Miguel Angelo Martins Moreira

Introduction: Cervical cancer (CC) arises as a result of chronic and persistent female infection by different oncogenic human papillomaviruses (HPV). The incidence of this disease is still high in developing countries, such as Brazil, where diagnosis is often made in advanced stages. HPV 16 is the most common Papillomavirus genotype in CC worldwide. Studies regarding the association of different HPV 16 lineages with overall and disease-free CC survival rates can contribute to further understanding the behavior of different HPV 16 lineages concerning the prognosis of CC cases. Objective: To assess the CC prognosis of patients treated in a Brazilian institution concerning HPV16 lineages. Methods: Data were obtained from a prospective cohort of 334 patients with CC recruited between July 2011 and March 2014 and treated at the Brazilian National Cancer Institute (INCA), in Rio de Janeiro, Brazil. HPV 16 lineages were identified in tumor tissue samples. Genetic HPV 16 diversity comprised 218 cases of lineage A, 10 of lineage B, 10 of lineage C, and 96 of lineage D. In addition to HPV 16 lineages, age, histopathological type, staging, and treatment completion were evaluated as predictors of CC prognosis. Results: The median patient age was 48 years. The most common histopathological type was squamous cell carcinoma (82.3%), followed by adenocarcinoma. Locally advanced disease staging was the most frequently detected, represented by similar stage II and III percentages (36.2% and 37.7%), followed by initial stage I (19.2%) and stage IV (6.9%). Two hundred two patients completed CC treatment. Age, histological type, staging, and treatment completion were associated with a higher risk of death, which was not observed for the HPV 16 lineage variable. With regard to age, an increase in each year of life led to approximately a 1% increase in the risk of death. Other histopathological types (poorly differentiated carcinoma, adenosquamous, neuroendocrine, and sarcoma) were associated with a higher risk of death compared with adenocarcinoma. Patients diagnosed in advanced stages exhibited a higher risk of death, and those who did not complete treatment exhibited an over 2-fold increased risk of death. Conclusion: This study found no associations between HPV 16 lineages A, B, C, and D and CC prognosis.

Endometrial cancer (EC) is the most common pelvic gynecologic malignancy in developed countries, and its incidence is also increasing in developing countries. Endometrioid endometrial carcinoma (EEC) is the most frequent subtype. EEC is often associated with favorable clinicopathological features and a good prognosis, especially when diagnosed in stage I. Although some patients have no signs to predict locally advanced or metastatic disease, they may present tumor relapse in the future. There is no biomarker capable of predicting the relapse of stage I EEC. The present study applied a transcriptome analysis to identify differentially expressed genes in stage I EEC, comparing relapsed with non-relapsed tumors. The estrogen receptor 1 gene (ESR1) was overexpressed in EEC stage I samples from patients who developed relapse by 4.3-fold compared to non-relapsed tumors. Subsequently, an independent set of 64 stage I EEC samples was used to validate ESR1 gene overexpression in relapsed tumors and assess estrogen receptor alpha (ERα) protein levels. ESR1 was confirmed to be overexpressed in samples from relapsed tumors, and its expression level was an independent prognostic variable for disease-free (hazard ratio=7.25) and overall survival (hazard ratio=5.15). In contrast, Erα did not show different values between relapsed and non-relapsed tumors. We concluded that ESR1 overexpression is a biomarker for poor prognosis in stage I EEC.

The difficulty of obtaining samples from certain human tissues has led to efforts to find accessible sources to analyze molecular markers derived from DNA. In this study, we look for DNA methylation patterns in blood samples and its association with the brain methylation pattern in neurodegenerative disorders. Using data from methylation databases, we selected 18,293 CpGs presenting correlated methylation levels between blood and brain (bb-CpGs) and compare their methylation level between blood samples from patients with neurodegenerative diseases (Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis, and X Fragile Syndrome) and healthy controls. Sixty-four bb-CpGs presented significant distinct methylation levels in patients, being: nine for Alzheimer’s disease, nine for Parkinson’s disease, 28 for Multiple Sclerosis, and 18 for Fragile X Syndrome. Similar differences in methylation pattern for the nine Alzheimer’s bb-CpGs was also observed when comparing brain tissue from patients vs. controls. The genomic regions of some of these 64 bb-CpGs are placed close to or inside genes previously associated with the respective condition. Our findings support the rationale of using blood DNA as a surrogate of brain tissue to analyze changes in CpG methylation level in patients with neurodegenerative diseases, opening the possibility for characterizing new biomarkers.

Background: Approximately 3/4 of ovarian cancers are diagnosed in advanced stages, with the high‐grade epithelial ovarian carcinoma (EOC) accounting for 90% of the cases. EOC present high genomic instability and somatic loss‐of‐function variants in genes associated with homologous recombination mutational repair pathway (HR), such as BRCA1 and BRCA2, and in TP53. The identification of germline variants in HR genes in EOC is relevant for treatment of platinum resistant tumors and relapsed tumors with therapies based in synthetic lethality such as PARP inhibitors. Patients with somatic variants in HR genes may also benefit from these therapies. In this work was analyzed the frequency of somatic variants in BRCA1, BRCA2, and TP53 in an EOC cohort of Brazilian patients, estimating the proportion of variants in tumoral tissue and their association with progression‐free survival and overall survival.Methods: The study was conducted with paired blood/tumor samples from 56 patients. Germline and tumoral sequences of BRCA1, BRCA2, and TP53 were obtained by massive parallel sequencing. The HaplotypeCaller method was used for calling germline variants, and somatic variants were called with Mutect2.Results: A total of 26 germline variants were found, and seven patients presented germline pathogenic or likely pathogenic variants in BRCA1 or BRCA2. The analysis of tumoral tissue identified 52 somatic variants in 41 patients, being 43 somatic variants affecting or likely affecting protein functionality. Survival analyses showed that tumor staging was associated with overall survival (OS), while the presence of somatic mutation in TP53 was not associated with OS or progression‐free survival.Conclusion: Frequency of pathogenic or likely pathogenic germline variants in BRCA1 and BRCA2 (12.5%) was lower in comparison with other studies. TP53 was the most altered gene in tumors, with 62.5% presenting likely non‐functional or non‐functional somatic variants, while eight 14.2% presented likely non‐functional or non‐functional somatic variants in BRCA1 or BRCA2.

Approximately 704,000 new cases of cancer are expected to occur per year in Brazil between 2023 and 2025, corresponding to an incidence of 222 per 100,000 in men and 186 per 100,000 in women. Data from the last decades show that an epidemiological transition is occurring and cancer will soon be the first cause of death by disease in Brazil¹ 1 Instituto Nacional de Câncer (INCA). Estimativa 2023: incidência de câncer no Brasil / Instituto Nacional de Câncer. Rio de Janeiro (RJ): INCA; 2022a. p. 160. ,² 2 Instituto Nacional de Câncer (INCA). A situação do câncer no Brasil/Ministério da Saúde, Secretaria de Atenção à Saúde, Instituto Nacional de Câncer, Coordenação de Prevenção e Vigilância. Rio de Janeiro (RJ): INCA; 2006. p. 120. , overtaking cardiovascular diseases. Consequently, it is estimated that the annual cost of cancer treatment in the Brazilian public health system (SUS) will practically double until 2040, reaching R$ 7.8 billion³ 3 Instituto Nacional de Câncer (INCA). Gastos federais atuais e futuros com os cânceres atribuíveis aos fatores de risco relacionados à alimentação, nutrição e atividade física no Brasil. Rio de Janeiro (RJ): INCA; 2022b. p. 50. . Therefore, cancer prevention and early detection are key strategies to lower incidence, mortality growth, and costs of cancer care in the public health system. Although most cancers are associated with environmental causes, approximately 5–10% are mainly due to hereditary predisposition, characterized by multiple cases in a family, early age of onset, and frequently, diagnosis of multiple primary cancers in one person. Hereditary syndromes have been well-established for the most common solid tumors such as breast and colorectal cancers. However, recent expansion of genetic testing has identified hereditary versions of practically all types of cancers, associated or not with specific phenotypic criteria. In Brazil, several studies carried out in the last decades characterized the prevalence of germline pathogenic genetic variants associated with distinct cancer types including breast, colorectal, retinoblastoma, adrenocortical carcinoma, and ovarian cancers⁴ 4 Ribeiro RC, Sandrini F, Figueiredo B, Zambetti GP, Michalkiewicz E, Lafferty AR, et al. An inherited p53 mutation that contributes in a tissue-specific manner to pediatric adrenal cortical carcinoma. Proc Natl Acad Sci USA. 2001;98(16):9330-5. https://doi.org/10.1073/pnas.161479898 https://doi.org/10.1073/pnas.161479898... 5 Giacomazzi J, Graudenz MS, Osorio CA, Koehler-Santos P, Palmero EI, Zagonel-Oliveira M, et al. Prevalence of the TP53 p.R337H mutation in breast cancer patients in Brazil. PLoS One. 2014;9(6):e99893. https://doi.org/10.1371/journal.pone.0099893 https://doi.org/10.1371/journal.pone.009... 6 Palmero EI, Carraro DM, Alemar B, Moreira MAM, Ribeiro-Dos-Santos Â, Abe-Sandes K, et al. The germline mutational landscape of BRCA1 and BRCA2 in Brazil. Sci Rep. 2018;8(1):9188. https://doi.org/10.1038/s41598-018-27315-2 https://doi.org/10.1038/s41598-018-27315... 7 Paula AE, Galvão HCR, Bonatelli M, Sabato C, Fernandes GC, Berardinelli GN, et al. Clinicopathological and molecular characterization of Brazilian families at risk for lynch syndrome. Cancer Genet. 2021;254-5:82-91. https://doi.org/10.1016/j.cancergen.2021.02.003 https://doi.org/10.1016/j.cancergen.2021... -⁸ 8 Guindalini RSC, Viana DV, Kitajima JPFW, Rocha VM, López RVM, Zheng Y, et al. Detection of germline variants in Brazilian breast cancer patients using multigene panel testing. Sci Rep. 2022;12(1):4190. https://doi.org/10.1038/s41598-022-07383-1 https://doi.org/10.1038/s41598-022-07383... . These studies showed a high prevalence of specific mutations also found in other populations as well as variants mostly or only observed in the Brazilian population⁹ 9 Achatz MI, Zambetti GP. The inherited p53 mutation in the Brazilian population. Cold Spring Harb Perspect Med. 2016;6(12):a026195. https://doi.org/10.1101/cshperspect.a026195 https://doi.org/10.1101/cshperspect.a026... and contributed to the growth of scientific knowledge in the field. They also helped insert oncogenetics and genetic cancer risk assessment (GCRA) in public and private health services and established in 2009 a network funded by the Brazilian National Council for Scientific and Technological Development (CNPq) and coordinated by the Brazilian National Cancer Institute (INCA) (Rede Nacional de Cancer Familial)¹⁰ 10 Instituto Nacional de Câncer (INCA). Rede nacional de câncer familial: manual operacional / Instituto Nacional de Câncer – Rio de Janeiro (RJ): INCA; 2009. p. 229. ,¹¹ 11 Ashton-Prolla P, Seuanez HN. The Brazilian hereditary cancer network: historical aspects and challenges for clinical cancer genetics in the public health care system in Brazil. Genet Mol Biol. 2016;39(2):163-5. https://doi.org/10.1590/1678-4685-GMB-2014-0373 https://doi.org/10.1590/1678-4685-GMB-20... . This network is now an autonomous organization (www.rebrach.org.br) intended to promote actions to optimize access, health care assistance, training of health care professionals, and scientific investigations in hereditary cancer.

Due to immunosuppressive cancer therapies, cancer patients diagnosed with COVID-19 have a higher chance of developing severe symptoms and present a higher mortality rate in comparison to the general population. Here we show a comparative analysis of the microbiome from naso-oropharyngeal samples of breast cancer patients with respect to SARS-CoV-2 status and identified bacteria associated with symptom severity. Total DNA of naso-oropharyngeal swabs from 74 women with or without breast cancer, positive or negative for SARS-CoV-2 were PCR-amplified for 16S-rDNA V3 and V4 regions and submitted to massive parallel sequencing. Sequencing data were analyzed with QIIME2 and taxonomic identification was performed using the q2-feature-classifier QIIME2 plugin, the Greengenes Database, and amplicon sequence variants (ASV) analysis. A total of 486 different bacteria were identified. No difference was found in taxa diversity between sample groups. Cluster analysis did not group the samples concerning SARS-CoV-2 status, breast cancer diagnosis, or symptom severity. Three taxa (Pseudomonas, Moraxella, and Klebsiella,) showed to be overrepresented in women with breast cancer and positive for SARS-CoV-2 when compared to the other women groups, and five bacterial groups were associated with COVID-19 severity among breast cancer patients: Staphylococcus, Staphylococcus epidermidis, Scardovia, Parasegitibacter luogiensis, and Thermomonas. The presence of Staphylococcus in COVID-19 breast cancer patients may possibly be a consequence of nosocomial infection.

The APOBEC3 (A3) proteins are cytidine deaminases that exhibit the ability to insert mutations in DNA and/or RNA sequences. APOBEC3B (A3B) has been evidenced as a DNA mutagen with consistent high expression in several cancer types. Data concerning the A3B influence on HPV infection and cervical cancer are limited and controversial. We investigated the role of A3B expression levels in cervical cancer in affected women positive for infection by different HPV types. Tumor biopsies from cancerous uterine cervix were collected from 216 women registered at Hospital do Câncer II of Instituto Nacional de Câncer, and infecting HPV was typed. A3B expression levels were quantified from RNA samples extracted from cervical biopsies using real-time quantitative PCR. Median A3B expression levels were higher among HPV18⁺ samples when compared to HPV16⁺ counterparts and were also increased compared to samples positive for other HPV types. In squamous cell carcinoma, HPV18⁺ samples also showed increased median A3B expression when compared to HPV Alpha-9 species or only to HPV16⁺ samples. Our findings suggest that A3B expression is differentially upregulated in cervical cancer samples infected with HPV18. A3B could be potentially used as a biomarker for HPV infection and as a prognostic tool for clinical outcomes in the context of cervical cancer.

Retroelements are expressed in diverse types of cancer and are related to tumorigenesis and to cancer progression. We characterized the expression of retroelements in cervical cancer and explored their interplay with HPV infection and their association with expression of neighboring genes. Forty biopsies of invasive cervical carcinoma (squamous cell carcinomas and adenocarcinomas) with genotyped HPV were selected and analyzed for human endogenous retrovirus (HERV) and long interspersed nuclear element 1 (L1) expression through RNA-seq data. We found 8060 retroelements expressed in the samples and a negative correlation of DNA methyltransferase 1 expression with the two most expressed L1 elements. A total of 103 retroelements were found differentially expressed between tumor histological types and between HPV types, including several HERV families (HERV-K, HERV-H, HERV-E, HERV-I and HERV-L). The comparison between HPV mono- and co-infections showed the highest proportion of differentially expressed L1 elements. The location of retroelements affected neighboring gene expression, such as shown for the interleukin-20 gene family. Three HERVs and seven L1 were located close to this gene family and two L1 showed a positive association with IL20RB expression. This study describes the expression of retroelements in cervical cancer and shows their association with HPV status and host gene expression.

Purpose This study aimed to identify and classify genetic variants in consensus moderate-to-high-risk predisposition genes associated with Hereditary Breast and Ovarian Cancer Syndrome (HBOC), in BRCA1/2-negative patients from Brazil. Methods The study comprised 126 index patients who met NCCN clinical criteria and tested negative for all coding exons and intronic flanking regions of BRCA1/2 genes. Multiplex PCR-based assays were designed to cover the complete coding regions and flanking splicing sites of six genes implicated in HBOC. Sequencing was performed on HiSeq2500 Genome Analyzer. Results Overall, we identified 488 unique variants. We identified five patients (3.97%) that harbored pathogenic or likely pathogenic variants in four genes: ATM (1), CHEK2 (2), PALB2 (1), and TP53 (1). One hundred and thirty variants were classified as variants of uncertain significance (VUS), 10 of which were predicted to disrupt mRNA splicing (seven non-coding variants and three coding variants), while other six missense VUS were classified as probably damaging by prediction algorithms. Conclusion A detailed mutational profile of non-BRCA genes is still being described in Brazil. In this study, we contributed to filling this gap, by providing important data on the diversity of genetic variants in a Brazilian high-risk patient cohort. ATM, CHEK2, PALB2 and TP53 are well established as HBOC predisposition genes, and the identification of deleterious variants in such actionable genes contributes to clinical management of probands and relatives.