil citogenético y molecular. Las duplicaciones internas en tándem del gen FLT3 (FLT3-ITD) representan las mutaciones más frecuentes en LAM y confieren un mal pronóstico en pacientes con riesgo citogenético intermedio. Se ha reportado que la presencia de un ratio FLT3-ITD elevado (relación entre cantidad de alelo portador de ITD y de alelo salvaje) confiere un mayor pronóstico adverso. Objetivo: estandarizar una técnica, no disponible en Uruguay, para determinar el ratio de FLT3-ITD en pacientes portadores de LAM de riesgo citogenético intermedio. Discutir los primeros casos de LAM FLT3+ a los que se realizó el ratio. Material y método: para la detección de FLT3-ITD se amplificó un fragmento correspondiente a los exones 14 y 15 del gen en muestras de médula ósea al debut de la enfermedad. En los casos positivos se determinó el ratio de FLT3-ITD mediante análisis de fragmentos por electroforesis capilar. Resultados: en este trabajo mostramos la estandarización de un método para la determinación del ratio de FLT3-ITD y los primeros casos analizados en nuestro país. Se estudiaron 12 pacientes y se detectó la presencia de FLT3-ITD en tres de ellos. El ratio de FLT3-ITD encontrado fue en dos casos menor a 0,8 y en un caso mayor o igual a 0,8. Conclusiones: disponemos de una técnica de determinación del ratio de FLT3-ITD con importante valor pronóstico para pacientes portadores de LAM.(AU)

Abstract Introduction: In recent years, significant progress has been made in the biological knowledge of acute myeloid leukemia (AML), which has been reflected on treatment of affected patients being guided by cytogenetics and molecular profiling. FLT3 internal tandem duplications (FLT3/ITDs) represent the most frequent mutations in AML and confer a bad prognosis in patients with intermediate cytogenetic risk. It has been reported that the presence of a high FLT3-ITD ratio (relationship between number of ITD carrier allele and wild type allele). Objective: To standardize a technique, still not available in Uruguay, to determine the FLT3-ITD ratio in patients carriers of AML of intermediate cytogenetic risk. To discuss the first cases of AML FLT3+ who underwent ratio analysis. Methods: In order to identify FLT3-ITD, the fragment corresponding to exons 14 and 15 of the gene was amplified in bone marrow samples upon debut of the disease. In the cases it was positive, the FLT3-ITD ratio was determined by the analysis of fragments with capillary electrophoresis. Results: This study presented the standardization of a method to determine the FLT3-ITD ratio and the first cases analysed in our country. Twelve patients were studied and the presence of FLT3-ITD was detected in three of them. In two cases, the FLT3-ITD ratio found was below 0.8 and in one case it was greater than or equal to 0.8. Conclusions: We have a technique to determine the FLT3-ITD ratio with an important prognostic value for patients carriers of AML.(AU)

Resumo Introdução: nos últimos anos observou-se um avanço significativo do conhecimento biológico da leucemia aguda mieloide (LAM) que fez com que o tratamento destes pacientes seja orientado por seus perfis citogenético e molecular. As duplicações internas no tandem do gen FLT3 (FLT3-ITD) são as mutações mais frequentes na LAM e conferem um mal prognóstico em pacientes com risco citogenético intermediário. Foi descrito que uma proporção de FLT3-ITD elevada (relação entre a quantidade do alelo portador de ITD e do alelo selvagem) está vinculada com um maior prognóstico adverso. Objetivo: padronizar uma técnica, não disponível no Uruguai, para determinar a proporção de FLT3-ITD em pacientes portadores de LAM com risco citogenético intermediário. Discutir os primeiros casos de LAM FLT3+ cuja proporção foi calculada. Materiais e métodos: para a detecção de FLT3-ITD, foi realizada a ampliação de um fragmento correspondente aos exons 14 e 15 do gen em amostras de medula óssea no inicio da doença. Nos casos positivos, a proporção de FLT3-ITD foi determinada usando análise de fragmentos por eletroforese capilar. Resultados: neste trabalho mostramos a padronização de um método para a determinação da proporção de FLT3-ITD e os primeiros casos estudados no nosso país. Foram estudados 12 pacientes e a presença de FLT3-ITD foi determinada em 3. Em dois casos a proporção de FLT3-ITD era menor que 0,8 e em 1 caso maior ou igual a 0,8. Conclusões: contamos com uma técnica de determinação da proporção de FLT3-ITD com importante valor prognóstico para pacientes portadores de LAM.(AU)

Estudio del ratio de FLT3-ITD como factor pronóstico en leucemias agudas mieloides: primeros casos estudiados en Uruguay / Analysis of the FLT3-ITD ratio as a prognostic factor in acute myeloid leukemias. First cases studied in Uruguay

Introducción: en los últimos años ha existido un avance significativo en el conocimiento biológico de la leucemia aguda mieloide (LAM) que se ha traducido en que el tratamiento de los pacientes afectados se realice guiado por el perfil citogenético y molecular. Las duplicaciones internas en tándem del gen FLT3 (FLT3-ITD) representan las mutaciones más frecuentes en LAM y confieren un mal pronóstico en pacientes con riesgo citogenético intermedio. Se ha reportado que la presencia de un ratio FLT3-ITD elevado (relación entre cantidad de alelo portador de ITD y de alelo salvaje) confiere un mayor pronóstico adverso. Objetivo: estandarizar una técnica, no disponible en Uruguay, para determinar el ratio de FLT3-ITD en pacientes portadores de LAM de riesgo citogenético intermedio. Discutir los primeros casos de LAM FLT3+ a los que se realizó el ratio. Material y método: para la detección de FLT3-ITD se amplificó un fragmento correspondiente a los exones 14 y 15 del gen en muestras de médula ósea al debut de la enfermedad. En los casos positivos se determinó el ratio de FLT3-ITD mediante análisis de fragmentos por electroforesis capilar. Resultados: en este trabajo mostramos la estandarización de un método para la determinación del ratio de FLT3-ITD y los primeros casos analizados en nuestro país. Se estudiaron 12 pacientes y se detectó la presencia de FLT3-ITD en tres de ellos. El ratio de FLT3-ITD encontrado fue en dos casos menor a 0,8 y en un caso mayor o igual a 0,8. Conclusiones: disponemos de una técnica de determinación del ratio de FLT3-ITD con importante valor pronóstico para pacientes portadores de LAM.

Abstract Introduction: In recent years, significant progress has been made in the biological knowledge of acute myeloid leukemia (AML), which has been reflected on treatment of affected patients being guided by cytogenetics and molecular profiling. FLT3 internal tandem duplications (FLT3/ITDs) represent the most frequent mutations in AML and confer a bad prognosis in patients with intermediate cytogenetic risk. It has been reported that the presence of a high FLT3-ITD ratio (relationship between number of ITD carrier allele and wild type allele). Objective: To standardize a technique, still not available in Uruguay, to determine the FLT3-ITD ratio in patients carriers of AML of intermediate cytogenetic risk. To discuss the first cases of AML FLT3+ who underwent ratio analysis. Methods: In order to identify FLT3-ITD, the fragment corresponding to exons 14 and 15 of the gene was amplified in bone marrow samples upon debut of the disease. In the cases it was positive, the FLT3-ITD ratio was determined by the analysis of fragments with capillary electrophoresis. Results: This study presented the standardization of a method to determine the FLT3-ITD ratio and the first cases analysed in our country. Twelve patients were studied and the presence of FLT3-ITD was detected in three of them. In two cases, the FLT3-ITD ratio found was below 0.8 and in one case it was greater than or equal to 0.8. Conclusions: We have a technique to determine the FLT3-ITD ratio with an important prognostic value for patients carriers of AML.

Resumo Introdução: nos últimos anos observou-se um avanço significativo do conhecimento biológico da leucemia aguda mieloide (LAM) que fez com que o tratamento destes pacientes seja orientado por seus perfis citogenético e molecular. As duplicações internas no tandem do gen FLT3 (FLT3-ITD) são as mutações mais frequentes na LAM e conferem um mal prognóstico em pacientes com risco citogenético intermediário. Foi descrito que uma proporção de FLT3-ITD elevada (relação entre a quantidade do alelo portador de ITD e do alelo selvagem) está vinculada com um maior prognóstico adverso. Objetivo: padronizar uma técnica, não disponível no Uruguai, para determinar a proporção de FLT3-ITD em pacientes portadores de LAM com risco citogenético intermediário. Discutir os primeiros casos de LAM FLT3+ cuja proporção foi calculada. Materiais e métodos: para a detecção de FLT3-ITD, foi realizada a ampliação de um fragmento correspondente aos exons 14 e 15 do gen em amostras de medula óssea no inicio da doença. Nos casos positivos, a proporção de FLT3-ITD foi determinada usando análise de fragmentos por eletroforese capilar. Resultados: neste trabalho mostramos a padronização de um método para a determinação da proporção de FLT3-ITD e os primeiros casos estudados no nosso país. Foram estudados 12 pacientes e a presença de FLT3-ITD foi determinada em 3. Em dois casos a proporção de FLT3-ITD era menor que 0,8 e em 1 caso maior ou igual a 0,8. Conclusões: contamos com uma técnica de determinação da proporção de FLT3-ITD com importante valor prognóstico para pacientes portadores de LAM.