BRCA1 and BRCA2 germline mutation analysis from a cohort of 1267 patients at high risk for breast cancer in Brazil.

We determined the frequency and mutational spectrum of BRCA1 and BRCA2 in a series of patients at high risk for developing breast cancer from Brazil. A total of 1267 patients were referred for BRCA genetic testing, and no obligation of fulfilling criteria of mutation probability methods for molecular screening was applied. Germline deleterious mutations in BRCA1/2 (i.e., pathogenic/likely pathogenic variants) were identified in 156 out of 1267 patients (12%). We confirm recurrent mutations in BRCA1/2, but we also report three novel mutations in BRCA2, not previously reported in any public databases or other studies. Variants of unknown significance (VUS) represent only 2% in this dataset and most of them were detected in BRCA2. The overall mutation prevalence in BRCA1/2 was higher in patients diagnosed with cancer at age > 35 years old, and with family history of cancer. The present data expand our knowledge of BRCA1/2 germline mutational spectrum, and it is a valuable clinical resource for genetic counseling and cancer management programs in the country.

An unusual long-term outcome of a child with primary myelofibrosis harboring a JAK2 mutation.

We report an extremely rare case of a female child who presented the onset of primary myelofibrosis (PMF) harboring JAK2 (Janus Kinase 2 gene) mutation (JAK2V617F) when she was 15 months old. She was monitored over 25 years, a period in which she was treated with spleen radiotherapy and recombinant interferon α. She also underwent splenectomy when she was 13 years old, due to massive splenomegaly, anemia and various infection disease episodes. The longstanding evolution of the patient enabled us to verify that there were no complications related to post-splenectomy events and/or blast transformation. To the best of our knowledge, this is the first reported case of severe PMF with JAK2 mutation in a child. We provide evidence that a better quality of life and long survival in pediatric PMF may be provided by splenectomy.

BCR-ABL Mutations in Chronic Myeloid Leukemia Treated With Tyrosine Kinase Inhibitors and Impact on Survival.

This is the largest Latin American study of BCR-ABL mutations in chronic myeloid leukemia (CML) patients, resistant to imatinib (IM). In 195/467 (41%) patients, mutations were detected. The most frequent mutation was T315I (n = 31, 16%). Progression-free (PFS) and overall survival (OS) at 5 years were lower in patients with BCR-ABL mutations (43% vs. 65%, p = 0.07 and 47% vs. 72%, p = 0.03, respectively) and in those with the T315I mutation (p = 0.003 and p = 0.03). OS and PFS were superior in subgroup who switched to second generation inhibitors (SGIs) after IM failure (OS: 50% vs. 39% p = 0.01; PFS: 48% vs. 30% p = 0.02). BCR-ABL mutations conferred a significant poor prognosis in CML patients.

BCR-ABL mutations in chronic myeloid leukemia treated with tyrosine kinase inhibitors and impact on survival.

This is the largest Latin American study of BCR-ABL mutations in chronic myeloid leukemia (CML) patients, resistant to imatinib (IM). In 195/467 (41%) patients, mutations were detected. The most frequent mutation was T315I (n = 31, 16%). Progression-free (PFS) and overall survival (OS) at 5 years were lower in patients with BCR-ABL mutations (43% vs. 65%, p = 0.07 and 47% vs. 72%, p = 0.03, respectively) and in those with the T315I mutation (p = 0.003 and p = 0.03). OS and PFS were superior in subgroup who switched to second generation inhibitors (SGIs) after IM failure (OS: 50% vs. 39% p = 0.01; PFS: 48% vs. 30% p = 0.02). BCR-ABL mutations conferred a significant poor prognosis in CML patients.

Molecular characterization of V(D)J rearrangements in immature acute leukemias.

Early T-cell Precursor Acute Lymphoblastic Leukemia (ETP-ALL), T-Lymphoid/Myeloid Mixed Phenotype Acute Leukemia (T/M-MPAL), and Acute Myeloid Leukemia with minimal differentiation (AML-M0) are immature acute leukemias (AL) that present overlapping T-cell lymphoid and myeloid features at different degrees, with impact to disease classification. An interesting strategy to assess lymphoid lineage commitment and maturation is the analysis of V(D)J gene segment recombination, which can be applied to investigate leukemic cells in immature AL. Herein, we revisited 19 ETP-ALL, 8 T/M-MPAL, and 12 AML-M0 pediatric patients to characterize V(D)J rearrangement (V(D)J-r) profiles associated with other somatic alterations. V(D)J-r were identified in 74 %, 25 %, and 25 % of ETP-ALL, T/M-MPAL, and AML-M0, respectively. Forty-six percent of ETP-ALL harbored ≥ 3 V(D)J-r, while there was no more than one V(D)J-r per patient in AML-M0 and T/M-MPAL. TCRD was the most rearranged locus in ETPALL, but it was not rearranged in other AL. In ETP-ALL, N/KRAS mutations were associated with absence of V(D)J-r, while NF1 deletion was most frequent in patients with ≥ 3 V(D)J-r. Relapse and death occurred mainly in patients harboring one or no rearranged locus. Molecular characterization of V(D)J-r in our cohort indicates a distinct profile of ETP-ALL, compared to T/M-MPAL and AML-M0. Our findings also suggest that the clinical outcome of ETP-ALL patients may be affected by blast cell maturity, inferred from the number of rearranged TCR loci.

Acute Myeloid Leukemia Expresses a Specific Group of Olfactory Receptors.

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults, with a 5-year overall survival rate of approximately 30%. Despite recent advances in therapeutic options, relapse remains the leading cause of death and poor survival outcomes. New drugs benefit specific small subgroups of patients with actionable therapeutic targets. Thus, finding new targets with greater applicability should be pursued. Olfactory receptors (ORs) are seven transmembrane G-protein coupled receptors preferentially expressed in sensory neurons with a critical role in recognizing odorant molecules. Recent studies have revealed ectopic expression and putative function of ORs in nonolfactory tissues and pathologies, including AML. Here, we investigated OR expression in 151 AML samples, 6400 samples of 15 other cancer types, and 11,200 samples of 51 types of healthy tissues. First, we identified 19 ORs with a distinct and major expression pattern in AML, which were experimentally validated by RT-PCR in an independent set of 13 AML samples, 13 healthy donors, and 8 leukemia cell lines. We also identified an OR signature with prognostic potential for AML patients. Finally, we found cancer-related genes coexpressed with the ORs in the AML samples. In summary, we conducted an extensive study to identify ORs that can be used as novel biomarkers for the diagnosis of AML and as potential drug targets.

TET2 mutations as a part of DNA dioxygenase deficiency in myelodysplastic syndromes.

Decrease in DNA dioxygenase activity generated by TET2 gene family is crucial in myelodysplastic syndromes (MDS). The general downregulation of 5-hydroxymethylcytosine (5-hmC) argues for a role of DNA demethylation in MDS beyond TET2 mutations, which albeit frequent, do not convey any prognostic significance. We investigated TETs expression to identify factors which can modulate the impact of mutations and thus 5-hmC levels on clinical phenotypes and prognosis of MDS patients. DNA/RNA-sequencing and 5-hmC data were collected from 1665 patients with MDS and 91 controls. Irrespective of mutations, a significant fraction of MDS patients exhibited lower TET2 expression, whereas 5-hmC levels were not uniformly decreased. In searching for factors explaining compensatory mechanisms, we discovered that TET3 was upregulated in MDS and inversely correlated with TET2 expression in wild-type cases. Although TET2 was reduced across all age groups, TET3 levels were increased in a likely feedback mechanism induced by TET2 dysfunction. This inverse relationship of TET2 and TET3 expression also corresponded to the expression of L-2-hydroxyglutarate dehydrogenase, involved in agonist/antagonist substrate metabolism. Importantly, elevated TET3 levels influenced the clinical phenotype of TET2 deficiency whereby the lack of compensation by TET3 (low TET3 expression) was associated with poor outcomes of TET2 mutant carriers.

Pediatric Acute Promyelocytic Leukemia: Epidemiology, Molecular Features, and Importance of GST-Theta 1 in Chemotherapy Response and Outcome.

Previous studies have suggested a variation in the incidence of acute promyelocytic leukemia (APL) among the geographic regions with relatively higher percentages in the Latin American population. We aimed to explore the population burden of pediatric APL, gathering information from the population-based cancer registry (PBCR) and the diagnosis of APL obtained through incident cases from a hospital-based cohort. The homozygous deletion in glutathione S-transferases (GSTs) leads to a loss of enzyme detoxification activity, possibly affecting the treatment response. Mutations in the RAS pathway genes are also considered to be a key component of the disease both in the pathogenesis and in the outcomes. We have assessed mutations in a RAS-MAP kinase pathway (FLT3, PTPN11, and K-/NRAS) and GST variant predisposition risk in the outcome. Out of the 805 children and adolescents with acute myeloid leukemia (AML) who are registered in the PBCR, 35 (4.3%) were APL cases. The age-adjusted incidence rate (AAIR) was 0.03 per 100,000 person-years. One-hundred and sixty-three patients with APL were studied out of 931 AML cases (17.5%) from a hospital-based cohort. Mutations in FLT3, KRAS, and NRAS accounted for 52.1% of the cases. Patients with APL presented a 5-year probability of the overall survival (OS) of 67.3 ± 5.8%. A GST-theta 1 (GSTT1) null genotype conferred adverse prognosis, with an estimated hazard ratio of 2.8, 95% confidence interval (CI) 1.2-6.9. We speculate that the GSTT1 polymorphism is associated with therapeutics and would allow better OS of patients with APL with a GSTT1 null genotype.

JAK2V617F mutation in patients with splanchnic vein thrombosis.

BACKGROUND: Splanchnic vein thrombosis can be the presenting manifestation of myeloproliferative neoplasms. However, the diagnosis of a myeloproliferative neoplasm in these patients is often problematic, and more objective criteria are needed. AIM: To determine the frequency of the mutation JAK2V617F in patients with splanchnic vein thromboses. METHODS: A consecutive series of 108 adult patients with portal vein thrombosis (n = 77) and Budd-Chiari syndrome (n = 31) referred for hemostasis evaluation was retrospectively studied, with a median follow-up of 51 months (1-104). RESULTS: One or more prothrombotic risk factors were present in 63% of the patients. Twenty-four (22%) out of the 108 patients presented the JAK2V617F, including 2 cirrhotic patients. Most had a low mutated allele burden (median 16.5%). JAK2V617F was present in all four patients with a previous diagnosis of a myeloproliferative neoplasm. In nine JAK2V617F-positive patients, the diagnosis of a myeloproliferative neoplasm was made at the thrombosis work-up, during follow-up or after JAK2V617F detection. Among the other 11 patients carrying the mutation, 2 patients have died, 4 had no evidence suggesting a myeloproliferative neoplasm, 1 had a normal bone marrow biopsy, and the other 4 could not be persuaded to undergo a biopsy. Among the patients without an overt myeloproliferative neoplasm, 15 out of 99 (15%) presented the JAK2V617F mutation. None of the JAK2V617F-negative patients have developed signs of a myeloproliferative neoplasm during follow-up. CONCLUSIONS: Our findings suggest that JAK2V617F occurs in a high proportion of patients with splanchnic vein thrombosis, and reinforces the diagnostic utility of JAK2V617F testing in this setting.

Detection of somatic TP53 mutations and 17p deletions in patients with chronic lymphocytic leukemia: a review of the current methods.

Chronic lymphocytic leukemia is the most common hematologic malignancy among adults in Western countries. Several studies show that somatic mutations in the TP53 gene are present in up to 50% of patients with relapsed or refractory chronic lymphocytic leukemia. This study aims to review and compare the methods used to detect somatic TP53 mutations and/or 17p deletions and analyze their importance in the chronic lymphocytic leukemia diagnosis and follow-up. In chronic lymphocytic leukemia patients with refractory or recurrent disease, the probability of clonal expansion of cells with the TP53 mutation and/or 17p deletion is very high. The studies assessed showed several methodologies able to detect these changes. For the 17p deletion, the chromosome G-banding (karyotype) and interphase fluorescence in situ hybridization are the most sensitive. For somatic mutations involving the TP53 gene, moderate or high-coverage read next-generation sequencing and Sanger sequencing are the most recommended ones. The TP53 gene mutations represent a strong adverse prognostic factor for patient survival and treatment resistance in chronic lymphocytic leukemia. Patients carrying low-proportion TP53 mutation (less than 20-25% of all alleles) remain a challenge to these tests. Thus, for any of the methods employed, it is essential that the laboratory conduct its analytical validation, documenting its accuracy, precision and sensitivity/limit of detection.

Multidisciplinary diagnostics of chronic lymphocytic leukemia: European Research Initiative on CLL - ERIC recommendations.

Recent advances in chronic lymphocytic leukemia (CLL) includes description of disease genomic landscape, inclusion of prognostic relevant genetic tests in CLL workflow and evaluation of minimal residual disease (MRD)1 in parallel with the increase availability of novel therapy agents. In this review, the theoretical and practical aspects of response assessment have been discussed. These are based on updated recommendations of the European Research Initiative on Chronic Lymphocytic Leukemia (ERIC) for genetic tests (TP53 mutation and IGHV status) and flow cytometry analysis for CLL. Methodological approaches and interpretation of results were also discussed.2,3.

miRNome profiling of LSC-enriched CD34+CD38-CD26+ fraction in Ph+ CML-CP samples from Argentinean patients: a potential new pharmacogenomic tool.

Chronic myeloid leukemia (CML) is a myeloid stem cell neoplasm characterized by an expansion of myeloid progenitor cells and the presence of BCR-ABL1 oncoprotein. Since the introduction of specific BCR-ABL1 tyrosine kinase inhibitors (TKI), overall survival has improved significantly. However, under long-term therapy patients may have residual disease that originates from TKI-resistant leukemic stem cells (LSC). In this work, we analyzed the miRNome of LSC-enriched CD34+CD38-CD26+ and normal hematopoietic stem cells (HSC) fractions obtained from the same chronic phase (CP) CML patients, and stem and progenitor cells obtained from healthy donors (HD) by next-generation sequencing. We detected a global decrease of microRNA levels in LSC-enriched CD34+CD38-CD26+ and HSC fractions from CML-CP patients, and decreased levels of microRNAs and snoRNAs from a genomic cluster in chromosome 14, suggesting a mechanism of silencing of multiple non-coding RNAs. Surprisingly, HSC from CML-CP patients, despite the absence of BCR-ABL1 expression, showed an altered miRNome. We confirmed by RT-qPCR that the levels of miR-196a-5p were increased more than nine-fold in CD26+ (BCR-ABL1 + ) vs. CD26- (BCR-ABL1 -) CD34+CD38- fractions from CML-CP patients at diagnosis, and in silico analysis revealed a significant association to lipid metabolism and hematopoiesis functions. In the light of recent descriptions of increased oxidative metabolism in CML LSC-enriched fractions, these results serve as a guide for future functional studies that evaluate the role of microRNAs in this process. Metabolic vulnerabilities in LSCs open the road for new therapeutic strategies. This is the first report of the miRNome of CML-CP CD34+CD38- fractions that distinguishes between CD26+ (BCR-ABL1 + ) and their CD26- (BCR-ABL1 - ) counterparts, providing valuable data for future studies.

Myeloid malignancies-related somatic mutations in aging individuals.

We search for the presence of somatic mutations in 12 genes related to MDS, MPN, and AML in a Brazilian cohort composed of 609 elderly individuals from a census-based sample.

Presence of CD34+ in Megakaryocytes in Association With p53 Expression Predicts Unfavorable Prognosis in Low-risk Myelodysplastic Syndrome Patients.

BACKGROUND/AIM: Although risk stratification using the Prognostic Scores Systems (IPSS, WPSS and IPSS-R) incorporate key information about prognosis of patients with Myelodysplastic syndromes (MDS), patients classified as low-risk may evolve rapidly and aggressively, despite a "favorable" prognostic stratification. The aim of this study was to identify biomarkers for predicting prognosis, and for better stratification and management of these patients. MATERIALS AND METHODS: Expression of CD34 and p53 in megakaryocytes was examined by immunohistochemistry in 71 MDS patients classified as low-risk. RESULTS: CD34 staining in megakaryocytes was associated with p53 expression (p=0.0166). CD34 and p53 expression were associated to worse overall survival in patients (p=0.0281). CONCLUSION: The presence of CD34 in megakaryocytes is associated with p53 expression and an adverse prognosis for MDS patients.

Clinical and demographic characteristics of Epstein-Barr virus-associated childhood Burkitt's lymphoma in Southeastern Brazil: epidemiological insights from an intermediate risk region.

We studied a group of 54 children with Burkitt's lymphoma from Southeastern Brazil, where epidemiological status of Burkitt's lymphoma is poorly understood. Epstein-Barr virus association showed an intermediate frequency (~60%) between endemic and sporadic subtypes. Median age was five years. Epstein-Barr virus infection was significantly associated to low age (Epstein-Barr virus(+) four years vs. Epstein-Barr virus(-) eight years). Sex ratio (M:F) was 2:1, with a significantly higher number of males in old age classes. Young age at diagnosis and excess of males at older ages, as well as a causal relationship between low age, epstein-barr virus and Burkitt's lymphoma risk, may characterize Burkitt's lymphoma in Brazil.

Clinical and laboratorial prediction of bone marrow involvement in children and adolescents with Hodgkin Lymphoma.

BACKGROUND: A risk group (RG) system to predict bone marrow involvement (BMI) based on clinical and laboratory parameters was previously shown to have prognostic value in adult patients with Hodgkin lymphoma (HL). Our aim was to test the applicability of BMI RG in an independent group of childhood and adolescent HL patients. PROCEDURE: Seventy-eight HL patients (range 3-18, median 14 years old) were retrospectively studied, including revision of histopathological diagnosis and bilateral BM biopsies. Patients were divided into BMI high-, standard- and low-RG. RESULTS: The high-RG included 29.5% of the patients, the standard-RG, 32%, and the low-RG, 38.5%. All the patients in the high-RG had stage III or IV. The five children (6.4%) with BMI were included in the high-RG (P = 0.001, chi(2) test) and had stage IV (P < 0.00001, chi(2) test). The BMI risk group out-performed the clinico-pathologic risk assessment in predicting BMI, showing 100% sensitivity, 75% specificity and a positive predictive value of 0.88. Neither BMI nor risk of BMI was statistically associated to age groups (14 years old), suggesting that age is not a risk factor for BMI in the pediatric population. CONCLUSIONS: The application of BMI RG score was able to efficiently foresee BMI in a pediatric group of HL patients, adding independent information to clinico-pathologic BMI risk assessment.