Comparative analysis between cytomorphology and flow cytometry methods in central nervous system infiltration assessment in oncohematological patients.

INTRODUCTION: Oncohematological patients require the evaluation for possible infiltration of the central nervous system (CNS) by neoplastic cells at diagnosis and/or during the monitoring of the chemotherapeutic treatment. Morphological analysis using conventional microscopy is considered the method of choice to evaluate the cerebrospinal fluid (CSF) samples, despite technical limitations. OBJECTIVE: This study aimed to compare the performance of the cytomorphology and flow cytometric immunophenotyping (FC) in the detection of CNS infiltration. METHOD: We evaluated 520 CSF samples collected from 287 oncohematological patients for whom the detection of neoplastic cells was simultaneously requested by cytomorphology and FC. RESULTS: Laboratory analyses revealed 435/520 (83.7%) conclusive results by the two methods evaluated, among which 385 (88.5%) were concordant. Discordance between the methods was observed in 50/435 (11.5%) samples, 45 (90%) being positive by FC. Furthermore, the FC defined the results in 69/72 (95.8%) inconclusive samples by cytomorphology. The positivity of FC was particularly higher among hypocellular samples. Among 431 samples with a cell count of < 5/µL, the FC identified neoplastic cells in 75 (17.4%), while the cytomorphology reported positive results in 26 (6%). Among the samples that presented adequate cell recovery for evaluation by both methods (506/520), the comparative analysis between FC and cytomorphology revealed a Kappa coefficient of 0.45 (CI: 0.37-0.52), interpreted as a moderate agreement. CONCLUSION: The data showed that the CSF analysis by FC helps in the definition of CNS infiltration by neoplastic cells, particularly in the cases with dubious morphological analysis or in the evaluation of samples with low cellularity.

Comparison between flow cytometry and standard PCR in the evaluation of MRD in children with acute lymphoblastic leukemia treated with the GBTLI LLA - 2009 protocol.

Minimal residual disease (MRD) monitoring is of prognostic importance in childhood acute lymphoblastic leukemia (ALL). The detection of immunoglobulin and T-cell receptor gene rearrangements by real-time quantitative PCR (RT-PCR) is considered the gold standard for this evaluation. However, more accessible methods also show satisfactory performance. This study aimed to compare MRD analysis by four-color flow cytometry (FC) and qualitative standard PCR on days 35 and 78 of chemotherapy and to correlate these data with patients' clinical characteristics. Forty-two children with a recent diagnosis of ALL, admitted to a public hospital in Brazil for treatment in accordance with the Brazilian Childhood Cooperative Group for ALL Treatment (GBTLI LLA-2009), were included. Bone marrow samples collected at diagnosis and on days 35 and 78 of treatment were analyzed for the immunophenotypic characterization of blasts by FC and for the detection of clonal rearrangements by standard PCR. Paired analyses were performed in 61/68 (89.7%) follow-up samples, with a general agreement of 88.5%. Disagreements were resolved by RT-PCR, which evidenced one false-negative and four false-positive results in FC, as well as two false-negative results in PCR. Among the prognostic factors, a significant association was found only between T-cell lineage and MRD by standard PCR. These results show that FC and standard PCR produce similar results in MRD detection of childhood ALL and that both methodologies may be useful in the monitoring of disease treatment, especially in regions with limited financial resources.

Cytogenetic abnormalities, WHO classification, and evolution of children and adolescents with acute myeloid leukemia.

OBJECTIVES: To describe cytogenetic and molecular abnormalities observed in children and adolescents with acute myeloid leukemia (AML), classify AML according to the World Health Organization (WHO) classifications from 2008 and 2016, and evaluate the prognosis according to clinical characteristics and cytogenetic abnormalities. METHODS: A retrospective longitudinal study was performed on a population of 98 patients with AML, aged up to 16 years, seen in a single hospital from 2004 to 2015. RESULTS: Among the 80 patients for whom it was possible to analyze the karyotype, 78.7% had chromosomal changes, the most frequent being t(15;17)(q22;q21). Of the 86 patients for whom we had cytogenetic or molecular data, making it possible to classify their AML according to the WHO classification, 52.3% belonged to the group with recurrent genetic abnormalities, 22% to the "AML not otherwise specified" group, 18.6% to the group with myelodysplasia-related cytogenetic changes, and 7% to the group with Down syndrome-related leukemia. Five-year overall survival (OS) for the whole group was 49.7%±5.2%. In the univariate and multivariate analyses, patients with myelodysplasia-related cytogenetic changes (OS 28.1%±12.2%) and those with "AML not otherwise specified" (OS 36.1%±11.2%) had an unfavorable prognosis when compared to patients with AML with recurrent genetic abnormalities (OS 71%±5.8%) and patients with Down syndrome-related AML (OS 83%±15.2%, p=0.011). CONCLUSIONS: The results corroborate the importance of cytogenetic abnormalities as a prognostic factor and indicate the need for cooperative and prospective studies to evaluate the applicability of the WHO classification in the pediatric population.

Immunophenotyping of the cerebrospinal fluid as a prognostic factor at diagnosis of acute lymphoblastic leukemia in children and adolescents.

This study aimed at evaluating the use of immunophenotyping (IMP) in the identification of blast cells in the cerebrospinal fluid (CSF) of children and adolescents with acute lymphoblastic leukemia (ALL). Sixty-seven patients aged 18 years or younger were included. Fifty-five CSF samples were analyzed at initial diagnosis and 17 at the time of relapse. A cytological analysis (CA) was performed in all 72 samples, while IMP was done in 63. Blasts were identified in only three samples by CA, whereas all three samples were found negative by IMP, one of which had no isolation of nucleated cells after centrifugation. Among the samples analyzed by IMP, 11 showed a positive blast count, two of which had been inconclusive using CA. No equivalence was found between CA and IMP results (p = 0.55). CSF IMP positivity was not associated with other risk factors for ALL relapse. Among the 55 patients included at the time of diagnosis of ALL, eight relapsed during follow-up. Considering the cases of central nervous system (CNS) relapse, one of the patients belonged to the CSF IMP-positive group (11%) at diagnosis, and the other two cases, to the IMP-negative (5%) group. Detection of CSF blast cells using IMP was associated with a worse overall (p < 0.0001) and event-free survival (p < 0.0001). These results show that CSF IMP may be a useful additional method to conventional CA in the diagnosis of CNS involvement in ALL, and for the identification of high-risk subgroups that would benefit from an intensified therapy.

Current Strategies for the Detection of Minimal Residual Disease in Childhood Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Current treatment strategies for childhood ALL result in long-term remission for approximately 90% of patients. However, the therapeutic response is worse among those who relapse. Several risk stratification approaches based on clinical and biological aspects have been proposed to intensify treatment in patients with high risk of relapse and reduce toxicity on those with a greater probability of cure. The detection of residual leukemic cells (minimal residual disease, MRD) is the most important prognostic factor to identify high-risk patients, allowing redefinition of chemotherapy. In the last decades, several standardized research protocols evaluated MRD using immunophenotyping by flow cytometry and/or real-time quantitative polymerase chain reaction at different time points during treatment. Both methods are highly sensitive (10(-3) a 10(-5)), but expensive, complex, and, because of that, require qualified staff and frequently are restricted to reference centers. The aim of this article was to review technical aspects of immunophenotyping by flow cytometry and real-time quantitative polymerase chain reaction to evaluate MRD in ALL.

Palliative care in pediatric hematological oncology patients: experience of a tertiary hospital.

OBJECTIVE: To evaluate the approach to palliative care for hematological oncology patients in the pediatric ward of a tertiary hospital. METHODS: This was a retrospective, descriptive study of 29 hematological oncology patients who died between 2009 and 2011. Data regarding the approach and prevalence of pain, prevalence of other symptoms, multidisciplinary team participation, communication between staff and family and limited invasive therapy were collected from the medical records. RESULTS: Twenty-seven (93.1%) patients displayed disease progression unresponsive to curative treatment. The median age at death was ten years old. Pain was the most prevalent symptom with all patients who reported pain receiving analgesic medications. The majority took weak (55.2%) and/or strong (65.5%) opioids. The patients were followed by pediatricians and a pediatric hematologist/oncologist. Participation of other professionals was also documented: 86.2% were followed by social services and 69% by psychologists, among others. There were explicit descriptions of limitation of invasive therapy in the medical records of 26 patients who died with disease progression. All these decisions were shared with the families. CONCLUSION: Although the hospital where this study was conducted does not have a specialized team in pediatric palliative care, it meets all the requirements for developing a specific program. The importance of approaching pain and other prevalent symptoms in children with cancer involving a comprehensive multidisciplinary team is evident. Discussions were had with most of the families on limiting invasive therapy, but no record of a well-defined and coordinated treatment plan for palliative care was found.

Hemophagocytic lymphohistiocytosis: a case series of a Brazilian institution.

OBJECTIVE: To describe the clinical and laboratory presentation of hemophagocytic lymphohistiocytosis in children treated at a referral institution. METHODS: A retrospective descriptive study was carried out of seven children diagnosed with hemophagocytic lymphohistiocytosis between 2010 and 2012. The criteria for diagnosis were those proposed by the Histiocyte Society. When indicated, immunochemotherapy was prescribed according to the HLH94 and HLH2004 protocols of the Histiocyte Society. RESULTS: The patients' ages at diagnosis ranged from one month to nine years. All patients had splenomegaly, fever, anemia, thrombocytopenia, hyperferritinemia and hypertriglyceridemia. Bone marrow hemophagocytosis was detected in six patients. In six cases, infectious diseases triggered the syndrome. In two cases, associated with visceral leishmaniasis, remission was achieved after treatment of the underlying infection. Three patients, who had Epstein-Barr-related hemophagocytic lymphohistiocytosis, required treatment with immunochemotherapy. They are alive and in remission; one patient had symptoms of juvenile rheumatoid arthritis and another, who was suspected of having primary hemophagocytic lymphohistiocytosis, entered into remission after bone marrow transplantation. Two deaths (28.6%) occurred in patients with suspected primary hemophagocytic lymphohistiocytosis; one whose clinical picture was triggered by cytomegalovirus infection did not respond to immunochemotherapy and the other died before any specific treatment was provided. CONCLUSION: As reported before, hemophagocytic lymphohistiocytosis has a multifaceted presentation with nonspecific signs and symptoms. In secondary forms, remission may be achieved by treating the underlying disease. In the primary forms, remission may be achieved with immunochemotherapy, but bone marrow transplantation is required for cure.

Gene rearrangement study for minimal residual disease monitoring in children with acute lymphocytic leukemia.

OBJECTIVE: To detect markers for minimal residual disease monitoring based on conventional polymerase chain reaction for immunoglobulin, T-cell receptor rearrangements and the Sil-Tal1 deletion in patients with acute lymphocytic leukemia. METHODS: Fifty-nine children with acute lymphocytic leukemia from three institutions in Minas Gerais, Brazil, were prospectively studied. Clonal rearrangements were detected by polymerase chain reaction followed by homo/heteroduplex clonality analysis in DNA samples from diagnostic bone marrow. Follow-up samples were collected on Days 14 and 28-35 of the induction phase. The Kaplan-Meier and multivariate Cox methods were used for survival analysis. RESULTS: Immunoglobulin/T-cell receptor rearrangements were not detected in 5/55 children screened (9.0%). For precursor-B acute lymphocytic leukemia, the most frequent rearrangement was IgH (72.7%), then TCRG (61.4%), and TCRD and IgK (47.7%); for T-acute lymphocytic leukemia, TCRG (80.0%), and TCRD and Sil-Tal deletion (20.0%) were the most common. Minimal residual disease was detected in 35% of the cases on Day 14 and in 22.5% on Day 28-35. Minimal residual disease on Day 28-35, T-acute lymphocytic leukemia, and leukocyte count above 50 x 10(9)/L at diagnosis were bad prognostic factors for leukemia-free survival in univariate analysis. Relapse risk for minimal residual disease positive relative to minimal residual disease negative children was 8.5 times higher (95% confidence interval: 1.02-70.7). CONCLUSION: Immunoglobulin/T-cell receptor rearrangement frequencies were similar to those reported before. Minimal residual disease is an independent prognostic factor for leukemia-free survival, even when based on a non-quantitative technique, but longer follow-ups are needed.

Incidence and risk factors for central nervous system relapse in children and adolescents with acute lymphoblastic leukemia.

BACKGROUND: Despite all the advances in the treatment of childhood acute lymphoblastic leukemia, central nervous system relapse remains an important obstacle to curing these patients. This study analyzed the incidence of central nervous system relapse and the risk factors for its occurrence in children and adolescents with acute lymphoblastic leukemia. METHODS: This study has a retrospective cohort design. The studied population comprised 199 children and adolescents with a diagnosis of acute lymphoblastic leukemia followed up at Hospital das Clinicas, Universidade Federal de Minas Gerais (HC-UFMG) between March 2001 and August 2009 and submitted to the Grupo Brasileiro de Tratamento de Leucemia da Infância - acute lymphoblastic leukemia (GBTLI-LLA-99) treatment protocol. RESULTS: The estimated probabilities of overall survival and event free survival at 5 years were 69.5% (± 3.6%) and 58.8% (± 4.0%), respectively. The cumulative incidence of central nervous system (isolated or combined) relapse was 11.0% at 8 years. The estimated rate of isolated central nervous system relapse at 8 years was 6.8%. In patients with a blood leukocyte count at diagnosis ≥ 50 x 10(9)/L, the estimated rate of isolated or combined central nervous system relapse was higher than in the group with a count < 50 x 10(9)/L (p-value = 0.0008). There was no difference in cumulative central nervous system relapse (isolated or combined) for the other analyzed variables: immunophenotype, traumatic lumbar puncture, interval between diagnosis and first lumbar puncture and place where the procedure was performed. CONCLUSIONS: These results suggest that a leukocyte count > 50 x 10(9)/L at diagnosis seems to be a significant prognostic factor for a higher incidence of central nervous system relapse in childhood acute lymphoblastic leukemia.

Isolated relapse in the oral cavity of a child with T-lineage acute lymphoblastic leukemia.

Despite high cure rates, approximately 20% of patients with acute lymphoblastic leukemia (ALL) have disease relapse. Isolated recurrence in oral cavity is extremely unusual. The aim of this paper is to report a case of an isolated relapse occurred in a child with T-lineage ALL. Clinical picture included swelling and pain in the right upper gingiva of the oral cavity, with no other clinical or hematological alterations. Diagnosis was confirmed by biopsy and immunohistochemical staining. Bone marrow aspiration was normal. Five months later leukemic infiltration of the bone marrow was detected and systemic chemotherapy was reintroduced. This case report highlights the relevance of dental care during and after chemotherapy, not only to treat lesions in the oral cavity resulting from the disease itself or from treatment side effects, but also to detect unusual sites of ALL relapse.

Compliance with a protocol for acute lymphoblastic leukemia in childhood.

BACKGROUND: Remission rates achieved after the initial treatment of acute lymphoblastic leukemia may be similar in both developed and developing countries, but relapse rates are much higher in the latter. Thus, other reasons are needed, in addition to biological characteristics of the leukemic cells themselves, to explain the unfavorable evolution of patients living in unfavorable socioeconomic and cultural conditions. OBJECTIVE: The aim of this study was to retrospectively evaluate compliance to an acute lymphoblastic leukemia treatment protocol. METHODS: MAIN ABSTRACTED DATA WERE: total duration and reasons for interruption of chemotherapy, prescribed doses of 6-mercaptopurine, and median white blood cell and neutrophil counts during the maintenance phase. Interruptions of chemotherapy were considered inappropriate if they did not follow predetermined criteria established in the protocol. RESULTS: Fourteen of 73 patients (19.2%) unduly interrupted chemotherapy by determination of their physicians. The median white blood cell count was higher when compared with the protocol recommendations; the median 6-MP dose was lower than the standard recommended dose. The estimated probability of event-free survival was higher for patients with lower median leukocyte counts and close to those predetermined by the protocol. Event-free survival was also higher for children with a higher percentage of days without chemotherapy due to bone marrow or liver toxicity excluding undue interruptions. In multivariate analysis, both factors remained statistically significant. These results suggest that the intensity of maintenance chemotherapy may not have been enough in some children, to achieve adequate myelosuppression, hence the observation of higher leukocyte counts and none or rare episodes of therapy interruption. CONCLUSIONS: Compliance to the therapeutic protocol by both doctors and patients should always be considered in the evaluation of therapeutic failure in acute lymphoblastic leukemia; strict adherence to treatment protocols contributes to better treatment results in acute lymphoblastic leukemia children.

Langerhans cell histiocytosis: 37 cases in a single brazilian institution.

OBJECTIVES: To improve the level of 'definitive' diagnosis of Langerhans cell histiocytosis by immunohistochemical investigation of the CD1a surface antigen and to compare outcomes in respect to age, gender, stage of the disease, treatment response and level of diagnostic accuracy. METHODS: A retrospective study was carried out of 37 children and adolescents with possible Langerhans cell histiocytosis between 1988 and 2008. The diagnoses were revisited using immunohistochemical investigations for CD1a, S-100 and CD68 in an attempt to reach definitive diagnoses for all cases. RESULTS: Before the study, only 13 of 37 patients (35.1%) had a 'definitive' diagnosis; by the end of the study, this number rose to 25 patients (67.6%). All reviewed cases were positive for the CD1a antigen. Overall survival was 88.5%. Multisystem disease (Stage 2; n=19) and absence of response at the 6th week of therapy (n=5) were associated to significantly lower overall survival (p-value = 0.04 and 0.0001, respectively). All deaths occurred in patients with multisystem disease and organ dysfunction at diagnosis. Other potential prognostic factors were not significant. Reactivation episodes occurred in 75% of the patients with multisystem disease. Diabetes insipidus was the most common sequel (21.6%). CONCLUSION: The level of diagnostic accuracy was increased through immunohistochemistry. The overall survival rate was similar to international multicentric studies. Multisystem disease and absence of response at six weeks of treatment were the most important unfavorable prognostic factors. The frequency of reactivation for patients with multisystem disease was higher than described in the literature, probably because maintenance chemotherapy was used only in two cases.

Thiopurine S-methyltransferase (TPMT) gene polymorphism in Brazilian children with acute lymphoblastic leukemia: association with clinical and laboratory data.

The frequency of allele variants of gene TPMT*2, *3A, *3B, and *3C was estimated in a population of 116 Brazilian children with acute lymphoblastic leukemia. The association between genotype and clinical and laboratory data obtained during chemotherapy maintenance phase and the correlation of intraerythrocyte concentration of 6-mercaptopurine metabolites (6-tioguanine nucleotide nucleotides and methylmercaptopurine) were analyzed. A multiplex amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) was used in DNA amplification. Twelve patients presented TPMT gene mutation, all in heterozygous form. The most frequent allele variation was TPMT*3A (3.9%), followed by *3C (0.9%), *2 (0.4%), and *3B (0%). There was no significant association between clinical and laboratory data and the presence of mutation in TPMT gene. Of the 36 patients who were monitored for 6-mercaptopurine metabolite levels, only 1 had the mutation. In this patient, high 6-tioguanine nucleotide and low methylmercaptopurine concentrations were found. Event-free survival (EFS) for the whole group was 73.4%. There was no significant difference in event-free survival in the comparison between the groups with and without mutation (P = 0.06).

Langerhans cell histiocytosis: a 16-year experience.

OBJECTIVES: To describe the clinical course of Langerhans cell histiocytosis and to compare its outcome according to age, staging of the disease and treatment response. METHODS: Retrospective analysis of data on 33 children with Langerhans cell histiocytosis followed at Hospital das Clínicas, Universidade Federal de Minas Gerais, Brazil, between 1988 and 2004. RESULTS: Age at diagnosis ranged from 2 months to 16 years (median: 2.5 years). Seventeen children were male. The follow-up period varied from 21 days to 16.2 years (median: 3.4 years). The most common clinical manifestations at diagnosis were osteolytic lesions, enlarged lymph nodes and skin lesions. The overall survival rate for the whole group was 86.1% at 16 years (95%CI 66.6-94.6%). Deaths occurred in patients with multisystem disease and organ dysfunction at diagnosis. Those patients who had a "better" response to treatment in the sixth week were likely to have a significantly higher overall survival rate than those who showed disease progression. Overall survival rate was significantly higher for patients with single-system disease. The disease-free survival rate for the whole group was 30.9% at 16 years (95%CI 15.6-47.5%), and was significantly higher for those with single-system disease. Age groups were not associated with different disease-free survival rates. Diabetes insipidus was the most common sequela. No cases of secondary neoplasms were observed. CONCLUSION: The clinical manifestations of Langerhans cell histiocytosis vary widely, with a high relapse rate and low mortality rate.