Immunophenotypic shift in the B-cell precursors from regenerating bone marrow samples: A critical consideration for measurable residual disease assessment in B-lymphoblastic leukemia.

Accurate knowledge of expression patterns/levels of commonly used MRD markers in regenerative normal-B-cell-precursors (BCP) is highly desirable to distinguish leukemic-blasts from regenerative-BCP for multicolor flow cytometry (MFC)-based measurable residual disease (MRD) assessment in B-lymphoblastic leukemia (B-ALL). However, the data highlighting therapy-related immunophenotypic-shift in regenerative-BCPs is scarce and limited to small cohort. Herein, we report the in-depth evaluation of immunophenotypic shift in regenerative-BCPs from a large cohort of BALL-MRD samples. Ten-color MFC-MRD analysis was performed in pediatric-BALL at the end-of-induction (EOI), end-of-consolidation (EOC), and subsequent-follow-up (SFU) time-points. We studied normalized-mean fluorescent intensity (nMFI) and coefficient-of-variation of immunofluorescence (CVIF) of CD10, CD19, CD20, CD34, CD38, and CD45 expression in regenerative-BCP (early, BCP1 and late, BCP2) from 200 BALL-MRD samples, and compared them with BCP from 15 regenerating control (RC) TALL-MRD samples and 20 treatment-naïve bone-marrow control (TNSC) samples. Regenerative-BCP1 showed downregulation in CD10 and CD34 expression with increased CVIF and reduced nMFI (p < 0.001), upregulation of CD20 with increased nMFI (p = 0.014) and heterogeneous CD45 expression with increased CVIF (p < 0.001). Immunophenotypic shift was less pronounced in the BCP2 compared to BCP1 compartment with increased CVIF in all but CD45 (p < 0.05) and reduced nMFI only in CD45 expression (p = 0.005). Downregulation of CD10/CD34 and upregulation of CD20 was higher at EOI than EOC and SFU time-points (p < 0.001). Regenerative-BCPs are characterized by the significant immunophenotypic shift in commonly used B-ALL-MRD markers, especially CD10 and CD34 expression, as compared to treatment-naïve BCPs. Therefore, the templates/database for BMRD analysis must be developed using regenerative-BCP.

Sobrevida global de pacientes con leucemia aguda en el Hospital de Especialidades Pediátricas de Chiapas, México / Childhood acute leukemia overall survival at the Hospital de Especialidades Pediátricas in Chiapas, México

Resumen: Introducción: A 10 años de la fundación del Hospital de Especialidades Pediátricas en Chiapas, México, es importante valorar la sobrevida global a 5 años de los pacientes con leucemia aguda bajo el régimen del Seguro Popular. Métodos: Estudio descriptivo y de sobrevida de 210 casos de leucemia aguda diagnosticados y tratados entre 2008 y 2012. Empleando curvas de Kaplan-Meier se analizó cada variedad de la enfermedad (B, T y mieloide), y para la leucemia B en función del grupo de riesgo, el sexo, la edad, los leucocitos al diagnóstico, los marcadores de superficie, el índice de DNA, el cariotipo y las translocaciones. Resultados: La edad, el sexo y la proporción de tipos de leucemia aguda (B = 85%; M = 10%; T = 5%) fueron similares al resto del país. El 20% de los pacientes estaban vivos a 5 años; el 53% habían fallecido y el 27% abandonaron el tratamiento. La sobrevida global a 5 años fue del 42% (B = 45%; T = 20%; M = 10%) (mediana: 38.8 meses; intervalo de confianza del 95%: 28.9-48.7). La mediana de «muy alto riesgo¼ fue de 7.7 contra 47 meses; no hubo diferencia entre riesgo habitual y alto riesgo. Los leucocitos < 50,000/µl al diagnóstico y CD10 positivo se asociaron con mejor sobrevida. En el momento del deceso, el 29% se encontraba en remisión. Conclusiones: La sobrevida de la leucemia aguda bajo el Seguro Popular fue desfavorable los primeros 5 años del Hospital de Especialidades Pediátricas. Se identificaron como contribuyentes la alta tasa de mortalidad temprana, de pacientes en remisión y el abandono. Además de revisar la atención médica, se requiere el estudio de elementos extrahospitalarios determinantes del abandono para mejorar el programa.

Abstract: Background: At the 10th anniversary of the Hospital de Especialidades Pediátricas in Chiapas, Mexico, it was important to assess the 5-year acute leukemia overall survival under the Seguro Popular program (Popular Insurance). Methods: A descriptive and survival study of 210 acute leukemia patients diagnosed and treated during 2008-2012 was performed. Kaplan-Meier survival curves were developed for all patients, each leukemia type (B, T and myeloid) and for B type related to risk group, age, sex, leukocytes, cell markers, DNA index, karyotype, and translocations. Results: Age, gender and proportion of leukemia types (B = 85%; M = 10%; T = 5%), were similar to other parts of the country. At the end of the 5-year treatment, 20% of the patients were alive, 53% had died and 27% had abandoned the treatment. Global survival was 42% (B = 45%; T = 20%; M = 10%) (median: 38.8 months; confidence interval of 95% = 28.9-48.7). Very high-risk median survival was 7.7 versus 47 months. There was no difference between standard and high-risk groups. The initial leukocyte count < 50,000/µL and CD10 positive were related to better B survival; no other variables were related. At the time of death, 29% of patients were in remission. Conclusions: Global survival of acute leukemia at Hospital de Especialidades Pediátricas under the Seguro Popular during its first 5 years was surprisingly poor given the medical resources available through the insurance. Early mortality, death during remission and high desertion rates contributed to these results. A detailed revision of treatment protocols and reasons for abandoning treatment is mandatory.

[Sobrevida global de pacientes con leucemia aguda en el Hospital de Especialidades Pediátricas de Chiapas, México]. / Childhood acute leukemia overall survival at the Hospital de Especialidades Pediátricas in Chiapas, México.

Background: At the 10th anniversary of the Hospital de Especialidades Pediátricas in Chiapas, Mexico, it was important to assess the 5-year acute leukemia overall survival under the Seguro Popular program (Popular Insurance). Methods: A descriptive and survival study of 210 acute leukemia patients diagnosed and treated during 2008-2012 was performed. Kaplan-Meier survival curves were developed for all patients, each leukemia type (B, T and myeloid) and for B type related to risk group, age, sex, leukocytes, cell markers, DNA index, karyotype, and translocations. Results: Age, gender and proportion of leukemia types (B = 85%; M = 10%; T = 5%), were similar to other parts of the country. At the end of the 5-year treatment, 20% of the patients were alive, 53% had died and 27% had abandoned the treatment. Global survival was 42% (B = 45%; T = 20%; M = 10%) (median: 38.8 months; confidence interval of 95% = 28.9-48.7). Very high-risk median survival was 7.7 versus 47 months. There was no difference between standard and high-risk groups. The initial leukocyte count < 50,000/mL and CD10 positive were related to better B survival; no other variables were related. At the time of death, 29% of patients were in remission. Conclusions: Global survival of acute leukemia at Hospital de Especialidades Pediátricas under the Seguro Popular during its first 5 years was surprisingly poor given the medical resources available through the insurance. Early mortality, death during remission and high desertion rates contributed to these results. A detailed revision of treatment protocols and reasons for abandoning treatment is mandatory.

Introducción: A 10 años de la fundación del Hospital de Especialidades Pediátricas en Chiapas, México, es importante valorar la sobrevida global a 5 años de los pacientes con leucemia aguda bajo el régimen del Seguro Popular. Métodos: Estudio descriptivo y de sobrevida de 210 casos de leucemia aguda diagnosticados y tratados entre 2008 y 2012. Empleando curvas de Kaplan-Meier se analizó cada variedad de la enfermedad (B, T y mieloide), y para la leucemia B en función del grupo de riesgo, el sexo, la edad, los leucocitos al diagnóstico, los marcadores de superficie, el índice de DNA, el cariotipo y las translocaciones. Resultados: La edad, el sexo y la proporción de tipos de leucemia aguda (B = 85%; M = 10%; T = 5%) fueron similares al resto del país. El 20% de los pacientes estaban vivos a 5 años; el 53% habían fallecido y el 27% abandonaron el tratamiento. La sobrevida global a 5 años fue del 42% (B = 45%; T = 20%; M = 10%) (mediana: 38.8 meses; intervalo de confianza del 95%: 28.9-48.7). La mediana de «muy alto riesgo¼ fue de 7.7 contra 47 meses; no hubo diferencia entre riesgo habitual y alto riesgo. Los leucocitos < 50,000/ml al diagnóstico y CD10 positivo se asociaron con mejor sobrevida. En el momento del deceso, el 29% se encontraba en remisión. Conclusiones: La sobrevida de la leucemia aguda bajo el Seguro Popular fue desfavorable los primeros 5 años del Hospital de Especialidades Pediátricas. Se identificaron como contribuyentes la alta tasa de mortalidad temprana, de pacientes en remisión y el abandono. Además de revisar la atención médica, se requiere el estudio de elementos extrahospitalarios determinantes del abandono para mejorar el programa.

Development of a cost-effective high-throughput process of microsatellite analysis involving miniaturized multiplexed PCR amplification and automated allele identification.

BACKGROUND: Microsatellites are nucleotide sequences of tandem repeats occurring throughout the genome, which have been widely used in genetic linkage analysis, studies of loss of heterozygosity, determination of lineage and clonality, and the measurement of genome instability or the emergence of drug resistance reflective of mismatch repair deficiency. Such analyses may involve the parallel evaluation of many microsatellite loci, which are often limited by sample DNA, are labor intensive, and require large data processing. RESULTS: To overcome these challenges, we developed a cost-effective high-throughput approach of microsatellite analysis, in which the amplifications of microsatellites are performed in miniaturized, multiplexed polymerase chain reaction (PCR) adaptable to 96 or 384 well plates, and accurate automated allele identification has been optimized with a collective reference dataset of 5,508 alleles using the GeneMapper software. CONCLUSIONS: In this investigation, we have documented our experience with the optimization of multiplex PCR conditions and automated allele identification, and have generated a unique body of data that provide a starting point for a cost-effective, high-throughput process of microsatellite analysis using the studied markers.

Comparison of different polymerase chain reaction-based approaches for clonality assessment of immunoglobulin heavy-chain gene rearrangements in B-cell neoplasia.

Several frequently applied polymerase chain reaction strategies for analysis of immunoglobulin heavy-chain gene rearrangements were compared by analyzing 70 B-cell lymphoproliferative disorders and 24 reactive lymphoid lesions. Southern blot analysis was used as the "gold standard" for clonality assessment. For polymerase chain reaction analysis, primers directed against framework (FR) 3 (FR3-A and FR3-B), FR2, and FR1 of the variable gene segments and against joining gene segments of the immunoglobulin heavy-chain gene were used. Polymerase chain reaction products were analyzed by high-resolution fingerprinting analysis using radiolabeled nucleotides, gene scanning using fluorochrome-labeled primers, and heteroduplex analysis. All of the assays generated polyclonal patterns in the reactive tissues. The sensitivity in detecting monoclonality as defined by Southern blotting varied between 60% (heteroduplex analysis with FR3 primers) and 77% (high-resolution fingerprinting analysis with FR2 primers). Comparison of the three FR3 assays revealed that gene scanning had the highest sensitivity (69%), probably because it could detect small aberrant monoclonal amplicons. False-negative results were especially frequent in follicular center lymphoma (n = 20), but also in diffuse large B-cell lymphoma (n = 18), both renowned for having mutated variable gene segments, potentially leading to primer mismatching. For example, in follicular center lymphoma, the FR3, FR2, and FR1 region primer sets detected clonality in only 35 to 40, 65, and 50%, respectively. Combining these techniques, 17 (85%) of 20 follicular center lymphoma samples showed monoclonality. Therefore, especially in follicular center lymphoma, diffuse large B-cell lymphoma, and, to a lesser extent, in other lymphomas, multiple variable gene segment primer sets must be used for a reliable assessment of clonality. Our results also suggest that gene scanning is somewhat more sensitive than other read-out systems. Heteroduplex analysis, however, is a reliable alternative within a diagnostic setting, avoiding the use of radioactivity or expensive automated sequencing equipment and fluorochrome-labeled (oligo)nucleotides.

Optimal primer selection for clonality assessment by polymerase chain reaction analysis. III. Intermediate and high-grade B-cell neoplasms.

Previous studies have reported that low-grade B-cell lymphoproliferative disorders have variable B-cell monoclonality detection rates by polymerase chain reaction (PCR) analysis. For instance, monoclonal B-cell populations from chronic lymphocytic leukemia/small lymphocytic leukemia and mantle cell lymphoma are most often readily amplified with a single primer pair, whereas follicular lymphomas and plasma cell neoplasms require alternative strategies to approach these higher diagnostic sensitivity standards. Because most published reports have not focused on the variation in PCR B-cell monoclonality detection among subtypes of intermediate and high-grade B-cell neoplasms, additional information is necessary to determine primer selection strategies and identify problematic tumor subtypes within this group. The current investigation, the third part in a series, was aimed at documenting the efficiency of B-cell monoclonality detection by PCR in 71 aggressive B-cell neoplasms of various types using a comprehensive approach. A predetermined panel of primer sets was used in an algorithmic fashion. Specifically, all samples were analyzed with the standard VH-FRIII/JH assay previously shown to have the highest efficiency of monoclonality detection within low-grade B-cell lymphoproliferative disorders. Negative samples were further evaluated with primer sets in the following order until a positive result was observed, or all primer sets were used: (1) bcl-2/JH, (2) VH-FRI family specific/JH, and (3) VH-FRI consensus/JH. Forty-three (61%) of the 71 B-cell neoplasms evaluated with VH-FRIII/JH showed monoclonal B-cell populations. Sequential use of the three reserve primer sets in samples negative with this initial primer pair resulted in an overall improvement in PCR detection from 61% to 82% (58 of 71 specimens) (P < .001). The VH-FRI family specific assay identified B-cell monoclonality in 11 (73%) of these 15 specimens and was the most productive reserve primer set. Individual categories exhibited the following initial (I) and final (F) PCR detection rates: acute lymphoblastic leukemia/lymphoblastic lymphoma, 11 specimens (I = 91% to F = 91%); small noncleaved cell lymphoma, 14 specimens (I = 79% to F = 86% [P > .25]); diffuse large cell lymphoma, 33 specimens (I = 52% to F= 85% [P < .005]) and large cell, immunoblastic lymphoma, 13 specimens (I = 38% to F = 62% [P < .01]). The authors have shown that comprehensive PCR analysis is capable of detecting B-cell monoclonality in a significant proportion of samples from each subtype of intermediate and high-grade B-cell neoplasm. The VH-FRIII/JH assay was an adequate initial primer set, but required augmentation with the reserve PCR assays to attenuate the false negative rate and improve diagnostic sensitivity. The B-cell clonality PCR assay is optimally used as a screening tool and when used in this fashion, the more laborious and time-consuming restriction fragment-Southern blot hybridization (RF-SBH) method for IgH gene rearrangement detection may be limited to a relatively small proportion of PCR-negative aggressive B-cell neoplasms.