Recomendaciones metodológicas para el monitoreo molecular BCR-ABL1 en pacientes con leucemia mieloide crónica por pcr cuantitativa en tiempo real / Guidelines for molecular monitoring of BCR-ABL1 in chronic myeloid leukemia patients by RT-qPCR

Actualmente las guías clínicas para el manejo de pacientes con leucemia mieloide crónica incluyen el monitoreo molecular de BCR-ABL1 por PCR cuantitativa en tiempo real; esta metodología permite definir la respuesta molecular. A pesar de la probada importancia pronóstica de la respuesta molecular, en muchos casos no se tiene en cuenta que la PCR cuantitativa puede producir datos muy variables, que pueden afectar la validez de los resultados, y hacer difícil la comparación entre diferentes laboratorios. Por lo tanto, para un manejo clínico óptimo, es absolutamente necesaria la estandarización de las metodologías de medición de BCR-ABL1. La estrategia para obtener valores de BCR-ABL1 comparables consiste en la adopción de la escala internacional. La conversión a la escala internacional se logra mediante la aplicación de un factor de conversión específico para cada laboratorio; este factor de conversión se puede obtener mediante el uso de calibradores secundarios validados, que hoy se producen en Argentina, en el marco del programa nacional de armonización. Por otra parte, con el objetivo de mitigar las diferencias entre laboratorios y facilitar criterios uniformes en la interpretación de los resultados y presentación de los informes, decidimos preparar estas guías de laboratorio. Esto permitirá además a los laboratorios poder evaluar su calidad de trabajo, tarea muy importante, en particular para aquellos centros más aislados, que no tienen fácil acceso a costosos kits comerciales o programas internacionales de intercambio de muestras.

Current clinical guidelines for managing chronic myeloid leukemia include molecular monitoring of BCR-ABL1 transcript quantitative reverse-transcription PCR. Despite the proven prognostic significance of molecular response, it is not widely appreciated that quantitative reverse-transcription PCR potentially produces highly variable data, which may affect the validity of results, making comparability between different laboratories difficult. Therefore, standardized reporting of BCR-ABL1 measurements is needed for optimal clinical management. An approach to achieve comparable BCR-ABL1 values is the use of an international reporting scale. Conversion to the international scale is achieved by the application of laboratory specific conversion factor that is obtained by using validated secondary reference calibrators. Moreover, with the aim to mitigate the interlaboratory imprecision of quantitative BCR-ABL1 measurements and to facilitate local laboratory results interpretation and reporting, we decide to prepare laboratory guidelines that will further facilitate interlaboratory comparative studies and independent quality-assessment programs, which are of paramount importance for worldwide standardization of BCR-ABL1 monitoring results, in particular for those most isolated laboratories, with not easy access to commercial kits or sample interchange programs.

Supervivencia a largo plazo de pacientes con síndromes mieloproliferativos crónicos filadelfia negativos y su relación con variables clínico-patológicas

RESUMEN: Los sindromes mieloproliferativos crónicos (SMP) Filadelfia negativosclásicos incluyen tres neoplasias hematológicas caracterizadas por exceso deproliferación de progenitores del linaje mieloide: policitemia vera (PV),trombocitemia esencial (TE) y mielofibrosis primaria (MFP).En el presente estudio multicéntrico y prospectivo se incluyeron 102pacientes con diagnóstico de SMP con el objetivo de estudiar variables clínicas ypatológicas asociadas con supervivencia a largo plazo. Al momento de la inclusiónse estudió la presencia de la mutación V617F del gen JAK2 y otras mutaciones delgen JAK2. En una mediana de seguimiento de 11,5 años, se registraron eventostrombóticos, hemorrágicos, progresión hematológica y otros tumores primarios.Los resultados obtenidos demostraron que los pacientes positivos para lamutación JAK2V617F tuvieron mayor edad, mayor prevalencia de hipertensiónarterial, mayor valor de glóbulos blancos y fosfatasa alcalina leucocitaria. Lamutación se asoció a mayor riesgo de trombosis y ello se trasladó a unasupervivencia libre de eventos trombóticos inferior para los pacientes JAK2V617Fpositivos. La incidencia de transformación fibrótica a 10 años para los pacientes conPV y TE fue de 5,9%. La incidencia de progresión a mielodisplasia y leucemia agudapara todos los pacientes fue de 2,5% a 10 años. Para todos los pacientes la edadmayor o igual a 60 años se asoció a inferior supervivencia global y en pacientes conPV también la trombosis arterial. Se demostró que las causas de muerte de estospacientes no sólo estuvieron relacionadas a la progresión de la malignidad primariasino también a causas no malignas (eventos cardiovasculares) o malignidadessecundarias no hematológicas.En conclusión, los SMP Filadelfia negativos son patologías complejas deevolución variable. Los datos obtenidos en este trabajo y las asociacionesencontradas permitirán mejorar las estrategias de seguimiento y tratamiento deestos pacientes.

SUMMARY: Philadelphia chromosome-negative chronic myeloproliferative neoplasms(MPN) are hematological malignancies characterized by excessive proliferation ofmyeloid progenitor cells. The three classical entities are: polycythemia vera (PV),essential thrombocythemia (ET) and primary mielofibrosis (PMF).In this multicenter and prospective study 102 patients with diagnosis ofclassical MPN were included. The purpose of this study was to analyze the impact ofclinical and pathological variable on long-term survival. At inclusion, JAK2V617F andothers JAK2 mutations were studied in all patients. In a median of follow-up of 11.5years, vascular events, hematological progression and others primary tumors wereregistered.The results showed that JAK2V617F mutation positive patients were olderand had a higher rate of arterial hypertension, white cell counts, and alkalinephosphatase score. JAK2V61F was associated with a higher risk of thrombosis and alower thrombotic event free survival. The 10-year cumulative incidence of fibrotictransformation for patients with PV and ET was 5.9% and the 10-year cumulativeincidence of myelodysplasia and leukemia transformation was 2.5%. For all patients,age equal or more than 60 years old was associated with inferior overall survival; inpatients with PV also the arterial thrombosis affected the overall survival. Causes ofdeath of this cohort were related not only to the progression of hematologicalmalignancy but also to vascular events and others primary tumors.In conclusion MPN are complex diseases with variable clinical outcome. Thepresent study offers important insights about the natural history of this group ofmalignancies and provides information for developing treatment and follow-upstrategies.

Circulating endothelial cells are increased in chronic myeloid leukemia blast crisis

We measured circulating endothelial precursor cells (EPCs), activated circulating endothelial cells (aCECs), and mature circulating endothelial cells (mCECs) using four-color multiparametric flow cytometry in the peripheral blood of 84 chronic myeloid leukemia (CML) patients and 65 healthy controls; and vascular endothelial growth factor (VEGF) by quantitative real-time PCR in 50 CML patients and 32 healthy controls. Because of an increase in mCECs, the median percentage of CECs in CML blast crisis (0.0146%) was significantly higher than in healthy subjects (0.0059%, P<0.01) and in the accelerated phase (0.0059%, P=0.01). There were no significant differences in the percentages of CECs in chronic- or active-phase patients and healthy subjects (P>0.05). In addition, VEGF gene expression was significantly higher in all phases of CML: 0.245 in blast crisis, 0.320 in the active phase, and 0.330 in chronic phase patients than it was in healthy subjects (0.145). In conclusion, CML in blast crisis had increased levels of CECs and VEGF gene expression, which may serve as markers of disease progression and may become targets for the management of CML.

Recuperación temprana de linfocitos como factor pronóstico en el trasplante hematopoyético / Early lymphocyte recovery as a prognostic factor after hematopoietic stem cell transplantation

Introducción: la recuperación temprana de linfocitos es un factor pronóstico que está relacionado con una mayor supervivencia libre de eventos y supervivencia global en pacientes sometidos a trasplante hematopoyético. Objetivo: determinar el valor pronóstico del recuento absoluto de linfocitos (RAL). Métodos: se realizó un estudio observacional analítico, transversal, ambispectivo, en pacientes pediátricos con hemopatías malignas trasplantados en el Instituto de Hematología e Inmunología de La Habana, Cuba, entre 1986 y 2008. Se estudiaron 36 pacientes: 15 con leucemia linfoide aguda, 13 con leucemia mieloide aguda, 6 con leucemia mieloide crónica y 2 con linfoma no hodgkiniano. Veintitrés trasplantes fueron autólogos y 13 alogénicos; 22 de médula ósea y 14 de sangre periférica. Resultados : de los trasplantes antólogos, el 60,9 por ciento alcanzó un RAL el día + 15 (RAL-15) = 500 x mm3, mientras en los alogénicos este se alcanzó en el 53,8 por ciento. La sangre periférica tuvo un RAL-15 mayor que la médula ósea y se obtuvo en el 78,6 por ciento y el 45,4 por ciento de los enfermos, respectivamente (p = 0.049). Los factores pronósticos asociados a una peor supervivencia global fueron la sepsis (p <0.001), el RAL-15 < 500 x mm3 ( p= 0.001) y la recaída (p = 0.03). Las curvas de Kapplan-Meier mostraron una mejor supervivencia global y libre de eventos a los cinco años, en los pacientes con RAL-15 = 500 x mm3 (85 por ciento vs 15 por ciento; p <0.001). Conclusiones: el RAL-15 = 500 x mm3 es una herramienta simple y útil para predecir un mejor resultado en pacientes pediátricos sometidos a trasplante hematopoyético

Introduction: early lymphocyte recovery is a prognostic factor related to a higher event-free survival and overall survival in patients who have received hematopoietic transplantation. Objective: eo determine the prognostic value of absolute lymphocyte count (ALC). Method: a study in pediatric patients with hematological malignancies transplanted at the Institute of Hematology and Immunology from 1986 to 2011 was performed. The study group included 36 patients: 15 with acute lymphoid leukemia, 13 with acute myeloid leukemia, 6 with chronic myeloid leukemia and 2 with non Hodgkin lymphoma. Twenty transplants were autologous and 13 allogeneic. As stem cell source, bone marrow was used in 22 patients and peripheral blood in 14. Results : 60,9 percent of the autologous transplants reached an absolute lymphocyte count = 500 x mm3 on day 15 (ALC-15), whereas in the allogeneic this was achieved in 53,8 percent. Peripheral blood had a higher ALC-15 than bone marrow, 78,6 percent and 45,4 percent, respectively (p = 0.049). Prognostic factors associated to worse overall survival were sepsis (p <0.001), ALC-15 <500 x mm3 (p = 0.001) and relapse (p = 0.03). Kapplan-Meier curves showed better overall survival and event-free survival after five years in patients with ALC-15 = 500 x mm3 (85 percent vs. 15 percent, p <0.001). Conclusions: the ALC-15 = 500 x mm3 is a simple and useful tool to predict a better outcome in pediatric patients undergoing hematopoietic transplantation

Hipoxemia espuria por hiperleucocitosis: Reporte de un caso y revisión de la literatura / Spurious hypoxemia due to hyperleukocytosis: Report of one case

Background: Arterial gasometry is considered the gold standard for establishing a diagnosis of respiratory failure of any etiology. However, there are some circumstances in which it loses specificity, making necessary to consider other tests such as pulse oximetry to adequately determine hypoxemia. We report a 67 years old patient with sudden hypoacusia, right hemiparesis and polypnea. His laboratory exams on admission, showed extreme hypoxemia in several readings, without correlation to the patient's clinical condition nor the pulse oximetry, and a leukocytosis of 800.000 cells x ml, with many immature cells. Chronic myeloid leukemia was diagnosed and treatment with hydroxyurea was initiated, achieving normalization in the arterial gases in accordance with the fall of the white cell count. Interpretation of laboratory findings according to the general clinical context of the patient allowed to suspect a spurious hypoxemia, saving the patient from unnecessary and risky interventions.

Analysis of imatinib in bone marrow and plasma samples of chronic myeloid leukaemia patients using solid phase extraction LC-ESI-MS

The LC-ESI-MS was developed and validated for the analysis of imatinib in plasma and bone marrow samples using deuterated imatinib [D[8]-IM] as an internal standard. The biological samples were extracted using Strata-X-C SPE cartridges and separated on C[8] column [50 x 3 mm, 3 microm], and methanol: 0.1% formic acid [70:30] was delivered at the rate of 0.7 ml/min as a mobile phase. Imatinib was quantified in samples by monitoring the ions m/z 494.3 for imatinib and 502.3 for D[8]-imatinib on mass spectrometer. The method was linear in the concentration range of 1-1500 ng/250 microl in spiked human plasma samples and limit of quantification was 5 ng/mL. Inter-day and intra-day variations in spiked human plasma spiked with 50, 250 and 500 ng /mL were less than 3.16%. The repeatability and reproducibility and other parameters of the methods were also validated. The method was employed for the analysis of the imatinib in human plasma and bone marrow samples. The drug levels in bone marrow and plasma samples were correlated to the degree of cytogenetic response. No significant difference of imatinib level between blood and bone marrow in IM-treated patients dosed to steady state was observed

Detection of molecular variants of BCR-ABL gene in bone marrow and blood of patients with chronic myeloid leukemia by reverse-transcriptase polymerase chain reaction (RT-PCR).

Very limited data exists in Thailand regarding the frequency of BCR-ABL leukemic gene and its prognostic implication in Thai CML patients. The objective of this study was to develop a rapid molecular assay for the detection of the two most commonly reported variants of BCR-ABL fusion gene, B2A2 and B3A2 in CML patients. Bone marrow or peripheral blood were used for RNA extraction and reverse-transcribed to cDNA for PCR amplification. 92 per cent of CML patients (91/99) were positive for BCR-ABL gene (61% B3A2 and 31% B2A2). 8/99 CML patients were BCR-ABL-negative. B3A2 and B2A2-positive patients did not have any different clinical and hematological features at presentation although B3A2 patients tended to be slightly older and had higher platelet counts. 71/71 non-CML including other MPD and leukemia cases were all negative for BCR-ABL gene. In conclusion, a rapid RT-PCR assay has now been developed for the detection of this hallmark gene in CML patients. It should be of great value in the differential diagnosis of CML from other diseases. Long-term follow-ups of CML patients with different variants are needed to determine the prognostic importance of each gene variant.

BCR/ABL rearrangement in Philadelphia chromosome negative CML patients.

Reverse transcription-polymerase chain reaction (RT-PCR) was used to study 34 patients with chronic myelogenous leukemia (CML) associated with negative Philadelphia (Ph) chromosome. This report showed evidence of a chimeric BCR/ABL transcript in 18 (52.9%) and 28 (82.4%) cases by first PCR and seminested PCR, respectively. In these BCR/ABL transcript positive cases, the incidence of BCR exon3/ABL exon2 (B3A2) and BCR exon 2/ABL exon2 rearrangement was 25 (89.3%) and 3 (10.7%) cases, respectively. The other 6 Ph negative patients showed no evidence of reciprocal translocation of BCR to chromosome 9. This data demonstrates that seminested PCR is sufficiently sensitive to detect BCR/ABL fusion transcript in Ph chromosome negative CML patients.

Serum vitamin E in chronic myeloid leukaemia.

OBJECTIVE: Experimental evidence suggests that free radicals can participate in tumour promotion and malignant conversion and free radical scavengers like alphatocopherol act as first line of defense against peroxidation of polyunsaturated fatty acids. The present study was planned to estimate serum vitamin E levels in patients of chronic myeloid leukaemia (CML). METHODS: Serum vitamin E levels were estimated in 25 CML patients before and after the treatment. Twenty five age and sex matched healthy individuals served as control. RESULTS: Mean serum vitamin E levels were significantly decreased in CML patients before starting the treatment as compared to control. Also, vitamin E levels increased significantly after the treatment, but, remained lower than the control. CONCLUSION: Vitamin E levels were lower in CML patients as compared to control and the levels increased significantly after treatment in these patients. This could be due to decrease in oxidative stress due to decrease in tumour load.

Serum vitamin E levels in patients of chronic myeloid leukaemia.

OBJECTIVE: To estimate vitamin E levels in sera of patients of chronic myeloid leukaemia (CML) before and after treatment with busulphan and hydroxyurea and to compare the levels with those in healthy controls. METHODS: The study was carried out in a total of 50 subjects (mean age 34 +/- 8.86 years; 25 were proved cases of CML and 25 were healthy controls. Vitamin E level was measured by spectrofluorometeric method. In patients of CML vitamin E level was estimated twice i.e. once when their total leucocyte count (TLC) was raised (i.e. before treatment) and then after treatment after bringing the TLC near normal. RESULTS: The mean serum vitamin E level in 25 normal healthy controls was 7.19 micrograms/ml and that in 25 patients of CML at presentation (before treatment) was 2.67 micrograms/ml. It increased to 3.61 micrograms/ml after treatment. CONCLUSION: The results show that patients of CML have a significantly lower (p < 0.001) level of serum vitamin E as compared to normal healthy adults and that the level of vitamin E increased significantly (p < 0.001) after treatment even without supplementing vitamin E.

Busulfán versus busulfán-interferón como terapia de mantenimiento en leucemia mieloide crónica / Busulfan versus busulfan-interferon as maintenance therapy in chronic myeloid leukemia

Se estudiaron 30 pacientes con el fin de evaluar la eficacia terapéutica y la toxicidad del interferón alfa-2b, asociado al busulfán, en el mantenimiento de la fase crónica con leucemia mieloide crónica de novo. Los pacientes recibieron busulfán hasta alcanzar la remisión hematológica completa, y se dividieron aleatoriamente en dos grupos: uno se mantuvo con busulfán el cual se administró cuando los leucocitos superaron los 5 x 10 9/L, y otro recibió interferón alfa a dosis de 5 millones UI SC tres veces por semana, agregándose busulfán cuando los leucocitos eran superiores a 15 x 10 9/L. El promedio de la duración de la fase crónica fue mayor en el grupo de busulfán-interferón, 31 vs 16 meses (p= 0.03) pero no hubo ningún caso de remisión citogenética. El interferón fue bien tolerado: ningún enfermo se eliminó por toxicidad del medicamento

Utilidad de la relación cobre/zinc en pacientes con linfoma o leucemias aguda o crónica / Serum copper and zinc levels and Cu/Zn ration in lymphoma and acute and chronic leukemia

Objetivo. Conocer si la relación cobre/zinc (cobre elevado, zinc bajo) se encuentra aumentada en pacientes con neoplasias malignas hematológicas comparada con sujetos controles sanos de edad y sexo similares. Metodología. Se estudiaron 44 pacientes con neoplasias hemato-concológicas de reciente diagnóstico, sin tratamiento previo: 17 linfomas (11 no-Hodgkin), 15 con leucemia aguda (10 mieloblásticas) y 12 con leucemia crónica (8 granulocíticas). También se incluyeron 95 sujetos controles sanos. Se utizó un espectrofotómetro de absorción atómica (Perkin Elmer modelo 2380) para la cuantificación de los niveles séricos de cobre y zinc. Resultados. Los niveles séricos de cobre (µg/dL) fueron significativamente menores en los sujetos controles(54.4 ñ 8.9, p< 0.05), en comparación con los pacientes con linfoma (93.7 ñ 37.5), con leucemia aguda (80.6 ñ 44.6) y con leucemia crónica (95.7 ñ 28.9) mientras que los niveles séricos de zinc (µg/dL) resultaron significativamente mayores en sujetos controles (100.4 ñ 14, p< 0.05) en comparación con los pacientes con linfoma (77.2 ñ 22.6), leucemia aguda (66 ñ 15.6) o leucemia crónica (74.8 ñ 14.7). La relación cobre/zinc resultó ser significativamente más baja en sujetos controles (0.54 ñ 0.13, p< 0.05) que en pacientes con linfoma (1.21 ñ 0.5), leucemia aguda (1.22 ñ 0.7) o leucemia crónica (1.28 ñ 0.4). Veintitrés pacientes falleciron durante el seguimiento (media de 13 meses) observándose que sus niveles séricos de zinc fueron significativamente más bajos (68 ñ 28) que en los pacientes que sobrevivieron (76 ñ 15, p< 0.05). Conclusión. La relación sobre/zinc se encuentra significativamente elevada en pacientes con neoplasias malignas hematológicas

Detection of BCR/ABL fusion gene in CML: a preliminary report.

The BCR/ABL fusion gene in 31 patients with chronic myeloid leukemia (CML) was detected by RT/PCR. In 18 cases of Ph' positive patients, 13 had BCR 3/ABL II rearrangement, 1 had BCR 2/ABL II rearrangement and 4 had both rearrangements. One case with complex translocation: 46,XY,t(9;9;22), had BCR 3/ABL II rearrangement. In 8 cases of Ph' negative patients, 4 had BCR 3/ABL II rearrangement, 3 had both rearrangements while 1 had no BCR/ABL rearrangement. Interestingly, in 4 patients who had no cytogenetic result, we could observe BCR 3/ABL II rearrangement in 3 cases and both rearrangements in 1 case. The results suggest that this procedure is sensitive and independent of the presence or absence of an identifiable Ph' chromosome.

Role of serum ferritin in assessment of disease activity in acute & chronic leukemia.

Serum ferritin (SF) was estimated using double antibody sandwich ELISA in 83 patients of acute and chronic leukemia at various stages of the disease. In 35 patients of acute lymphoblastic leukemia (ALL) in remission, the SF levels fell significantly from 550.63 ng/ml at presentation to 319.56 ng/ml but remained significantly higher than the control values of 46.14 ng/ml. In 28 patients of acute myeloid leukemia (AML), the SF values at 775.0 ng/ml were much higher than those in ALL patients and showed no decline with remission. This pattern was also seen in patients of chronic myeloid leukemia in blast crisis (CML-BC) with SF levels of 804.03 ng/ml at presentation and 717.43 ng/ml at partial remission. The values of SF were lowest in patients of CML in chronic phase ranging from 271.5 ng/ml to 332.12 ng/ml and showed no relationship with variation in total leucocyte count. No correlation was found between SF values and various clinical and laboratory parameters such as age, sex, fever, organomegaly, haemoglobin and total leucocyte count. Thus, while there appeared to be a correlation between remission and SF values in ALL, no such correlation existed between the activity of the disease and SF in other types of leukemia.

Platelet function in acute leukemias.

Bleeding time, clot retraction, platelet factor 3 availability and platelet aggregation in response to ADP, epinephrine, collagen and ristocetin were studied in 13 cases of acute leukemia which included 5 cases of acute myeloid leukemia, 2 of chronic myeloid leukemia in blast crisis and 6 of acute lymphoblastic leukemia. More than one abnormality was seen in all the patients. Defects in bleeding time, clot retraction and platelet factor 3 availability were encountered in 43% of cases. Platelet aggregation responses to all the reagents were significantly impaired. There was, however, no consistency in the pattern of the defects.

Esplenectomía en fase crónica de leucemia granulocítica crónica

El presente trabajo hace una revisión de la literatura y determina el valor de la esplenectomía en la fase crónica de 5 pacientes con diagnóstico establecido de LGC, contribuyéndose, al igual que varios autores, que no se mejora con ella la supervivencia, ni se retarda la transformación blástica.

Chronic granulocytic leukaemia. A study of 160 cases.

Chronic granulocytic leukaemia (CGL) is the commonest leukaemia among adults in India. Case records of 183 CGL patients diagnosed between 1975 and 1985 were reviewed. The median age at diagnosis was 40.5 years. Most patients presented with weakness, fullness in the left upper abdomen and fever. Splenomegaly and hepatomegaly were present in 90% and 48% respectively. Patients were treated with oral, intermittent busulphan with monitoring of total leucocyte count. Overall, 87 patients expired, including 63 (72%) due to blast crisis. The median survival was 33 months from diagnosis and 44 months from the onset of symptoms.