Avaliação molecular de lesões pré-invasivas de carcinoma ductal de mama para detectar candidatos a biomarcadores de predição de progressão de carcinoma ductal in situ / Molecular assessment of preinvasive lesions of breast ductal carcinoma for detecting potential biomarkers of ductal carcinoma in situ progression

INTRODUÇÃO: O câncer de mama é o tipo de câncer mais frequente na população feminina mundial. Cerca de 75% dos pacientes são do tipo carcinoma ductal, podendo se apresentar como carcinoma ductal in situ (CDIS puro) ou carcinoma mamário invasivo tipo não especial (CMI-TNE), sendo que este último pode conter o componente in situ associado (componente in situ de CMI-TNE). O número de pacientes de CDIS diagnosticados tem aumentado nas últimas décadas devido aos programas de rastreamento, estimulando a busca por biomarcadores capazes de estratificar adequadamente as lesões com maior ou menor risco de progredir ou recidivar, e consequentemente poupar as pacientes com menor risco do tratamento convencional, atualmente considerado excessivo para essas pacientes. OBJETIVO: Assim, o objetivo do presente estudo foi caracterizar molecularmente lesões pré-invasivas de carcinoma ductal de mama a fim de identificar candidatos a biomarcadores de predição de progressão ou recidiva de CDIS. METODOLOGIA: Amostras de RNA amplificado de células epiteliais neoplásicas e mioepiteliais capturadas por microdissecção a laser dos dois grupos de lesões pré-invasivas (CDIS puro e componente in situ de CMI-TNE) foram submetidas a uma análise de expressão gênica com ensaios TLDA customizados e o perfil de expressão dos genes diferencialmente expressos (GDE) (fold change ≥ 2) foi submetido a uma análise de clusterização para avaliar a capacidade em distinguir os dois tipos de lesões pré-invasivas. Na análise mutacional, dois painéis multigenes (409 genes inteiros e regiões hotspots de 50 genes) foram utilizados no sequenciamento de DNA (plataforma Ion Torrent) de uma casuística composta por pacientes com laudo anatomopatológico de CDIS puro na biópsia e que apresentaram diferentes desfechos: grupo 1 - diagnóstico de CDIS puro na biópsia e peça cirúrgica e que não manifestaram progressão ou recidiva da doença num período mínimo de 3 anos de seguimento; grupo 2 - CDIS puro na biópsia e que foi subdividido em dois subgrupos: grupo 2.1 - diagnóstico de CMI-TNE com componente in situ na peça cirúrgica; grupo 2.2 - diagnóstico de CDIS puro na peça cirúrgica também e que apresentaram progressão ou recidiva da doença no período de seguimento. RESULTADOS: Na análise de expressão gênica foram analisados 11 pacientes representados por 15 amostras de células epiteliais neoplásicas ou mioepiteliais (5 CDIS puro e 10 componente in situ de CMI-TNE). Confirmamos a diferença de expressão de 49% (26/53) dos genes analisados. O perfil de expressão de 12 genes (ANAPC13, BAALC, DNAJB6, ERCC8, FLCN, NCF4, RARRES3, RBM47, REC8, RRP1, SRA1 e TMEM45A) foi capaz de discriminar 80% (4/5) das amostras de CDIS puro de 100% (10/10) das amostras de componente in situ de CMI-TNE. Na análise mutacional foram avaliados 34 pacientes (43 amostras) divididos em dois grupos. O grupo 1 (CDIS sem progressão) com 20 pacientes e o grupo 2 (CDIS com progressão) com 14 pacientes, sendo que no grupo 2.1 e 2.2 foram 7 pacientes cada. Dos 34 pacientes, 23 pacientes tiveram 29 amostras com sequenciamento qualificado para a análise mutacional (15 e 8 pacientes dos grupos 1 e 2, respectivamente). Dos 23 pacientes, 16 apresentaram variantes potencialmente somáticas, 53% (8/15) e 100% (8/8) dos grupos 1 e 2, respectivamente. Das 106 variantes potencialmente somáticas detectadas, 45 foram em 40 oncogenes ou genes supressores tumorais, os quais apresentaram variantes oncogênicas ou provavelmente oncogênicas em 33% (5/15) e 75% (6/8) dos grupos 1 e 2, respectivamente. Variantes potencialmente somáticas no gene TP53 foram identificadas em 6,6% (1/15) e 25% (2/8) dos grupos 1 e 2, respectivamente; e em PIK3CA em 0% e 25% (2/8) dos grupos 1 e 2, respectivamente. CONCLUSÃO: A assinatura gênica baseada nos 12 genes tem potencial de predizer a progressão de CDIS para a doença invasiva. Além disso, variantes oncogênicas em oncogenes e genes supressores de tumor foram identificadas em maior frequência nos pacientes com diagnóstico de CDIS puro na biópsia e que sofreram progressão/recidiva ou discordância com o diagnóstico da peça cirúrgica.

INTRODUCTION: Breast cancer is the most common cancer in women worldwide. Ductal carcinoma accounts for approximately 80% of all breast cancers and is represented by ductal carcinoma in situ (DCIS) and invasive breast carcinoma of no special type (IBCNST), the last can be detected concurrent with an in situ component (in situ component of IBC). The diagnosis of DCIS has been increased in the last decades due to an increase in screening mammography programs, encouraging the seek for molecular markers able to stratify patients according to risk of progression or relapse, and consequently to identify patients at a lower risk who could avoid an overtreatment. OBJECTIVE: The aim of this study was to perform a molecular characterization of preinvasive lesions of breast ductal carcinoma, to identify potential biomarkers of DCIS progression or relapse. METHODOLOGY: Amplified RNA from epithelial and myoepithelial cells captured by laser microdissection of both groups of preinvasive lesions (pure DCIS and in situ component of IBC) were submitted to gene expression analysis by TLDA assays and gene expression profile It was considered genes differentially expressed those with fold change ≥ 2. It was performed an unsupervised hierarchical clustering analysis with Euclidian distance and average linkage to evaluate the ability of the expression profile of GDE to correctly discriminate between preinvasive lesions (DCIS and in situ component of IBC). Somatic mutational profiling was performed by 2 multigene panels (409-gene panel and 50-gene hotspot regions panel) for NGS sequencing on Ion Torrent platform. The cohort was composed essentially by cases diagnosed as pure DCIS at anatomopathological report of biopsy and presenting different outcomes afterwards. Group 1: pure DCIS ­ pure DCIS at anatomopathological report of both biopsy and surgical specimen and with no manifestation of progression or relapse after at least 3-year follow-up. Group 2 ­ pure DCIS at anatomopathological report of biopsy and it was divided into 2 groups. Group 2.1 - pure DCIS at anatomopathological report of biopsy and invasive ductal carcinoma of no special type with concurrent in situ component at anatomopathological report of surgical specimen (DCIS/invasive). Group 2.2 ­ pure DCIS at anatomopathological report of both biopsy and surgical specimen and with manifestation of progression or relapse during the follow-up. RESULTS: Gene expression analysis was performed for 11 patients which were represented by 15 samples of epithelial or myoepithelial cells (5 DCIS and 10 in situ component of IBC). We confirmed a differential expression for 49% (26/53) of genes. The expression profile of 12 selected genes (ANAPC13, BAALC, DNAJB6, ERCC8, FLCN, NCF4, RARRES3, RBM47, REC8, RRP1, SRA1 e TMEM45A) was able to discriminate 80% (4/5) of pure DCIS samples from 100% (10/10) of in situ component of IBC samples. Somatic mutational profiling was performed for 34 patients (43 samples) divided into 2 groups. Group 1 (DCIS with no progression) was composed by 20 patients and group 2 (DCIS with progression) by 14 patients, of which 7 patients were from each of group 2.1 and group 2.2. From 34 patients, 23 patients (29 samples) presented sequencing data qualified for single nucleotide variant analysis (15 and 8 patients from groups 1 and 2, respectively). From 23 patients, a total of 16 patients was found carrying at least one potentially somatic variant, 53% (8/15) and 100% (8/8) of the patients from groups 1 and 2, respectively. A total of 106 potentially somatic variants were found, of which 45 variants were found in 40 oncogenes or tumor suppressor genes. Oncogenic and probably oncogenic variants were found in 33% (5/15) and 75% (6/8) of patients from groups 1 and 2, respectively. Potentially somatic variants in gene TP53 were found in 6,6% (1/15) and 25% (2/8) of patients from groups 1 and 2, respectively; and in PIK3CA in 0% and 25% (2/8) of patients from groups 1 and 2, respectively. CONCLUSION: The gene signature based on the 12 genes shows a potential to predict DCIS progression to invasive disease. Additionally, oncogenic variants in oncogenes and tumor suppressor genes were found in a higher proportion in patients who were diagnosed with pure DCIS at anatomopathological report of biopsy and manifested progression/relapse or invasion at anatomopathological report of surgical specimen.

Epithelial cells captured from ductal carcinoma in situ reveal a gene expression signature associated with progression to invasive breast cancer.

Breast cancer biomarkers that can precisely predict the risk of progression of non-invasive ductal carcinoma in situ (DCIS) lesions to invasive disease are lacking. The identification of molecular alterations that occur during the invasion process is crucial for the discovery of drivers of transition to invasive disease and, consequently, biomarkers with clinical utility. In this study, we explored differences in gene expression in mammary epithelial cells before and after the morphological manifestation of invasion, i.e., early and late stages, respectively. In the early stage, epithelial cells were captured from both pre-invasive lesions with distinct malignant potential [pure DCIS as well as the in situ component that co-exists with invasive breast carcinoma lesions (DCIS-IBC)]; in the late stage, epithelial cells were captured from the two distinct morphological components of the same sample (in situ and invasive components). Candidate genes were identified using cDNA microarray and rapid subtractive hybridization (RaSH) cDNA libraries and validated by RT-qPCR assay using new samples from each group. These analyses revealed 26 genes, including 20 from the early and 6 from the late stage. The expression profile based on the 20 genes, marked by a preferential decrease in expression level towards invasive phenotype, discriminated the majority of DCIS samples. Thus, this study revealed a gene expression signature with the potential to predict DCIS progression and, consequently, provides opportunities to tailor treatments for DCIS patients.

Effects of Oligo dT-T7 RNA Primer in RNA Amplification from Paraffin-Embedded Tissue for Microarray Experiments

Introduction: Formalin-Fixed Paraffin-Embedded Tissue samples (FFPET) represent a valuable source for studies of geneexpression comparisons, since a great number of these samples is available in archive and presents a long time of clinicalfollow-up. However, the quality of total RNA of these samples is known to be inferior to frozen samples, being many timesinadequate for studies of gene expression using conventional methodologies. Objective: This study aims to establish a protocolfor amplification of messenger RNA (mRNA) derived from FFPET samples for using in microarray experiments. Material andMethods: 4 tumoral samples of invasive ductal breast carcinoma FFPET-buffered 10% were used. Total RNA was extracted andthe mRNA was linearly amplified in two rounds based on T7 RNA polymerase methodology using different concentrations ofoligo dT-T7 Primer for first strand cDNA (1st-cDNA) synthesis. Amplified antisense RNA (aRNA) was labeled with cianine-Cy3through reverse transcription in the presence of random primers and co-hybridized with reference RNA (HB4a) labeled withcianine-Cy5 in a customized platform containing 4,608 cDNAs corresponding to human genes. Results: The amplified RNAquality was influenced by the relative amount of oligo dT-T7, showing better results for ratio of 1:0.1 (total RNA : oligo dT-T7).Hybridizations showed value of intensity signals for the most of cDNAs immobilized in the platform. Conclusion: This studyshowed that the control of the relative amounts of RNA derived from FFPET material and oligo dT-T7 is extremely important toobtain high-quality amplified RNA, allowing its use in microarray experiments.