RESUMO
N6-methyladenosine (m6A) in mRNA regulates almost every stage in the mRNA life cycle, and the development of methodologies for the high-throughput detection of methylated sites in mRNA using m6A-specific methylated RNA immunoprecipitation with next-generation sequencing (MeRIPSeq) or m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) have revolutionized the m6A research field. Both of these methods are based on immunoprecipitation of fragmented mRNA. However, it is well documented that antibodies often have nonspecific activities, thus verification of identified m6A sites using an antibody-independent method would be highly desirable. We mapped and quantified the m6A site in the chicken ß-actin zipcode based on the data from chicken embryo MeRIPSeq results and our RNA-Epimodification Detection and Base-Recognition (RedBaron) antibody-independent assay. We also demonstrated that methylation of this site in the ß-actin zipcode enhances ZBP1 binding in vitro, while methylation of a nearby adenosine abolishes binding. This suggests that m6A may play a role in regulating localized translation of ß-actin mRNA, and the ability of m6A to enhance or inhibit a reader protein's RNA binding highlights the importance of m6A detection at nucleotide resolution.
Assuntos
Actinas , Galinhas , Animais , Embrião de Galinha , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Actinas/genética , Galinhas/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Anticorpos , Nucleotídeos/metabolismoRESUMO
Cyclin-dependent kinase 2 (CDK2) is a key cell cycle regulator, with essential roles during G1/S transition. The clinicopathological significance of CDK2 in ductal carcinomas in situ (DCIS) and early-stage invasive breast cancers (BCs) remains largely unknown. Here, we evaluated CDK2's protein expression in 479 BC samples and 216 DCIS specimens. Analysis of CDK2 transcripts was completed in the METABRIC cohort (n = 1980) and TCGA cohort (n = 1090), respectively. A high nuclear CDK2 protein expression was significantly associated with aggressive phenotypes, including a high tumour grade, lymph vascular invasion, a poor Nottingham prognostic index (all p-values < 0.0001), and shorter survival (p = 0.006), especially in luminal BC (p = 0.009). In p53-mutant BC, high nuclear CDK2 remained linked with worse survival (p = 0.01). In DCIS, high nuclear/low cytoplasmic co-expression showed significant association with a high tumour grade (p = 0.043), triple-negative and HER2-enriched molecular subtypes (p = 0.01), Comedo necrosis (p = 0.024), negative ER status (p = 0.004), negative PR status (p < 0.0001), and a high proliferation index (p < 0.0001). Tumours with high CDK2 transcripts were more likely to have higher expressions of genes involved in the cell cycle, homologous recombination, and p53 signaling. We provide compelling evidence that high CDK2 is a feature of aggressive breast cancers. The clinical evaluation of CDK2 inhibitors in early-stage BC patients will have a clinical impact.
Assuntos
Neoplasias da Mama , Carcinoma Intraductal não Infiltrante , Quinase 2 Dependente de Ciclina , Humanos , Feminino , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 2 Dependente de Ciclina/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Carcinoma Intraductal não Infiltrante/patologia , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Intraductal não Infiltrante/metabolismo , Prognóstico , Pessoa de Meia-Idade , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Estadiamento de Neoplasias , Carcinoma Ductal de Mama/patologia , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/mortalidade , Idoso , Regulação Neoplásica da Expressão Gênica , Invasividade Neoplásica , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
Cyclin-dependent kinases (CDK2, CDK4, CDK6), cyclin D1, cyclin E1 and phosphorylated retinoblastoma (pRB1) are key regulators of the G1/S cell cycle checkpoint and may influence platinum response in ovarian cancers. CDK2/4/6 inhibitors are emerging targets in ovarian cancer therapeutics. In the current study, we evaluated the prognostic and predictive significance of the CDK2/4/6-cyclin D1/E1-pRB1 axis in clinical ovarian cancers (OC). The CDK2/4/6, cyclin D1/E1 and RB1/pRB1 protein expression were investigated in 300 ovarian cancers and correlated with clinicopathological parameters and patient outcomes. CDK2/4/6, cyclin D1/E1 and RB1 mRNA expression were evaluated in the publicly available ovarian TCGA dataset. We observed nuclear and cytoplasmic staining for CDK2/4/6, cyclins D1/E1 and RB1/pRB1 in OCs with varying percentages. Increased nuclear CDK2 and nuclear cyclin E1 expression was linked with poor progression-free survival (PFS) and a shorter overall survival (OS). Nuclear CDK6 was associated with poor OS. The cytoplasmic expression of CDK4, cyclin D1 and cyclin E1 also has predictive and/or prognostic significance in OCs. In the multivariate analysis, nuclear cyclin E1 was an independent predictor of poor PFS. Tumours with high nuclear cyclin E1/high nuclear CDK2 have a worse PFS and OS. Detailed bioinformatics in the TCGA cohort showed a positive correlation between cyclin E1 and CDK2. We also showed that cyclin-E1-overexpressing tumours are enriched for genes involved in insulin signalling and release. Our data not only identified the prognostic/predictive significance of these key cell cycle regulators but also demonstrate the importance of sub-cellular localisation. CDK2 targeting in cyclin-E1-amplified OCs could be a rational approach.
Assuntos
Neoplasias Ovarianas , Neoplasias da Retina , Retinoblastoma , Feminino , Humanos , Carcinoma Epitelial do Ovário , Ciclina D1/genética , Neoplasias Ovarianas/genética , Quinase 2 Dependente de Ciclina/genética , Ubiquitina-Proteína Ligases , Proteínas de Ligação a Retinoblastoma/genéticaRESUMO
BACKGROUND: The routine assessment of progesterone receptor (PR) expression in breast cancer (BC) remains controversial. This study aimed to evaluate the role of PR expression in luminal BC, with emphasis on the definition of positivity and its prognostic significance as compared to Ki67 expression. METHODS: A large cohort (n = 1924) of estrogen receptor (ER)-positive/HER2-negative BC was included. PR was immunohistochemically (IHC) stained on full face sections and core needle biopsies (CNB) where the optimal scoring cutoff was evaluated. In addition, the association of PR with other clinicopathological factors, cellular proliferation, disease outcome, and response to adjuvant therapy were analyzed. RESULTS: Although several cutoffs showed prognostic significance, the optimal cutoff to categorize PR expression into two clinically distinct prognostic groups on CNB was 10%. PR negativity showed a significant association with features of aggressive tumor behavior and poor outcome. Multivariate analyses indicated that the association between PR negativity and poor outcome was independent of tumor grade, size, node stage, and Ki67. PR negativity showed independent association with shorter survival in patients who received endocrine therapy whereas Ki67did not. CONCLUSION: PR IHC expression provides independent prognostic value superior to Ki67. Routine assessment of PR expression in BC using 10% cutoff in the clinical setting is recommended. PLAIN LANGUAGE SUMMARY: In this study, we have established an optimal approach to determine the prognostic value of progesterone receptor expression in estrogen receptor-positive breast cancer patients. To do this, the levels of progesterone receptor were measured in a large cohort of estrogen receptor-positive breast cancer patients. We have refined the definition of progesterone receptor positivity in estrogen receptor-positive breast cancer. We show that progesterone receptor expression adds prognostic and predictive value of endocrine therapy in estrogen receptor-positive breast cancer patients, and our results show that the absence of progesterone receptor is associated with poorer outcomes independent of tumor grade, size, node stage, and Ki67 expression.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/patologia , Receptores de Progesterona/metabolismo , Progesterona/uso terapêutico , Antígeno Ki-67/metabolismo , Receptores de Estrogênio/metabolismo , Seguimentos , Receptor ErbB-2/metabolismo , Prognóstico , Biomarcadores TumoraisRESUMO
BACKGROUND: Tumour infiltrating lymphocytes (TILs) are a prognostic parameter in triple-negative and human epidermal growth factor receptor 2 (HER2)-positive breast cancer (BC). However, their role in luminal (oestrogen receptor positive and HER2 negative (ER + /HER2-)) BC remains unclear. In this study, we used artificial intelligence (AI) to assess the prognostic significance of TILs in a large well-characterised cohort of luminal BC. METHODS: Supervised deep learning model analysis of Haematoxylin and Eosin (H&E)-stained whole slide images (WSI) was applied to a cohort of 2231 luminal early-stage BC patients with long-term follow-up. Stromal TILs (sTILs) and intratumoural TILs (tTILs) were quantified and their spatial distribution within tumour tissue, as well as the proportion of stroma involved by sTILs were assessed. The association of TILs with clinicopathological parameters and patient outcome was determined. RESULTS: A strong positive linear correlation was observed between sTILs and tTILs. High sTILs and tTILs counts, as well as their proximity to stromal and tumour cells (co-occurrence) were associated with poor clinical outcomes and unfavourable clinicopathological parameters including high tumour grade, lymph node metastasis, large tumour size, and young age. AI-based assessment of the proportion of stroma composed of sTILs (as assessed visually in routine practice) was not predictive of patient outcome. tTILs was an independent predictor of worse patient outcome in multivariate Cox Regression analysis. CONCLUSION: AI-based detection of TILs counts, and their spatial distribution provides prognostic value in luminal early-stage BC patients. The utilisation of AI algorithms could provide a comprehensive assessment of TILs as a morphological variable in WSIs beyond eyeballing assessment.
Assuntos
Neoplasias da Mama , Neoplasias de Mama Triplo Negativas , Humanos , Feminino , Neoplasias da Mama/patologia , Linfócitos do Interstício Tumoral/patologia , Inteligência Artificial , Prognóstico , Neoplasias de Mama Triplo Negativas/patologia , Biomarcadores Tumorais/metabolismoRESUMO
Estrogen receptor (ER) status in breast cancer (BC) is determined using immunohistochemistry (IHC) with nuclear expression in ≥1% of cells defined as ER-positive. BC with 1%-9% expression (ER-low-positive), is a clinically and biologically unique subgroup. In this study, we hypothesized that ER-low-positive BC represents a heterogeneous group with a mixture of ER-positive and ER-negative tumor, which may explain their divergent clinical behavior. A large BC cohort (n = 8171) was investigated and categorized into 3 groups: ER-low-positive (1%-9%), ER-positive (≥10%), and ER-negative (<1%) where clinicopathological and outcome characteristics were compared. A subset of ER-low-positive cases was further evaluated using IHC, RNAscope, and RT-qPCR. PAM50 subtyping and ESR1 mRNA expression levels were assessed in ER-low-positive cases within The Cancer Genome Atlas data set. The reliability of image analysis software in assessment of ER expression in the ER-low-positive category was also assessed. ER-low-positive tumors constituted <2% of BC cases examined and showed significant clinicopathological similarity to ER-negative tumors. Most of these tumors were nonluminal types showing low ESR1 mRNA expression. Further validation of ER status revealed that 45% of these tumors were ER-negative with repeated IHC staining and confirmed by RNAscope and RT-qPCR. ER-low-positive tumors diagnosed on needle core biopsy were enriched with false-positive ER staining. BCs with 10% ER behaved similar to ER-positive, rather than ER-negative or low-positive BCs. Moderate concordance was found in assessment of ER-low-positive tumors, and this was not improved by image analysis. Routinely diagnosed ER-low-positive BC includes a proportion of ER-negative cases. We recommend repeat testing of BC showing 1%-9% ER expression and using a cutoff ≥10% expression to define ER positivity to help better inform treatment decisions.
RESUMO
Tumor-associated stroma in breast cancer (BC) is complex and exhibits a high degree of heterogeneity. To date, no standardized assessment method has been established. Artificial intelligence (AI) could provide an objective morphologic assessment of tumors and stroma, with the potential to identify new features not discernible by visual microscopy. In this study, we used AI to assess the clinical significance of (1) stroma-to-tumor ratio (S:TR) and (2) the spatial arrangement of stromal cells, tumor cell density, and tumor burden in BC. Whole-slide images of a large cohort (n = 1968) of well-characterized luminal BC cases were examined. Region and cell-level annotation was performed, and supervised deep learning models were applied for automated quantification of tumor and stromal features. S:TR was calculated in terms of surface area and cell count ratio, and the S:TR heterogeneity and spatial distribution were also assessed. Tumor cell density and tumor size were used to estimate tumor burden. Cases were divided into discovery (n = 1027) and test (n = 941) sets for validation of the findings. In the whole cohort, the stroma-to-tumor mean surface area ratio was 0.74, and stromal cell density heterogeneity score was high (0.7/1). BC with high S:TR showed features characteristic of good prognosis and longer patient survival in both the discovery and test sets. Heterogeneous spatial distribution of S:TR areas was predictive of worse outcome. Higher tumor burden was associated with aggressive tumor behavior and shorter survival and was an independent predictor of worse outcome (BC-specific survival; hazard ratio: 1.7, P = .03, 95% CI, 1.04-2.83 and distant metastasis-free survival; hazard ratio: 1.64, P = .04, 95% CI, 1.01-2.62) superior to absolute tumor size. The study concludes that AI provides a tool to assess major and subtle morphologic stromal features in BC with prognostic implications. Tumor burden is more prognostically informative than tumor size.
RESUMO
BACKGROUND: Encapsulated papillary carcinoma (EPC) is surrounded by a thick fibrous capsule-like structure, which is interpreted as a thickened basement membrane (BM). This study aimed to describe the geometric characteristics of the EPC capsule and to refine whether it is an expansion of the BM or a stromal reactive process. MATERIAL AND METHODS: In all, 100 cases were divided into four groups: EPC, ductal carcinoma in situ (DCIS), normal breast tissue and invasive tumours, with an additional encapsulated papillary thyroid carcinoma (EPTC) control group. Representative slides from each case were stained with picrosirius red (PSR) stain and examined using polarised microscopy. Images were analysed using ImageJ, CT-FIRE, and Curve align image analysis programmes. RESULTS: Compared to the normal and DCIS BM, the EPC group showed a significant increase of collagen fibre width, straightness, and density, and a decrease of fibre length. The EPC capsule showed less alignment of fibres with a more perpendicular arrangement, and it was enriched with disorganised collagen type I (stromal collagen) fibres. Compared to other groups, the EPC capsule showed significant variation in the thickness, evenness, distribution of collagen fibres, and significant intracapsular heterogeneity. Compared to BM-like material in the invasive group, the EPC capsule showed a higher density of collagen fibres with longer, straighter, and more aligned fibres, but there was no difference in the distribution of both collagen types I and III. Conversely, compared to EPTC, there were no differences between both EPC and EPTC capsules except that the fibres in the EPC capsule were straighter. Although differences between normal ducts and lobules and DCIS BM collagen fibre density, straightness, orientation, and alignment were detected, both were significantly different from EPC capsule. CONCLUSION: This study provided evidence that the EPC capsule is a reactive process rather than a thickened native BM characteristic of normal and in situ lesions, which provides further evidence that EPC is an indolent invasive carcinoma based on capsule characteristics.
Assuntos
Neoplasias da Mama , Carcinoma Intraductal não Infiltrante , Carcinoma Papilar , Neoplasias da Glândula Tireoide , Humanos , Feminino , Carcinoma Intraductal não Infiltrante/patologia , Carcinoma Papilar/patologia , Membrana Basal , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide/patologia , ColágenoRESUMO
AIM: Polo-like kinase-1 (PLK1) plays a crucial role in cell cycle progression, and it is considered a potential therapeutic target in many cancers. Although the role of PLK1 is well established in triple-negative breast cancer (TNBC) as an oncogene, its role in luminal BC is still controversial. In this study, we aimed to evaluate the prognostic and predictive role of PLK1 in BC and its molecular subtypes. METHODS: A large BC cohort (n = 1208) were immunohistochemically stained for PLK1. The association with clinicopathological, molecular subtypes, and survival data was analysed. PLK1 mRNA was evaluated in the publicly available datasets (n = 6774), including The Cancer Genome Atlas and the Kaplan-Meier Plotter tool. RESULTS: 20% of the study cohort showed high cytoplasmic PLK1 expression. High PLK1 expression was significantly associated with a better outcome in the whole cohort, luminal BC. In contrast, high PLK1 expression was associated with a poor outcome in TNBC. Multivariate analyses indicated that high PLK1 expression is independently associated with longer survival in luminal BC, and in poorer prognosis in TNBC. At the mRNA levels, PLK1 expression was associated with short survival in TNBC consistent with the protein expression. However, in luminal BC, its prognostic value significantly varies between cohorts. CONCLUSION: The prognostic role of PLK1 in BC is molecular subtype-dependent. As PLK1 inhibitors are introduced to clinical trials for several cancer types, our study supports evaluation of the pharmacological inhibition of PLK1 as an attractive therapeutic target in TNBC. However, in luminal BC, PLK1 prognostic role remains controversial.
Assuntos
Neoplasias da Mama , Neoplasias de Mama Triplo Negativas , Humanos , Feminino , Neoplasias da Mama/patologia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , PrognósticoRESUMO
This study aimed to identify microRNAs associated with histological grade using comprehensive microRNA analysis data obtained by next-generation sequencing from early-stage invasive breast cancer. RNA-seq data from normal breast and breast cancer samples were compared to identify candidate microRNAs with differential expression using bioinformatics. A total of 108 microRNAs were significantly differentially expressed in normal breast and breast cancer tissues. Using clinicopathological information and microRNA sequencing data of 430 patients with breast cancer from The Cancer Genome Atlas (TCGA), the differences in candidate microRNAs between low- and high-grade tumors were identified. Comparing the expression of the 108 microRNAs between low- and high-grade cases, 25 and 18 microRNAs were significantly upregulated and downregulated, respectively, in high-grade cases. Clustering analysis of the TCGA cohort using these 43 microRNAs identified two groups strongly predictive of histological grade. miR-3677 is a microRNA upregulated in high-grade breast cancer. The outcome analysis revealed that patients with high miR-3677 expression had significantly worse prognosis than those with low miR-3677 expression. This study shows that microRNAs are associated with histological grade in early-stage invasive breast cancer. These findings contribute to the elucidation of a new mechanism of breast cancer growth regulated by specific microRNAs.
Assuntos
Neoplasias da Mama , MicroRNAs , Humanos , Feminino , MicroRNAs/genética , Neoplasias da Mama/genética , Mama , Análise por Conglomerados , Biologia ComputacionalRESUMO
The MRE11 nuclease is essential during DNA damage recognition, homologous recombination, and replication. BRCA2 plays important roles during homologous recombination and replication. Here, we show that effecting an MRE11 blockade using a prototypical inhibitor (Mirin) induces synthetic lethality (SL) in BRCA2-deficient ovarian cancer cells, HeLa cells, and 3D spheroids compared to BRCA2-proficient controls. Increased cytotoxicity was associated with double-strand break accumulation, S-phase cell cycle arrest, and increased apoptosis. An in silico analysis revealed Mirin docking onto the active site of MRE11. While Mirin sensitises DT40 MRE11+/- cells to the Top1 poison SN-38, it does not sensitise nuclease-dead MRE11 cells to this compound confirming that Mirin specifically inhibits Mre11 nuclease activity. MRE11 knockdown reduced cell viability in BRCA2-deficient PEO1 cells but not in BRCA2-proficient PEO4 cells. In a Mirin-resistant model, we show the downregulation of 53BP1 and DNA repair upregulation, leading to resistance, including in in vivo xenograft models. In a clinical cohort of human ovarian tumours, low levels of BRCA2 expression with high levels of MRE11 co-expression were linked with worse progression-free survival (PFS) (p = 0.005) and overall survival (OS) (p = 0.001). We conclude that MRE11 is an attractive SL target, and the pharmaceutical development of MRE11 inhibitors for precision oncology therapeutics may be of clinical benefit.
Assuntos
Proteínas de Ligação a DNA , Neoplasias Ovarianas , Humanos , Feminino , Proteínas de Ligação a DNA/metabolismo , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Células HeLa , Medicina de Precisão , Proteína BRCA2/metabolismo , Reparo do DNA , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Linhagem Celular TumoralRESUMO
BACKGROUND: The Ubiquitin-conjugating enzyme 2C (UBE2C) is essential for the ubiquitin-proteasome system and is involved in cancer cell migration and apoptosis. This study aimed to determine the prognostic value of UBE2C in invasive breast cancer (BC). METHODS: UBE2C was evaluated using the Molecular Taxonomy of Breast Cancer International Consortium (n = 1980), The Cancer Genome Atlas (n = 854) and Kaplan-Meier Plotter (n = 3951) cohorts. UBE2C protein expression was assessed using immunohistochemistry in the BC cohort (n = 619). The correlation between UBE2C, clinicopathological parameters and patient outcome was assessed. RESULTS: High UBE2C mRNA and protein expressions were correlated with features of poor prognosis, including high tumour grade, large size, the presence of lymphovascular invasion, hormone receptor negativity and HER2 positivity. High UBE2C mRNA expression showed a negative association with E-cadherin, and a positive association with adhesion molecule N-cadherin, matrix metalloproteinases and cyclin-related genes. There was a positive correlation between high UBE2C protein expression and cell cycle-associated biomarkers, p53, Ki67, EGFR and PI3K. High UBE2C protein expression was an independent predictor of poor outcome (p = 0.011, HR = 1.45, 95% CI; 1.10-1.93). CONCLUSION: This study indicates that UBE2C is an independent prognostic biomarker in BC. These results warrant further functional validation for UBE2C as a potential therapeutic target in BC.
Assuntos
Neoplasias da Mama , Enzimas de Conjugação de Ubiquitina , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Humanos , Imuno-Histoquímica , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Atypical mitosis is considered a feature of malignancy, however, its significance in breast cancer (BC) remains elusive. Here, we aimed to assess the clinical value of atypical mitoses in BC and to explore their underlying molecular features. Atypical and typical mitotic figures were quantified and correlated with clinicopathological variables in a large cohort of primary BC tissue sections (n = 846) using digitalized hematoxylin and eosin whole-slide images (WSIs). In addition, atypical mitoses were assessed in The Cancer Genome Atlas (TCGA) BC dataset (n = 1032) and were linked to the genetic alterations and pathways. In this study, the median of typical mitoses was 17 per 3 mm2 (range 0-120 mitoses), while the median of atypical mitoses was 4 (range 0-103 mitoses). High atypical mitoses were significantly associated with parameters characteristic of aggressive tumor behavior. The total number of mitoses, and a high atypical-to-typical mitoses ratio (>0.27) were associated with poor BC specific survival (BCSS), (p = 0.04 and 0.01, respectively). The atypical-to-typical mitoses ratio dichotomized triple negative-BC (TNBC) patients into two distinct groups in terms of the association with the outcome, while the overall number of mitoses was not. Moreover, TNBC patients with high atypical-to-typical mitoses ratio treated with adjuvant chemotherapy were associated with shorter survival (p = 0.003). Transcriptomic analysis of the TCGA-BRCA cohort dichotomized based on atypical mitoses identified 2494 differentially expressed genes. These included genes linked to pathways involved in chromosomal localization and segregation, centrosome assembly, spindle and microtubule formation, regulation of cell cycle and DNA repair. To conclude, the atypical-to-typical mitoses ratio has prognostic value independent of the overall mitotic count in BC patients and could predict the response to chemotherapy in TNBC.
Assuntos
Neoplasias da Mama , Neoplasias de Mama Triplo Negativas , Neoplasias da Mama/patologia , Amarelo de Eosina-(YS) , Feminino , Hematoxilina , Humanos , Mitose , Prognóstico , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
BACKGROUND: Ki67 reflects the proliferation activity in breast cancer (BC). However, an optimal method for its assessment in clinical settings has yet to be robustly defined. In this study we compared several methods to score Ki67 to identify a reliable and reproducible method for routine practice. METHODS: Sections from luminal BC cohort (n = 1662) were immunohistochemically stained with Ki67 and were assessed for the percentage, pattern, and intensity of expression. Ki67 positivity was evaluated using three methods: (i) quantification of Ki67-positive cells among 1000 invasive tumour cells within hotspot, (ii) average estimation of Ki67 within a defined hotspot, and (iii) average estimation of Ki67 positivity within the whole section. Time required for scoring, interobserver agreement and association with outcome were determined. RESULTS: The mean percentage of Ki67 expression per 1000 cells method was 16%, while the mean value of Ki67 scores using the average estimation within hotspot and whole slide were 14% and 12%, respectively. Quantification of Ki67-positive cells within 1000 cells had the highest degree of consistency between observers, and the highest hazard ratio predicting patient outcome when compared to using different common Ki67 cutoffs, which was independent of the other two methods. Granular pattern of Ki67 expression was associated with poorer outcome as compared to the other patterns. CONCLUSION: Assessment of Ki67 expression using quantification positive cells among 1000 tumour cells is an optimal method to achieve high reliability and reproducibility. Comment on the predominant Ki67 expression pattern would add prognostic and predictive value in luminal BC.
Assuntos
Neoplasias da Mama , Feminino , Humanos , Neoplasias da Mama/patologia , Antígeno Ki-67/metabolismo , Prognóstico , Reprodutibilidade dos Testes , Projetos de PesquisaRESUMO
BACKGROUND: Emerging evidence indicates that breast cancer (BC) patients whose tumours express HER2 protein without HER2 gene amplification (HER2-low), can benefit from antibody-drug conjugates (ADC). However, the current definition of HER2-low BC remains incomplete with low rates of concordance. This study aims to refine HER2-low definition with emphasis on distinguishing HER2 score 0 from score 1+ to identify patients who are eligible for ADC. METHODS: A BC cohort (n = 363) with HER2 IHC scores 0, 1+ and 2+ (without HER2 gene amplification) and available HER2 mRNA was included. HER2 staining intensity, pattern and subcellular localisation were reassessed. Artificial neural network analysis was applied to cluster the cohort and to distinguish HER2 score 0 from 1+. Reproducibility and reliability of the refined criteria were tested. RESULTS: HER2 IHC score 1+ was refined as membranous staining in invasive cells as either: (1) faint intensity in ≥ 20% of cells regardless the circumferential completeness, (2) weak complete staining in ≤ 10%, (3) weak incomplete staining in > 10% and (4) moderate incomplete staining in ≤ 10%. Based on this, 63% of the HER2-negative cases were reclassified as positive (HER2-low). The refined score showed perfect observer agreement compared to the moderate agreement in the original clinical scores. Similar results were generated when the refined score was applied on the independent BC cohorts. A proposal to refine the definition of other HER2 classes is presented. CONCLUSION: This study refined the definition of HER2-low BC based on correlation with HER2 mRNA and distinguished between HER2 IHC score 1+ and score 0 tumours.
Assuntos
Neoplasias da Mama , Imunoconjugados , Humanos , Feminino , Receptor ErbB-2/análise , Neoplasias da Mama/patologia , Imuno-Histoquímica , Reprodutibilidade dos Testes , Hibridização in Situ Fluorescente , RNA MensageiroRESUMO
N6-methyladenosine (m6A) is the most common internal modification of eukaryotic messenger RNA (mRNA) and is decoded by YTH domain proteins. The mammalian mRNA m6A methylosome is a complex of nuclear proteins that includes METTL3 (methyltransferase-like 3), METTL14, WTAP (Wilms tumour 1-associated protein) and KIAA1429. Drosophila has corresponding homologues named Ime4 and KAR4 (Inducer of meiosis 4 and Karyogamy protein 4), and Female-lethal (2)d (Fl(2)d) and Virilizer (Vir). In Drosophila, fl(2)d and vir are required for sex-dependent regulation of alternative splicing of the sex determination factor Sex lethal (Sxl). However, the functions of m6A in introns in the regulation of alternative splicing remain uncertain. Here we show that m6A is absent in the mRNA of Drosophila lacking Ime4. In contrast to mouse and plant knockout models, Drosophila Ime4-null mutants remain viable, though flightless, and show a sex bias towards maleness. This is because m6A is required for female-specific alternative splicing of Sxl, which determines female physiognomy, but also translationally represses male-specific lethal 2 (msl-2) to prevent dosage compensation in females. We further show that the m6A reader protein YT521-B decodes m6A in the sex-specifically spliced intron of Sxl, as its absence phenocopies Ime4 mutants. Loss of m6A also affects alternative splicing of additional genes, predominantly in the 5' untranslated region, and has global effects on the expression of metabolic genes. The requirement of m6A and its reader YT521-B for female-specific Sxl alternative splicing reveals that this hitherto enigmatic mRNA modification constitutes an ancient and specific mechanism to adjust levels of gene expression.
Assuntos
Adenosina/análogos & derivados , Processamento Alternativo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Caracteres Sexuais , Processos de Determinação Sexual/genética , Regiões 5' não Traduzidas/genética , Adenosina/metabolismo , Animais , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Mecanismo Genético de Compensação de Dose , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/metabolismo , Feminino , Íntrons/genética , Masculino , Metiltransferases/deficiência , Metiltransferases/genética , Metiltransferases/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/biossíntese , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Precursores de RNA/química , Precursores de RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
BACKGROUND AND AIMS: Proliferation is an important indicator of breast cancer (BC) prognosis, but is assessed using different approaches. Not all cells in the cell cycle are committed to division. This study aimed to characterise quantitative differences between BC cells in the cell cycle and those in mitosis and assess their relationship with other pathological parameters. METHODS AND RESULTS: A cohort of BC sections (n = 621) was stained with haematoxylin and eosin and immunohistochemistry for Ki-67. The proportion of mitotic cells and Ki-67-positive cells was assessed in the same areas. The Cancer Genome Atlas (TCGA) BC cohort was used to assess MKI-67 transcriptome level and its association with the mitotic counts. The mean proportion of BC cells in the cell cycle was 24% (range = 1-90%), while the mean proportion of BC cells in mitosis was 5% (range = 0-73%). A low proportion of mitoses to whole cycling cells was associated with low histological grade tumours and the luminal A molecular subtype, while tumours with a high proportion of mitoses to the overall cycling cells were associated with triple-negative subtype, larger tumour size, grade 3 tumours and lymph node metastasis. The high mitosis/low Ki-67-positive cells tumours showed a significant association with variables of poor prognosis, including high-grade and triple-negative subtypes. CONCLUSION: The proportion of BC cells in the cell cycle and mitosis is variable. We show that not only the number of cells in the cell cycle or mitosis, but also the difference between them, provides valuable information on tumour aggressiveness.
Assuntos
Neoplasias da Mama/patologia , Ciclo Celular , Proliferação de Células , Mitose , Adulto , Idoso , Feminino , Humanos , Pessoa de Meia-Idade , Índice Mitótico , PrognósticoRESUMO
BACKGROUND: Invasive lobular carcinoma (ILC) accounts for 10-15% of primary breast cancers and is typically estrogen receptor alpha positive (ER+) and ERBB2 non-amplified. Somatic mutations in ERBB2/3 are emerging as a tractable mechanism underlying enhanced human epidermal growth factor 2 (HER2) activity. We tested the hypothesis that therapeutically targetable ERBB2/3 mutations in primary ILC of the breast associate with poor survival outcome in large public datasets. METHODS: We performed in silico comparison of ERBB2 non-amplified cases of ER+ stage I-III primary ILC (N = 279) and invasive ductal carcinoma (IDC, N = 1301) using METABRIC, TCGA, and MSK-IMPACT information. Activating mutations amenable to HER2-directed therapy with neratinib were identified using existing functional data from in vitro cell line and xenograft experiments. Multivariate analysis of 10-year overall survival (OS) with tumor size, grade, and lymph node status was performed using a Cox regression model. Differential gene expression analyses by ERBB2 mutation and amplification status was performed using weighted average differences and an in silico model of response to neratinib derived from breast cancer cell lines. RESULTS: ILC tumors comprised 17.7% of all cases in the dataset but accounted for 47.1% of ERBB2-mutated cases. Mutations in ERBB2 were enriched in ILC vs. IDC cases (5.7%, N = 16 vs. 1.4%, N = 18, p < 0.0001) and clustered in the tyrosine kinase domain of HER2. ERBB3 mutations were not enriched in ILC (1.1%, N = 3 vs. 1.8%, N = 23; p = 0.604). Median OS for patients with ERBB2-mutant ILC tumors was 66 months vs. 211 months for ERBB2 wild-type (p = 0.0001), and 159 vs. 166 months (p = 0.733) for IDC tumors. Targetable ERBB2 mutational status was an independent prognostic marker of 10-year OS-but only in ILC (hazard ratio, HR = 3.7, 95% CI 1.2-11.0; p = 0.021). Findings were validated using a novel ERBB2 mutation gene enrichment score (HR for 10-year OS in ILC = 2.3, 95% CI 1.04-5.05; p = 0.040). CONCLUSIONS: Targetable ERBB2 mutations are enriched in primary ILC and their detection represents an actionable strategy with the potential to improve patient outcomes. Biomarker-led clinical trials of adjuvant HER-targeted therapy are warranted for patients with ERBB2-mutated primary ILC.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/patologia , Mutação , Receptor ErbB-2/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Lobular/genética , Carcinoma Lobular/metabolismo , Simulação por Computador , Bases de Dados Genéticas/estatística & dados numéricos , Feminino , Humanos , Pessoa de Meia-Idade , Prognóstico , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
PURPOSE: MMP9 is a matricellular protein associated with extracellular matrix (ECM) remodelling, that promotes tumour progression, and modulates the activity of cell adhesion molecules and cytokines. This study aims to assess the prognostic value of MMP9 and its association with cytoskeletal modulators in early-stage invasive breast cancer (BC). METHODS: MMP9 expression was evaluated by immunohistochemistry using a well-characterised series of primary BC patients with long-term clinical follow-up. Association with clinicopathological factors, patient outcome and ECM remodelling BC-biomarkers were investigated. METABRIC dataset, BC-GenExMiner v4.0 and TCGA were used for the external validation of MMP9 expression. GSEA gene enrichment analyses were used to evaluate MMP9 associated pathways. RESULTS: MMP9 immunopositivity was observed in the stroma and cytoplasm of BC cells. Elevated MMP9 protein levels were associated with high tumour grade, high Nottingham Prognostic Index, and hormonal receptor negativity. Elevated MMP9 protein expression correlated significantly with cytokeratin 17 (Ck17), Epidermal Growth Factor Receptor (EGFR), proliferation (Ki67) biomarkers, cell surface adhesion receptor (CD44) and cell division control protein 42 (CDC42). Cytoplasmic MMP9 expression was an independent prognostic factor associated with shorter BC-specific survival. In the external validation cohorts, MMP9 expression was also associated with poor patients' outcome. Transcriptomic analysis confirmed a positive association between MMP9 and ECM remodelling biomarkers. GSEA analysis supports MMP9 association with ECM and cytoskeletal pathways. CONCLUSION: This study provides evidence for the prognostic value of MMP9 in BC. Further functional studies to decipher the role of MMP9 and its association with cytoskeletal modulators in BC progression are warranted.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/mortalidade , Mama/metabolismo , Mama/patologia , Metaloproteinase 9 da Matriz/metabolismo , Biomarcadores Tumorais/análise , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Progressão da Doença , Feminino , Seguimentos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Metaloproteinase 9 da Matriz/análise , Metaloproteinase 9 da Matriz/genética , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Prognóstico , Estudos Prospectivos , RNA Mensageiro/metabolismo , Fatores de Tempo , Análise Serial de TecidosRESUMO
BACKGROUND: KN motif and ankyrin repeat domains 1 (KANK1) plays an important role in cytoskeleton maintenance and contributes to the regulation of cell proliferation, adhesion and apoptosis. KANK1 is involved in progression of a variety of solid tumours; however, its role in invasive breast cancer (BC) remains unknown. This study aims to evaluate the clinicopathological and prognostic value of KANK1 expression in operable BC. METHODS: KANK1 expression was assessed at the transcriptomic level using multiple BC cohorts; the Molecular Taxonomy of BC International Consortium cohort (METABRIC; n = 1980), The Cancer Genome Atlas BC cohort (TCGA; n = 949) and the publicly available BC transcriptomic data hosted by BC Gene-Expression Miner (bc-GenExMiner v4.0) and Kaplan-Meier plotter?. The Nottingham BC cohort (n = 1500) prepared as tissue microarrays was used to assess KANK1 protein expression using immunohistochemistry (IHC). The association between clinicopathological variables and patient outcome was investigated. RESULTS: In the METABRIC cohort, high expression of KANK1 mRNA was associated with characteristics of good prognosis including lower grade, absence of lymphovascular invasion and HER2 negativity (all; p < 0.001) and with better outcome [p = 0.006, Hazards ratio, (HR) 0.70, 95% CI 0.54-0.91]. High KANK1 protein expression was correlated with smaller tumour size and HER2 negativity, and better outcome in terms of longer breast cancer-specific survival [p = 0.013, HR 0.7, 95% CI 0.536-0.893] and time to distant metastasis [p = 0.033, HR 0.65, 95% CI 0.51-0.819]. CONCLUSION: These results supported that upregulation of KANK1 works as a tumour suppressor gene in BC and is associated with improved patients' outcomes.