Elaine Sobral da Costa

Automated pattern-guided principal component analysis vs expert-based immunophenotypic classification of B-cell chronic lymphoproliferative disorders: a step forward in the standardization of clinical immunophenotyping

Immunophenotypic characterization of B-cell chronic lymphoproliferative disorders (B-CLPD) is becoming increasingly complex due to usage of progressively larger panels of reagents and a high number of World Health Organization (WHO) entities. Typically, data analysis is performed separately for each stained aliquot of a sample; subsequently, an expert interprets the overall immunophenotypic profile (IP) of neoplastic B-cells and assigns it to specific diagnostic categories. We constructed a principal component analysis (PCA)-based tool to guide immunophenotypic classification of B-CLPD. Three reference groups of immunophenotypic data files—B-cell chronic lymphocytic leukemias (B-CLL; n=10), mantle cell (MCL; n=10) and follicular lymphomas (FL; n=10)—were built. Subsequently, each of the 175 cases studied was evaluated and assigned to either one of the three reference groups or to none of them (other B-CLPD). Most cases (89%) were correctly assigned to their corresponding WHO diagnostic group with overall positive and negative predictive values of 89 and 96%, respectively. The efficiency of the PCA-based approach was particularly high among typical B-CLL, MCL and FL vs other B-CLPD cases. In summary, PCA-guided immunophenotypic classification of B-CLPD is a promising tool for standardized interpretation of tumor IP, their classification into well-defined entities and comprehensive evaluation of antibody panels.

Overview of clinical flow cytometry data analysis: recent advances and future challenges

Major technological advances in flow cytometry (FC), both for instrumentation and reagents, have emerged over the past few decades. These advances facilitate simultaneous evaluation of more parameters in single cells analyzed at higher speed. Consequently, larger and more complex data files that contain information about tens of parameters for millions of cells are generated. This increasing complexity has challenged pre-existing data analysis tools and promoted the development of new algorithms and tools for data analysis and visualization. Here, we review the currently available (conventional and newly developed) data analysis and visualization strategies that aim for easier, more objective, and robust interpretation of FC data both in biomedical research and clinical diagnostic laboratories.

Generation of flow cytometry data files with a potentially infinite number of dimensions.

Immunophenotypic characterization of B‐cell chronic lymphoproliferative disorders (B‐CLPD) is associated with the use of increasingly larger panels of multiple combinations of 3 to ≥6 monoclonal antibodies (Mab), data analysis being separately performed for each of the different stained sample aliquots. Here, we describe and validate an automated method for calculation of flow cytometric data from several multicolor stainings of the same cell sample—i.e., the merging of data from different aliquots stained with partially overlapping combinations of Mab reagents (focusing on ≥1 cell populations)—into one data file as if it concerned a single “super” multicolor staining. Evaluation of the performance of the method described was done in a group of 60 B‐CLPD studied at diagnosis with 18 different reagents in a panel containing six different 3‐ and 4‐color stainings, which systematically contained CD19 for the identification of B‐cells. Our results show a high degree of correlation and agreement between originally measured and calculated data about cell surface stainings, providing a basis for the use of this approach for the generation of flow cytometric data files containing information about a virtually infinite number of stainings for each individual cellular event measured in a sample, using a limited number of fluorochrome stainings.

A probabilistic approach for the evaluation of minimal residual disease by multiparameter flow cytometry in leukemic B-cell chronic lymphoproliferative disorders.

Multiparameter flow cytometry has become an essential tool for monitoring response to therapy in hematological malignancies, including B‐cell chronic lymphoproliferative disorders (B‐CLPD). However, depending on the expertise of the operator minimal residual disease (MRD) can be misidentified, given that data analysis is based on the definition of expert‐based bidimensional plots, where an operator selects the subpopulations of interest. Here, we propose and evaluate a probabilistic approach based on pattern classification tools and the Bayes theorem, for automated analysis of flow cytometry data from a group of 50 B‐CLPD versus normal peripheral blood B‐cells under MRD conditions, with the aim of reducing operator‐associated subjectivity. The proposed approach provided a tool for MRD detection in B‐CLPD by flow cytometry with a sensitivity of ≤8 × 10−5 (median of ≤2 × 10−7). Furthermore, in 86% of B‐CLPD cases tested, no events corresponding to normal B‐cells were wrongly identified as belonging to the neoplastic B‐cell population at a level of ≤10−7. Thus, this approach based on the search for minimal numbers of neoplastic B‐cells similar to those detected at diagnosis could potentially be applied with both a high sensitivity and specificity to investigate for the presence of MRD in virtually all B‐CLPD. Further studies evaluating its efficiency in larger series of patients, where reactive conditions and non‐neoplastic disorders are also included, are required to confirm these results.

Analysis of the immune system of multiple myeloma patients achieving long-term disease control by multidimensional flow cytometry

Multiple myeloma remains largely incurable. However, a few patients experience more than 10 years of relapse-free survival and can be considered as operationally cured. Interestingly, long-term disease control in multiple myeloma is not restricted to patients with a complete response, since some patients revert to having a profile of monoclonal gammopathy of undetermined significance. We compared the distribution of multiple compartments of lymphocytes and dendritic cells in the bone marrow and peripheral blood of multiple myeloma patients with long-term disease control (n=28), patients with newly diagnosed monoclonal gammopathy of undetermined significance (n=23), patients with symptomatic multiple myeloma (n=23), and age-matched healthy adults (n=10). Similarly to the patients with monoclonal gammopathy of undetermined significance and symptomatic multiple myeloma, patients with long-term disease control showed an expansion of cytotoxic CD8+ T cells and natural killer cells. However, the numbers of bone marrow T-regulatory cells were lower in patients with long-term disease control than in those with symptomatic multiple myeloma. It is noteworthy that B cells were depleted in patients with monoclonal gammopathy of undetermined significance and in those with symptomatic multiple myeloma, but recovered in both the bone marrow and peripheral blood of patients with long-term disease control, due to an increase in normal bone marrow B-cell precursors and plasma cells, as well as pre-germinal center peripheral blood B cells. The number of bone marrow dendritic cells and tissue macrophages differed significantly between patients with long-term disease control and those with symptomatic multiple myeloma, with a trend to cell count recovering in the former group of patients towards levels similar to those found in healthy adults. In summary, our results indicate that multiple myeloma patients with long-term disease control have a constellation of unique immune changes favoring both immune cytotoxicity and recovery of B-cell production and homing, suggesting improved immune surveillance.

Standardized flow cytometry for highly sensitive MRD measurements in B-cell acute lymphoblastic leukemia

A fully-standardized EuroFlow 8-color antibody panel and laboratory procedure was stepwise designed to measure minimal residual disease (MRD) in B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) patients with a sensitivity of ≤10-5, comparable to real-time quantitative polymerase chain reaction (RQ-PCR)-based MRD detection via antigen-receptor rearrangements. Leukocyte markers and the corresponding antibodies and fluorochromes were selected based on their contribution in separating BCP-ALL cells from normal/regenerating BCP cells in multidimensional principal component analyses. After 5 multicenter design-test-evaluate-redesign phases with a total of 319 BCP-ALL patients at diagnosis, two 8-color antibody tubes were selected, which allowed separation between normal and malignant BCP cells in 99% of studied patients. These 2 tubes were tested with a new erythrocyte bulk-lysis protocol allowing acquisition of high cell numbers in 377 bone marrow follow-up samples of 178 BCP-ALL patients. Comparison with RQ-PCR-based MRD data showed a clear positive relation between the percentage concordant cases and the number of cells acquired. For those samples with >4 million cells acquired, concordant results were obtained in 93% of samples. Most discordances were clarified upon high-throughput sequencing of antigen-receptor rearrangements and blind multicenter reanalysis of flow cytometric data, resulting in an unprecedented concordance of 98% (97% for samples with MRD < 0.01%). In conclusion, the fully standardized EuroFlow BCP-ALL MRD strategy is applicable in >98% of patients with sensitivities at least similar to RQ-PCR (≤10-5), if sufficient cells (>4 × 106, preferably more) are evaluated.

Overweight as a Prognostic Factor in Children With Acute Lymphoblastic Leukemia

Our purpose was to investigate the prognostic impact of overweight/obesity in 5‐year event‐free survival (EFS) in a cohort of children with acute lymphoblastic leukemia (ALL). We retrospectively analyzed 181 newly diagnosed ALL children enrolled between 1990 and 2009 and treated with Berlin‐Frankfurt‐Munich (BFM) protocols. The majority of children in our cohort were <10 years‐old. Our data clearly indicated that overweight/obesity is an independent predictor of relapse risk, mainly in the intermediate‐ and high‐risk groups (HR) of children. These results could be explained by changes in the chemotherapy pharmacokinetics in overweight/obese patients and by the antiapoptotic effects in leukemic cells caused by adipocytes.

Birth weight patterns by gestational age in Brazil

BACKGROUND AND OBJECTIVES We present an updated birth weight-for-gestational-age portrait, based on nearly 8 million observations of an ethnic-mixed population. It comprises the first comprehensive charts with Brazilian data. This contribution intends to assist clinicians in classifying fetal growth, to provide a reference for investigations of predictors and to show the consequences of small and large patterns for gestational age delivery. Most of the reference data for assessing birth weight for gestational age deal with insufficient sample size, especially at low gestational age. Population-based studies with considerably large sample size refer to data collected more than 15 years ago. METHODS We accomplished a population-based study on births in all the Brazilian states from 2003 to 2005. Results were based on 7,993,166 singletons. We constructed the 3(rd), 5(th), 10(th), 25(th), 50(th), 90(th), 95(th) and 97(th) smoothed percentiles curves and gender-specific tables from 22 to 43 completed weeks. RESULTS The resulting tables and graphical representation provide a gender-specific reference to access the birth weights distribution according to the gestational age in the Brazilian population. CONCLUSIONS This is the first population-based reference constructed on a developing country data. These charts could provide an important tool to improve clinical assessment of growth in newborns.

Contribution of Multiparameter Flow Cytometry Immunophenotyping to the Diagnostic Screening and Classification of Pediatric Cancer

Pediatric cancer is a relatively rare and heterogeneous group of hematological and non-hematological malignancies which require multiple procedures for its diagnostic screening and classification. Until now, flow cytometry (FC) has not been systematically applied to the diagnostic work-up of such malignancies, particularly for solid tumors. Here we evaluated a FC panel of markers for the diagnostic screening of pediatric cancer and further classification of pediatric solid tumors. The proposed strategy aims at the differential diagnosis between tumoral vs. reactive samples, and hematological vs. non-hematological malignancies, and the subclassification of solid tumors. In total, 52 samples from 40 patients suspicious of containing tumor cells were analyzed by FC in parallel to conventional diagnostic procedures. The overall concordance rate between both approaches was of 96% (50/52 diagnostic samples), with 100% agreement for all reactive/inflammatory and non-infiltrated samples as well as for those corresponding to solid tumors (n = 35), with only two false negative cases diagnosed with Hodgkin lymphoma and anaplastic lymphoma, respectively. Moreover, clear discrimination between samples infiltrated by hematopoietic vs. non-hematopoietic tumor cells was systematically achieved. Distinct subtypes of solid tumors showed different protein expression profiles, allowing for the differential diagnosis of neuroblastoma (CD56hi/GD2+/CD81hi), primitive neuroectodermal tumors (CD271hi/CD99+), Wilms tumors (>1 cell population), rhabdomyosarcoma (nuMYOD1+/numyogenin+), carcinomas (CD45−/EpCAM+), germ cell tumors (CD56+/CD45−/NG2+/CD10+) and eventually also hemangiopericytomas (CD45−/CD34+). In summary, our results show that multiparameter FC provides fast and useful complementary data to routine histopathology for the diagnostic screening and classification of pediatric cancer.

Retinoic Acid-Treated Pluripotent Stem Cells Undergoing Neurogenesis Present Increased Aneuploidy and Micronuclei Formation

The existence of loss and gain of chromosomes, known as aneuploidy, has been previously described within the central nervous system. During development, at least one-third of neural progenitor cells (NPCs) are aneuploid. Notably, aneuploid NPCs may survive and functionally integrate into the mature neural circuitry. Given the unanswered significance of this phenomenon, we tested the hypothesis that neural differentiation induced by all-trans retinoic acid (RA) in pluripotent stem cells is accompanied by increased levels of aneuploidy, as previously described for cortical NPCs in vivo. In this work we used embryonal carcinoma (EC) cells, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells undergoing differentiation into NPCs. Ploidy analysis revealed a 2-fold increase in the rate of aneuploidy, with the prevalence of chromosome loss in RA primed stem cells when compared to naïve cells. In an attempt to understand the basis of neurogenic aneuploidy, micronuclei formation and survivin expression was assessed in pluripotent stem cells exposed to RA. RA increased micronuclei occurrence by almost 2-fold while decreased survivin expression by 50%, indicating possible mechanisms by which stem cells lose their chromosomes during neural differentiation. DNA fragmentation analysis demonstrated no increase in apoptosis on embryoid bodies treated with RA, indicating that cell death is not the mandatory fate of aneuploid NPCs derived from pluripotent cells. In order to exclude that the increase in aneuploidy was a spurious consequence of RA treatment, not related to neurogenesis, mouse embryonic fibroblasts were treated with RA under the same conditions and no alterations in chromosome gain or loss were observed. These findings indicate a correlation amongst neural differentiation, aneuploidy, micronuclei formation and survivin downregulation in pluripotent stem cells exposed to RA, providing evidence that somatically generated chromosomal variation accompanies neurogenesis in vitro.