ESR1 Mutations Are Not a Common Mechanism of Endocrine Resistance in Patients With Estrogen Receptor-Positive Breast Cancer Treated With Neoadjuvant Aromatase Inhibitor Therapy.

Introduction: Mutations in the ESR1 gene (ESR1m) are important mechanisms of resistance to endocrine therapy in estrogen receptor-positive (ER+) metastatic breast cancer and have been studied as a potential therapeutic target, as well as a predictive and prognostic biomarker. Nonetheless, the role of ESR1m as a possible mechanism of primary endocrine resistance, as well as whether it also occurs in tumors that are resistant to ET administered in early-stage disease as (neo)adjuvant, has not been adequately studied. In this study, we evaluated the prevalence of ESR1m in tumor samples from patients with ER+ breast cancer resistant to neoadjuvant aromatase inhibitor therapy. Methods: We followed a prospective cohort of patients with ER+ HER2- stages II and III breast cancer treated with neoadjuvant endocrine therapy (NET). Tumor samples from patients with a pattern of primary endocrine resistance [defined as a Preoperative Endocrine Prognostic Index (PEPI) score of ≥4] were identified and analyzed for the presence of ESR1m. Results: One hundred twenty-seven patients were included in the cohort, of which 100 (79%) had completed NET and underwent surgery. Among these patients, the PEPI score ranged from 0 to 3 in 70% (70/100), whereas 30% (30/100) had a PEPI score of 4 or more. Twenty-three of these patients were included in the analysis. ESR1 mutations were not identified in any of the 23 patients with early-stage ER+ breast cancer resistant to NET. Discussion: Growing evidence supports the notion that there are different mechanisms for primary and secondary endocrine resistance. Our study suggests that ESR1 mutations do not evolve rapidly and do not represent a common mechanism of primary endocrine resistance in the neoadjuvant setting. Therefore, ESR1m should be considered a mechanism of acquired endocrine resistance in the context of advanced disease. Further research should be conducted to identify factors associated with intrinsic resistance to ET.

Association of ESR1 Mutations and Visceral Metastasis in Patients with Estrogen Receptor-Positive Advanced Breast Cancer from Brazil.

Mutations in the ESR1 gene (ESR1m) are important mechanisms of resistance to endocrine therapy in estrogen receptor-positive advanced breast cancer and have been recognized as a prognostic and predictive biomarker as well as a potential therapeutic target. However, the prevalence of ESR1m in real-world patients has not been adequately described. Therefore, we sought to evaluate the prevalence of ESR1m in metastatic samples from Brazilian patients with estrogen receptor-positive (ER+) advanced breast cancer previously treated with endocrine therapy. The presence of ESR1m was evaluated in formalin-fixed paraffin-embedded (FFPE) breast cancer tissue using real-time quantitative polymerase chain reaction (RT-qPCR). Mutations in codons 380, 537, and 538 of the ESR1 gene were analyzed. Out of 77 breast cancer samples, 11 (14.3%) showed mutations in the ESR1 gene. ESR1m were detected in a variety of organs, and the D538G substitution was the most common mutation. In visceral metastasis, ESR1m were detected in 25% (8/32) of the samples, whereas in nonvisceral metastasis, ESR1m were detected in 6.7% (3/45) of the samples. The odds of a sample with visceral metastasis having an ESR1 mutation is 4.66 times the odds of a sample of nonvisceral metastasis having an ESR1 mutation (95% CI: 1.13-19.27; p value = 0.0333). Our study indicates that the prevalence of ESR1m in samples from Brazilian patients with metastatic ER+ breast cancer is similar to that described in patients included in clinical trials. We observed an association of ESR1m with visceral metastasis.

Human papillomavirus among women with atypical squamous cells of undetermined significance in southern Brazil.

OBJECTIVE: To determine the prevalence of atypical squamous cells of undetermined significance (ASCUS) and human papillomavirus (HPV) genotypes in a population in southern Brazil. METHODS: In a retrospective cross-sectional study, the prevalence of ASCUS was determined among women aged 20-60 years who were referred to a private medical center in Caxias do Sul by a gynecologist for assessment of a cervical condition between January 1, 2010, and September 30, 2011. Histologic and cytologic samples were tested for HPV, and polymerase chain reaction (PCR) was used to genotype any HPV DNA identified. RESULTS: Among the 250 included women, 25 (10.0%) had ASCUS. HPV DNA was found in 15 (60.0%) women with ASCUS and 115 (51.1%) of the 225 without ASCUS. Viral typing showed that 7 (46.7%) HPV-positive women with ASCUS had multiple infections with up to five different genotypes. Both low- and high-risk HPV genotypes were found in ASCUS samples; the most prevalent genotypes were HPV6/HPV11 (affecting 10 [66.7%] women), HPV51 (6 [40.0%]), and HPV16 (6 [40.0%]). CONCLUSION: ASCUS is not an indication of HPV infection. HPV screening and genotyping would benefit women with ASCUS, because treatment can be planned according to risk of carcinogenesis.

Perinatal transmission of human papilomavirus DNA.

The purpose was to study the perinatal transmission of human papillomavirus DNA (HPV-DNA) in 63 mother-newborn pairs, besides looking at the epidemiological factors involved in the viral DNA transmission. The following sampling methods were used: (1) in the pregnant woman, when was recruited, in cervix and clinical lesions of the vagina, vulva and perineal region; (2) in the newborn, (a) buccal, axillary and inguinal regions; (b) nasopharyngeal aspirate, and (c) cord blood; (3) in the children, buccal was repeated in the 4th week and 6th and 12th month of life. HPV-DNA was identified using two methodologies: multiplex PCR (PGMY09 and MY11 primers) and nested-PCR (genotypes 6/11, 16, 18, 31, 33, 42, 52 and 58). Perinatal transmission was considered when concordance was found in type-specific HPV between mother/newborn or mother/child. HPV-DNA genital was detected in 49 pregnant women submitted to delivery. Eleven newborns (22.4%, n = 11/49) were HPV-DNA positive. In 8 cases (16.3%, n = 8/49) there was type specific HPV concordance between mother/newborn samples. At the end of the first month of life three children (6.1%, n = 3/49) became HPV-DNA positive, while two remained positive from birth. In 3 cases (100%, n = 3/3) there was type specific HPV concordance between mother/newborn samples. In the 6th month, a child (2%, n = 1/49) had become HPV-DNA positive between the 1st and 6th month of life, and there was type specific HPV concordance of mother/newborn samples. All the HPV-DNA positive children (22.4%, n = 11/49) at birth and at the end first month of life (6.1%, n = 3/49) became HPV-DNA negative at the age of 6 months. The HPV-DNA positive child (2%, n = 1/49) from 1st to the 6th month of life became HPV-DNA negative between the 6th and 12th month of life and one child had anogenital warts. In the twelfth month all (100%, n = 49/49) the children studied were HPV-DNA negative. A positive and significant correlation was observed between perinatal transmission of HPV-DNA and the immunodepression of maternal variables (HIV, p = 0.007). Finally, the study suggests that perinatal transmission of HPV-DNA occurred in 24.5% (n = 12/49) of the cases studied.

Transplacental transmission of Human Papillomavirus.

This paper aimed at studying the transplacental transmission of HPV and looking at the epidemiological factors involved in maternal viral infection. The following sampling methods were used: (1) in the pregnant woman, (a) genital; (b) peripheral blood; (2) in the newborn, (a) oral cavity, axillary and inguinal regions; (b) nasopharyngeal aspirate, and (c) cord blood; (3) in the placenta. The HPV DNA was identified using two methods: multiplex PCR of human beta-globin and of HPV using the PGMY09 and PGMY11 primers; and nested-PCR, which combines degenerated primers of the E6/E7 regions of the HPV virus, that allowed the identification of genotypes 6/11, 16, 18, 31, 33, 42, 52 and 58. Transplacental transmission was considered when type-specific HPV concordance was found between the mother, the placenta and the newborn or the mother and cord blood. The study included 49 HPV DNA-positive pregnant women at delivery. Twelve placentas (24.5%, n = 12/49) had a positive result for HPV DNA. Eleven newborn were HPV DNA positive in samples from the nasopharyngeal or buccal and body or cord blood. In 5 cases (10.2%, n = 5/49) there was HPV type-specific agreement between genital/placenta/newborn samples. In one case (2%, n = 1/49) there was type specific HPV concordance between genital/cord blood and also suggested transplacental transmission. A positive and significant correlation was observed between transplacental transmission of HPV infection and the maternal variables of immunodepression history (HIV, p = 0.011). In conclusion the study suggests placental infection in 23.3% of the cases studied and transplacental transmission in 12.2%. It is suggested that in future HPV DNA be researched in the normal endometrium of women of reproductive age. The possible consequence of fetal exposure to HPV should be observed.

Effect of CuCl2, NaCl and EDTA on the enzyme alpha-L-iduronidase in the plasma of normal individuals and heterozygotes for MPS I.

BACKGROUND: It has been previously demonstrated that the enzyme alpha-L-iduronidase (IDUA) of patients with MPS I shows a different biochemical behavior in each of the three clinical forms of these. In heterozygotes, its biochemical behavior has been recently established in leukocyte and plasma samples, demonstrating that it is possible to distinguish individuals heterozygous for MPS I within an unselected population. METHODS: We evaluated the effect of copper chloride, EDTA and sodium chloride on the activity of the enzyme alpha-L-iduronidase in the plasma of normal individuals and of MPS I heterozygotes and observed the type of inhibition caused, the Ki, the apparent Km and the apparent Vmax for each inhibitor. RESULTS: Sodium chloride inhibited the enzyme in normal individuals and in 40% of the heterozygotes evaluated and activated it in 60% of heterozygotes. The remaining compounds inhibited IDUA in both heterozygotes and normal individuals. CONCLUSIONS: We detected significant differences capable of differentiating MPS I heterozygotes from normal individuals by simply adding sodium chloride, EDTA or copper chloride to the incubation medium at the time of IDUA activity determination, with a potential use in carrier detection protocols.

Detection of mucopolysaccharidosis type I heterozygotes based on the biochemical characteristics of leukocyte alpha-L-iduronidase.

BACKGROUND: In the present study, we biochemically characterized the enzyme alpha-L-iduronidase (IDUA) of leukocytes from normal individuals and from mucopolysaccharidosis I (MPS I) heterozygotes, and compared these characteristics to discriminate for inclusion into two different groups. METHODS: We fluorimetrically measured IDUA activity in leukocytes using 4-methylumbelliferyl-alpha-L-iduronide as an artificial substrate. Optimum pH, Km, Vmax, and thermostability of the enzyme at 50 degrees C were determined. RESULTS: Based on leukocyte IDUA activity, we divided the heterozygotes into two groups, one (group 1) with activity below that detected in controls, and the other with activity similar to that of normal individuals (group 2). The optimum pH for IDUA was 2.7 for normal individuals and 2.6-2.8 for heterozygotes. With respect to Km, there was a difference only between the value for normal IDUA (0.60 mM) and the value for group 2 (0.38 mM), while group 1 showed a statistically similar value (0.49 mM). The Vmax of the reaction was discriminated in the three groups in a highly effective manner. The IDUA of normal individuals had a higher Vmax (60.98 nmoL/h x mg protein) than the enzyme of group 1 heterozygotes (28.66 nmoL/h x mg protein) and the enzyme of group 2 (31.78 nmoL/h x mg protein). When the IDUA from the three groups was pre-incubated at 50 degrees C, we observed that the IDUA of both group 1 and group 2 was significantly more thermostable than the IDUA of normal individuals. CONCLUSIONS: Determination of IDUA activity alone is not sufficient to discriminate between MPS I heterozygotes and normal individuals because a considerable overlap occurs between them. Our study showed that leukocyte IDUA from MPS I heterozygotes differed from the normal enzyme in terms of optimum pH, Km, and Vmax of the reaction and thermostability at 50 degrees C. These parameters provide a simple and reliable tool for the detection of carriers for MPS I.