Bárbara Amélia Aparecida Santana

Association of the estrogen receptor gene Pvu II restriction polymorphism with expected progeny differences for reproductive and performance traits in swine herds in Brazil

Estrogen has an important function in swine reproduction and growth. A Pvu II restriction enzyme polymorphism has been proven to be an important genetic variation in the estrogen receptor gene (ESR) and may be considered as a candidate gene for use in pig production but there is no data regarding the prevalence of this polymorphism in the Brazilian pig population. We used DNA samples from the following three purebred pig breeds: Large White (336 females and 26 males), Landrace (304 females and 27 males) and Pietrain (125 females and 11 males). The ESR genotyping was performed using PCR-RFLP. For each breed, genotypes for the ESR gene were compared independently for expected progeny differences (EPD) in litter size (LS), average daily weight gain (DWG) (g/day) and back fat thickness (BT) as measured in mm by ultrasound. In the Large White breed, but not the other breeds, the ESR genotype was significantly (p < 0.05) associated to LS, DWG and BT. Large Whites genotyped as AA or AB had higher EPD values for the LS and BT traits compared to BB Large Whites, while AA Large Whites had higher DWG EPD values than BB Large Whites. Our results for the Large White population showed that the A allele has a beneficial effect on LS, DWG and BT expected progeny differences.

Asynchronous expression of myeloid antigens in leukemic cells in a PML/RARalpha transgenic mouse model

Acute promyelocytic leukemia (APL) is characterized by the expansion of blasts that resemble morphologically promyelocytes and harbor a chromosomal translocation involving the retinoic acid receptor alpha (RARalpha) and the promyelocytic leukemia (PML) genes on chromosomes 17 and 15, respectively. The expression of the PML/RARalpha fusion gene is essential for APL genesis. In fact, transgenic mice (TM) expressing PML/RARalpha develop a form of leukemia that mimics the hematological findings of human APL. Leukemia is diagnosed after a long latency (approximately 12 months) during which no hematological abnormality is detected in peripheral blood (pre-leukemic phase). In humans, immunophenotypic analysis of APL blasts revealed distinct features; however, the precise immunophenotype of leukemic cells in the TM model has not been established. Our aim was to characterize the expression of myeloid antigens by leukemic cells from hCG-PML/RARalpha TM. In this study, TM (N = 12) developed leukemia at the mean age of 13.1 months. Morphological analysis of bone marrow revealed an increase of the percentage of immature myeloid cells in leukemic TM compared to pre-leukemic TM and wild-type controls (48.63 +/- 16.68, 10.83 +/- 8.11, 7.4 +/- 5.46%, respectively; P < 0.05). Flow cytometry analysis of bone marrow and spleen from leukemic TM identified the asynchronous co-expression of CD34, CD117, and CD11b. This abnormal phenotype was rarely detected prior to the diagnosis of leukemia and was present at similar frequencies in hematologically normal TM and wild-type controls of different ages. The present results demonstrate that, similarly to human APL, leukemic cells from hCG-PML/RARalpha TM present a specific immunophenotype.

Telomere length correlates with disease severity and inflammation in sickle cell disease

Background Telomeres, the ends of linear chromosomes, shorten during mitotic cell division and erosion may be aggravated by inflammation or proliferative and oxidative stress. As the bone marrow is under hyperproliferative pressure in sickle cell disease and several tissues are submitted to chronic inflammation, this study sought to determine the telomere length of patients with sickle cell disease. Methods The mean telomere length was measured in peripheral blood leukocytes by quantitative polymerase chain reaction. The age-adjusted telomere to single copy gene ratio was compared between 91 adult sickle cell disease patients and 188 controls. Results Sickle cell disease patients had significantly shorter telomeres than the controls (p-value < 0.0001). Moreover, among sickle cell disease genotypes, Hb SS patients had significantly shorter telomeres compared to Hb SC and Hb Sβ patients (p-value < 0.0001). Patients on hydroxyurea also had shorter telomeres in comparison to those off the drug (p-value = 0.02). A positive correlation was observed between telomere length and hemoglobin level (r = 0.3; p-value = 0.004), whereas negative correlations were detected between telomere length and lymphocyte count (r = −0.3; p-value = 0.005) and interleukin-8 serum levels (r = −0.4; p-value = 0.02). Conclusions The findings of this study indicate that telomeres are short in sickle cell disease patients and that telomere erosion directly correlates with disease genotype, inflammation markers, and the use of hydroxyurea.

Comparison of microRNA expression in high-count monoclonal B-cell lymphocytosis and Binet A chronic lymphocytic leukemia

Background Evidence suggests that monoclonal B-cell lymphocytosis precedes all chronic lymphocytic leukemia cases, although the molecular mechanisms responsible for disease progression are not understood. Aberrant miRNA expression may contribute to the pathogenesis of chronic lymphocytic leukemia. The objective of this study was to compare miRNA expression profiles of patients with Binet A chronic lymphocytic leukemia with those of subjects with high-count monoclonal B-cell lymphocytosis and healthy volunteers (controls). Methods Twenty-one chronic lymphocytic leukemia patients, 12 subjects with monoclonal B-cell lymphocytosis and ten healthy volunteers were enrolled in this study. Flow cytometry CD19+CD5+-based cell sorting was performed for the chronic lymphocytic leukemia and monoclonal B-cell lymphocytosis groups and CD19+ cells were sorted to analyze the control group. The expressions of miRNAs (miR-15a, miR-16-1, miR-29b, miR-34a, miR-181a, miR-181b and miR-155) were determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Results Significant differences between the expressions in the chronic lymphocytic leukemia and monoclonal B-cell lymphocytosis groups were restricted to the expression of miR-155, which was higher in the former group. A comparison between healthy controls and monoclonal B-cell lymphocytosis/chronic lymphocytic leukemia patients revealed higher miR-155 and miR-34a levels and lower miR-15a, miR-16-1, miR-181a and miR-181b in the latter group. Conclusions Our results show a progressive increase of miR-155 expression from controls to monoclonal B-cell lymphocytosis to chronic lymphocytic leukemia. The role of miR-155 in the development of overt chronic lymphocytic leukemia in individuals with monoclonal B-cell lymphocytosis must be further analyzed.

THPO gene variants in patients with acquired aplastic anemia

Background Human aplastic anemia is a hematologic disease characterized by low peripheral blood cell counts associated with reduced numbers of hematopoietic stem and progenitor cells and a hypocellular bone marrow. Thrombopoietin (THPO) regulates megakaryocytes, but it also stimulates hematopoietic stem and progenitor cells. Biallelic mutations in the THPO gene have been reported in a family with recessive inherited aplastic anemia. Methods This study screened 83 patients diagnosed with acquired aplastic anemia and 92 paired healthy controls for germline variants in the THPO gene using Sanger sequencing. Results Three common single nucleotide polymorphisms were identified in patients and controls at comparable allele frequencies. There was no correlation between the single nucleotide polymorphism carrier status and platelet counts at diagnosis. Conclusion The presence of THPO polymorphisms is comparable between patients with acquired aplastic anemia and healthy individuals.

Skewed X-chromosome inactivation and shorter telomeres associate with idiopathic premature ovarian insufficiency

OBJECTIVE To analyze whether telomere length, X-chromosome inactivation (XCI), and androgen receptor (AR) GAG polymorphism are related to idiopathic premature ovarian insufficiency (POI). DESIGN Case-control study. SETTING University hospital. PATIENT(S) A total of 121 women, including 46 nonsyndromic POI and 75 controls. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Age, weight, height, body mass index (BMI), systolic and diastolic arterial pressure, E2, androstenedione, T, and C-reactive protein were assessed. Telomere length was estimated by quantitative real-time polymerase chain reaction, XCI was measured using the Human Androgen Receptor and X-linked retinitis pigmentosa 2 (RP2) methylation assays. AR and FMR1 polymorphism was assessed by quantitative fluorescent polymerase chain reaction and sequencing. RESULT(S) Premature ovarian insufficiency women had a higher mean age, weighed less, and exhibited lower C-reactive protein, E2, and androstenedione levels. The AR polymorphism did not differ between the groups. Four patients had premutation (55-200 CGG repeats), and none displayed a full mutation in the FMR1 gene. However, patients with POI showed shorter telomere length and higher frequency of skewed XCI. Extreme skewing (≥90%) was observed in 15% of women with POI, and shorter telomeres correlated with XCI skewing in both groups. CONCLUSION(S) Skewed XCI and shortened telomere length were associated with idiopathic POI, despite no alterations in the AR and FMR1 genes. Additionally, there is a tendency for women with short telomeres to exhibit skewed XCI.

Abstract B33: Synergistic effect of gefitinib and arsenic trioxide in acute promyelocytic leukemia

Gefitinib and erlotinib are well-known epidermal growth factor receptor (EGFR) inhibitors approved by the Food and Drug Administration for the treatment of non-small cell lung cancer (NSCLC). Interestingly, patients with synchronous NSCLC and acute myeloid leukemia (AML) treated with erlotinib presented regression of both neoplasms, although AML myeloblasts were shown to lack expression of EGFR. In addition, preclinical studies have shown that gefitinib alone or combined with arsenic trioxide (ATO) or all-trans retinoic acid (ATRA) was able to induce differentiation in the EGFR-negative cell lines of acute promyelocytic leukemia (APL), which is a subtype of AML characterized by the t(15;17)/PML/RARA rearrangement. Our previous study demonstrated that EGFR gene expression levels were associated with prognostic outcomes of APL patients treated according to the International Consortium on APL protocol. However, the EGFR levels were not assessed at protein level. Two phase 3 trials reported that ATRA and ATO therapy was associated with excellent outcomes in APL. The two drugs induced the degradation of the PML/RARA oncoprotein through different mechanisms, with ATRA acting through the proteasome pathway, and ATO functioning through the PML-transformation related protein 53 (Trp53) axis. The purpose of this study was to investigate whether gefitinib or erlotinib has synergistic effects with ATO or ATRA in the treatment of APL. To this end, the APL cell lines NB4 and NB4-R2 were treated with gefitinib (C22H24ClFN4O; Selleck Chemicals, #S7786) and erlotinib (C22H23N3O4; Selleck-Chemicals, #S1025) alone or combined with ATO (As2O3; Sigma-Aldrich, #202673). The stock solutions of gefitinib and erlotinib were prepared in DMSO while ATO was dissolved in 1M NaOH solution. Then each drug was diluted with RPMI medium to the desired final concentration. The median effective dose (ED50) for gefitinib was calculated to be 20.97 and 27.06 µM for NB4 and NB4-R2, respectively. ATO was a potent inducer of apoptosis with an ED50 of 2.27 µM for NB4 and 1.73 µM for NB4-R2 cells. ED50 of ATRA (C20H28O2; Sigma-Aldrich, #R2625) was not calculated because ATRA is a potent differentiation inducer but did not induce cell death of APL blasts. Due to the high value of ED50 for NB4 (78.97 µM) and NB4-R2 (111.36 µM) after 24 h of erlotinib treatment, the analysis of this drug was discontinued. The interaction between gefitinib and ATO demonstrated a moderate synergism for NB4 and NB4-R2 with a combination index values of 0.88 and 0.83, respectively. These in vitro results encouraged us to investigate the in vivo effects of gefitinib alone or in combination with ATO and/or ATRA. To this second end, we developed a syngeneic transplant murine model using leukemic cells obtained from transgenic mice hCG-PML/RARA, which develop a form of leukemia that closely recapitulates the human disease. The engraftment of leukemic cells from hCG-PML-RARα transgenic mice transplanted to wild-type littermate recipients was evaluated by morphology and flow cytometry analysis of the peripheral blood (PB), bone marrow (BM), and spleen. Recipient mice were euthanized and evaluated for signs of engraftment 14 (n=3) and 20 (n=3) days after transplantation. Of note, the morphologic analysis revealed >20% of blasts among nucleated cells in the BM samples. The flow cytometric analysis showed the presence of CD11b+ CD117+ cells in PB (median 10.50, range 8.81-12.20), BM (median 18.30, range 17.80-18.80), and spleen (median 16.45, range 16.00-16.90). These results demonstrate that this rapid and robust murine model is useful to assess the efficacy of EGFR inhibitors in APL. Taken together, our preliminary in vitro results suggest that gefitinib and ATO may have potential application for the APL treatment and that this current syngeneic transplant mouse model of APL is suitable to test this hypothesis. Citation Format: Luciana Yamamoto Almeida, Cleide Lucia Araujo Silva, Isabel Weinhauser, Larissa Ananias Cândido, Priscila Santos Scheucher, Camila Cristina Oliveira Menezes Bonaldo, Barbara Amelia Aparecida Santana, Ana Silvia Gouvea Lima Yamada, Eduardo Magalhaes Rego. Synergistic effect of gefitinib and arsenic trioxide in acute promyelocytic leukemia [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; Sao Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B33.

Telomere biology and telomerase mutations in cirrhotic patients with hepatocellular carcinoma

Telomeres are repetitive DNA sequences at linear chromosome termini, protecting chromosomes against end-to-end fusion and damage, providing chromosomal stability. Telomeres shorten with mitotic cellular division, but are maintained in cells with high proliferative capacity by telomerase. Loss-of-function mutations in telomere-maintenance genes are genetic risk factors for cirrhosis development in humans and murine models. Telomerase deficiency provokes accelerated telomere shortening and dysfunction, facilitating genomic instability and oncogenesis. Here we examined whether telomerase mutations and telomere shortening were associated with hepatocellular carcinoma (HCC) secondary to cirrhosis. Telomere length of peripheral blood leukocytes was measured by Southern blot and qPCR in 120 patients with HCC associated with cirrhosis and 261 healthy subjects. HCC patients were screened for telomerase gene variants (in TERT and TERC) by Sanger sequencing. Age-adjusted telomere length was comparable between HCC patients and healthy subjects by both Southern blot and qPCR. Four non-synonymous TERT heterozygous variants were identified in four unrelated patients, resulting in a significantly higher mutation carrier frequency (3.3%) in patients as compared to controls (p = 0.02). Three of the four variants (T726M, A1062T, and V1090M) were previously observed in patients with other telomere diseases (severe aplastic anemia, acute myeloid leukemia, and cirrhosis). A novel TERT variant, A243V, was identified in a 65-year-old male with advanced HCC and cirrhosis secondary to chronic hepatitis C virus (HCV) and alcohol ingestion, but direct assay measurements in vitro did not detect modulation of telomerase enzymatic activity or processivity. In summary, constitutional variants resulting in amino acid changes in the telomerase reverse transcriptase were found in a small proportion of patients with cirrhosis-associated HCC.