Breast Cancer Specimens Research Articles

Assessment of Inter-observer Reproducibility of the Residual Cancer Burden Index and Neoadjuvant Chemotherapy Response in Breast Carcinoma

Background : Locally advanced breast cancers are nowadays treated with neoadjuvant chemotherapy (NAC) to downstage the tumor. One way to assess the NAC response is to use “Residual cancer burden index (RCB).” The aim of the study was to assess inter-pathologist reproducibility of residual cancer burden index and to evaluate different clinico-pathological parameters that determine the pathological response. Methods: In this retrospective cohort study, surgically excised specimens of 49 NAC treated breast cancer were examined by histopathology and immunohistochemistry over a period of one and half years. Four pathologists with different level of experience reviewed the reports (unaware of original RCB index) and slides and assigned the RCB indices for each case. Clinical, histopathological and immunohistochemical parameters were evaluated.. Fleiss- Kappa statistics was applied to assess interobserver reproducibility of RCB index. Results: No significant relationship was observed between RCB index (of original report) with age, largest tumor dimension and number of chemotherapy cycles . The RCB index of 49 cases as assigned by four pathologists was calculated by Fleiss-kappa statistics and it showed good overall agreement (82.6%). Conclusion: It has been observed that there is no significant relation between pathological response to NAC with age, largest tumor dimension and number of chemotherapy cycles of breast carcinoma. It can also be concluded that RCB can be reliably used to report neoadjuvant chemotherapy treated specimens of breast cancer.

Comprehensive single-cell and bulk transcriptomic analyses to develop an NK cell-derived gene signature for prognostic assessment and precision medicine in breast cancer

BackgroundNatural killer (NK) cells play crucial roles in mediating anti-cancer activity in breast cancer (BRCA). However, the potential of NK cell-related molecules in predicting BRCA outcomes and guiding personalized therapy remains largely unexplored. This study focused on developing a prognostic and therapeutic prediction model for BRCA by incorporating NK cell-related genes.MethodsThe data analyzed primarily originated from the TCGA and GEO databases. The prognostic role of NK cells was evaluated, and marker genes of NK cells were identified via single-cell analysis. Module genes closely associated with immunotherapy resistance were identified by bulk transcriptome-based weighted correlation network analysis (WGCNA). Following taking intersection and LASSO regression, NK-related genes (NKRGs) relevant to BRCA prognosis were screened, and the NK-related prognostic signature was subsequently constructed. Analyses were further expanded to clinicopathological relevance, GSEA, tumor microenvironment (TME) analysis, immune function, immunotherapy responsiveness, and chemotherapeutics. Key NKRGs were screened by machine learning and validated by spatial transcriptomics (ST) and immunohistochemistry (IHC).ResultsTumor-infiltrating NK cells are a favorable prognostic factor in BRCA. By combining scRNA-seq and bulk transcriptomic analyses, we identified 7 NK-related prognostic NKRGs (CCL5, EFHD2, KLRB1, C1S, SOCS3, IRF1, and CCND2) and developed an NK-related risk scoring (NKRS) system. The prognostic reliability of NKRS was verified through survival and clinical relevance analyses across multiple cohorts. NKRS also demonstrated robust predictive power in various aspects, including TME landscape, immune functions, immunotherapy responses, and chemotherapeutic sensitivity. Additionally, KLRB1 and CCND2 emerged as key prognostic NKRGs identified through machine learning and external validation, with their expression correlation with NK cells confirmed in BRCA specimens by ST and IHC.ConclusionsWe developed a novel NK-related gene signature that has proven valuable for evaluating prognosis and treatment response in BRCA, expecting to advance precision medicine of BRCA.

Method for co-registration of high-resolution specimen PET-CT with histopathology to improve insight into radiotracer distributions

BackgroundAs the spatial resolution of positron emission tomography (PET) scanners improves, understanding of radiotracer distributions in tissues at high resolutions is important. Hence, we propose a method for co-registration of high-resolution ex vivo specimen PET images, combined with computed tomography (CT) images, and the corresponding specimen histopathology.MethodsWe applied our co-registration method to breast cancer (BCa) specimens of patients who were preoperatively injected with 0.8 MBq/kg [18\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$^{18}$$\\end{document}F]fluorodeoxyglucose ([18F]FDG). The method has two components. First, we used an image acquisition scheme that minimises and tracks tissue deformation: (1) We acquired sub-millimetre (micro)-PET-CT images of ±2 mm-thick lamellas of the fresh specimens, enclosed in tissue cassettes. (2) We acquired micro-CT images of the same lamellas after formalin fixation to visualise tissue deformation. (3) We obtained 1 hematoxylin and eosin (H&E) stained histopathology section per lamella of which we captured a digital whole slide image (WSI). Second, we developed an automatic co-registration algorithm to improve the alignment between the micro-PET-CT images and WSIs, guided by the micro-CT of the fixated lamellas. To estimate the spatial co-registration error, we calculated the distance between corresponding microcalcifications in the micro-CTs and WSIs. The co-registered images allowed to study standardised uptake values (SUVs) of different breast tissues, as identified on the WSIs by a pathologist.ResultsWe imaged 22 BCa specimens, 13 cases of invasive carcinoma of no special type (NST), 6 of invasive lobular carcinoma (ILC), and 3 of ductal carcinoma in situ (DCIS). While the cassette framework minimised tissue deformation, the best alignment between the micro-PET-CT images and WSIs was achieved after deformable co-registration. We found an overall average co-registration error of 0.74 ± 0.17 mm between the micro-PET images and WSIs. (Pre)malignant tissue (including NST, ILC, and DCIS) generally showed higher SUVs than healthy tissue (including healthy glandular, connective, and adipose tissue). As expected, inflamed tissue and skin also showed high uptake.ConclusionsWe developed a method to co-register micro-PET-CT images of surgical specimens and WSIs with an accuracy comparable to the spatial resolution of the micro-PET images. While currently, we only applied this method to BCa specimens, we believe this method is applicable to a wide range of specimens and radiotracers, providing insight into distributions of (new) radiotracers in human malignancies at a sub-millimetre resolution.

Ex vivo fluorescence-guided resection margin assessment in breast cancer surgery using a topically applied, cathepsin-activatable imaging agent

Up to 40 % of breast cancer patients have a tumor-positive resection margin (TPRM) – defined as cancer cells at the surface of the resected specimen – after breast-conserving surgery (BCS), necessitating re-resection or boost radiation. To prevent these additional treatments, intraoperative near-infrared (NIR) fluorescence imaging with the topically applied, cathepsin-activatable imaging agent AKRO-6qcICG might be used to detect TPRMs and guide additional resection. Here, to validate its performance, the agent is topically applied to all surfaces of freshly resected breast cancer specimens (n = 11 patients) and to 3–5 mm thick tissue slices of the specimens (n = 26 patients). NIR fluorescence images of the resection surfaces and tissue slices are acquired and correlated to final histopathology. AKRO-6qcICG detects TPRMs with a sensitivity, specificity, PVV, and NPV of 100 %, 67 %, 10 %, and 100 %, respectively. On the tissue slices, the fluorescence signal has a median tumor-to-background ratio of 1.8. These findings indicate that topically applied AKRO-6qcICG can visualize TPRMs ex vivo with a high sensitivity and NPV, with sufficient contrast to adjacent healthy breast tissue.

Expression of Ki-67 in Invasive Breast Carcinoma and Its Correlation With Different Clinicopathological Features.

Introduction Worldwide, breast cancer is still the most common cancer that affects women. Breastcancer prognosis is based on a number of clinical and pathological indicators. Further features are needed to predict early metastasis andprognosis of patients with breast carcinoma, as the current pathological characteristics, such as tumor differentiation, vascular infiltration, and TNM (tumor, node, and metastasis) staging, cannot fully describe the early metastatic biological behavior in breast carcinoma. High Ki-67 expression is seen to be associated with worse survival in cancer patients. Hence, this study was conducted to study the utility of Ki-67as a prognostic marker in invasive breast carcinoma and its association with known clinicopathological factors. Methodology The study was a hospital-based cross-sectional study carried out in the Histopathology Section of the Department of Pathology, Shri BM Patil Medical CollegeHospital and Research Centre, Bijapur Lingayat District Education (BLDE) (Deemed to be University), Vijayapura. The study was conducted between September 1, 2022, and April 30, 2024. The study population consisted of 55 cases of mastectomy specimens of primary breast cancer admitted to our hospital. Data regarding the patient's age, tumor size, histological type, histological grade, lymph node status, and vascular invasion were noted from medical records. Immunohistochemical staining for estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2/neu (HER2/neu) proto-oncogene, and Ki-67 markers was performed according to standard protocol. The relationship of Ki-67with these clinicopathological parameters was analyzed statistically. Results In the current study, high-grade Ki-67 nuclear positivity was seen in 30 cases out of 55 cases, and low-grade Ki-67 was seen in the remaining 25 cases. The association of Ki-67 with tumor size and histological grade showed statistical significancewith a p-value of less than 0.05 and 0.001, respectively. However, no statistical significance was seen with lymph node status, vascular invasion, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2/neu (HER2/neu)proto-oncogene status with p-values greater than 0.05. Conclusion The expression of Ki-67 was statistically significant with histological grading and tumor size in our study. Immunohistochemical determination of the Ki-67 proliferation index should be performed in routine cases of breast cancerto obtain clinically useful information on tumor aggressiveness as reflected in their proliferative rate. Hence, Ki-67 can be used as a predictive and prognostic marker in managing breast cancer patients.

Testing of rapid evaporative mass spectrometry for histological tissue classification and molecular diagnostics in a multi-site study

BackgroundWhile REIMS technology has successfully been demonstrated for the histological identification of ex-vivo breast tumor tissues, questions regarding the robustness of the approach and the possibility of tumor molecular diagnostics still remain unanswered. In the current study, we set out to determine whether it is possible to acquire cross-comparable REIMS datasets at multiple sites for the identification of breast tumors and subtypes.MethodsA consortium of four sites with three of them having access to fresh surgical tissue samples performed tissue analysis using identical REIMS setups and protocols. Overall, 21 breast cancer specimens containing pathology-validated tumor and adipose tissues were analyzed and results were compared using uni- and multivariate statistics on normal, WT and PIK3CA mutant ductal carcinomas.ResultsStatistical analysis of data from standards showed significant differences between sites and individual users. However, the multivariate classification models created from breast cancer data elicited 97.1% and 98.6% correct classification for leave-one-site-out and leave-one-patient-out cross validation. Molecular subtypes represented by PIK3CA mutation gave consistent results across sites.ConclusionsThe results clearly demonstrate the feasibility of creating and using global classification models for a REIMS-based margin assessment tool, supporting the clinical translatability of the approach.

MDia2 is an important mediator of MRTF-A-dependent regulation of breast cancer cell migration.

Dysregulated actin cytoskeleton gives rise to aberrant cell motility and metastatic spread of tumor cells. This study evaluates the effect of overexpression of wild-type versus functional mutants of MRTF-A on migration and invasion of breast cancer (BC) cells. Our studies indicate that SRF's interaction is critical for MRTF-A-induced promotion of both two-dimensional and three-dimensional cell migration, while the SAP-domain function is important selectively for three-dimensional cell migration. Increased MRTF-A activity is associated with more effective membrane protrusion, a phenotype that is attributed predominantly to SRF's interaction with MRTF. We demonstrate formin-family protein mDia2 as an important mediator of MRTF-stimulated actin polymerization at the leading edge and cell migration. Multiplexed quantitative immunohistochemistry and transcriptome analyses of clinical BC specimens further demonstrate a positive correlation between nuclear localization of MRTF with malignant traits of cancer cells and enrichment of MRTF-SRF gene signature in pair-matched distant metastases versus primary tumors. In conclusion, this study establishes a novel mechanism of MRTF-dependent regulation of cell migration and provides evidence for the association between MRTF activity and increased malignancy in human BC, justifying future development of specific small molecule inhibitors of the MRTF-SRF transcriptional complex as potential therapeutic agents in BC.

Effects of Degreasing Pretreatment on Immunohistochemistry and Molecular Analysis of Gastrointestinal and Breast Cancer Samples

Lymph node status is a key factor in determining stage, treatment, and prognosis in cancers. Small lymph nodes in fat-rich gastrointestinal and breast cancer specimens are easily missed in conventional sampling methods. This study examined the effectiveness of the degreasing pretreatment with dimethyl sulfoxide (DMSO) in lymph node detection and its impact on the analysis of clinical treatment–related proteins and molecules. Thirty-three cases of gastrointestinal cancer specimens from radical gastrectomy and 63 cases of breast cancer specimens from modified radical mastectomy were included. After routine sampling of lymph nodes, the specimens were immersed in DMSO for 30 minutes for defatting. We assessed changes in the number of detected lymph nodes and pN staging in 33 gastrointestinal cancer specimens and 37 breast cancer specimens. In addition, we analyzed histologic characteristics, Masson trichrome special staining, and immunohistochemistry (gastrointestinal cancer: MMR, HER2, and PD-L1; breast cancer: ER, PR, AR, HER2, Ki-67, and PD-L1). Molecular status was evaluated for colorectal cancer (KRAS, NRAS, BRAF, and microsatellite instability) and breast cancer (HER2) in gastrointestinal cancer specimens and the remaining 26 breast cancer specimens. Compared with conventional sampling, DMSO pretreatment increased the detection rate of small lymph nodes (gastrointestinal cancer: P < .001; breast cancer: P < .001) and improved pN staging in 1 case each of gastric cancer, colon cancer, and rectal cancer (3/33; 9.1%). No significant difference in the morphology, special staining, protein, and molecular status of cancer tissue after DMSO treatment was found. Based on these results and our institutional experience, we recommend incorporating DMSO degreasing pretreatment into clinical pathologic sampling practices.

Zinc transporter ZnT5 is associated with epithelial mesenchymal transition via SMAD1 in breast cancer.

Zinc levels in breast cancer tissues have been reported to be higher than those in normal tissues. In addition, the expression levels of zinc transporters, including ZnT5 and ZnT6, are reportedly higher in breast cancer than in normal breast tissues. ZnT5 and ZnT6 also contribute to heterodimer formation and are involved in several biological functions. However, the functions of ZnT5 and ZnT6 heterodimers in breast cancer remain unknown. Therefore, we first investigated the immunolocalization of ZnT5 and ZnT6 in pathological breast cancer specimens and in MCF-7 and T-47D breast cancer cells. Next, we used small interfering RNA to assess cell viability and migration in ZnT5 knockdown MCF-7 and T-47D cells. Immunohistochemical analysis showed that the number of ZnT5-positive breast cancer cells was inversely correlated with the pathologic N factor status. ZnT5 knockdown had no effect on cell viability in the presence of 100 μM ZnCl2 in MCF-7 and T-47D cells. In a wound healing assay, 100 μM ZnCl2 treatment inhibited cell migration of MCF-7 and T-47D cells, whereas ZnT5 knockdown promoted cell migration, decreased E-cadherin expression and increased vimentin, slug and matrix metalloproteinase 9 expression. Antibody arrays showed that ZnT5 knockdown increased the expression of SMAD1, and that dorsomorphin treatment inhibited the promotion of migratory ability induced by ZnT5 knockdown. The results of this study revealed that both ZnT5 may be involved in less aggressive breast cancer subtypes, possibly through inhibition of cell migration.

Predictive analysis of breast cancer metastasis and identification of genetic markers using machine learning.

4 Background: The study devises a framework integrating machine learning (ML) paradigms with explainable artificial intelligence (XAI) to prognosticate the metastatic trajectory of breast cancer (BC) and delineate critical genomic indicators pertinent to metastasis. Methods: An examination was conducted on 98 initial BC specimens, which included 34 instances evolving into distant metastasis within a quintennial monitoring phase and 44 instances evincing no recurrence for a minimum pentad post-diagnosis. Genomic datasets underwent rigorous biostatistical scrutiny, followed by the implementation of an elastic net algorithm for feature discernment, thereby constraining the scope to a salient subset of genomic markers implicated in BC metastasis. An ensemble of advanced predictive models encompassing Light Gradient Boosting Machine (LightGBM), Categorical Boosting (CatBoost), Extreme Gradient Boosting (XGBoost), Gradient Boosting Trees (GBT), and Adaptive Boosting (AdaBoost) was deployed. Model efficacy was gauged through metrics such as accuracy, F1 score, precision, recall, the Area Under the Receiver Operating Characteristic Curve (AUC), and the Brier score. To elucidate the reasoning behind the ML predictions and to navigate the opacity inherent in such models, a SHapley Additive exPlanations (SHAP) approach was invoked. Results: The predictive acumen of the LightGBM model was superior, evidenced by a striking accuracy of 96% and an AUC of 99.3%. Parallel to biostatistical assessments, SHAP analysis leveraging XAI illuminated that augmented expression levels of specific genes, namely TSPYL5, ATP5E, CA9, NUP210, SLC37A1, ARIH1, PSMD7, UBQLN1, PRAME, and UBE2T (with statistical significance p ≤ 0.05), correlated with an escalated risk of BC metastasis. Conversely, diminished expression of CACTIN, TGFB3, SCUBE2, ARL4D, OR1F1, ALDH4A1, PHF1, and CROCC (p ≤ 0.05) was similarly associated with heightened metastatic susceptibility in BC. Conclusions: The insights garnered from this investigation may catalyze preventative strategies against BC progression and metastatic dissemination, thereby enhancing therapeutic efficacy through personalized intervention modalities for BC patients.

TRNA-derived fragment 3′tRF-AlaAGC modulates cell chemoresistance and M2 macrophage polarization via binding to TRADD in breast cancer

BackgroundDrug resistance, including Adriamycin-based therapeutic resistance, remains a challenge in breast cancer (BC) treatment. Studies have revealed that macrophages could play a pivotal role in mediating the chemoresistance of cancer cells. Accumulating evidence suggests that tRNA-Derived small RNAs (tDRs) are associated the physiological and pathological processes in multiple cancers. However, the underlying mechanisms of tDRs on chemoresistance of BC in tumor-associated macrophages remain largely unknown.MethodsThe high-throughput sequencing technique was used to screen tDRs expression profile in BC cells. Gain- and loss-of-function experiments and xenograft models were performed to verify the biological function of 3′tRF-Ala-AGC in BC cells. The CIBERSORT algorithm was used to investigate immune cell infiltration in BC tissues. To explore the role of 3′tRF-Ala-AGC in macrophages, M2 macrophages transfected with 3′tRF-Ala-AGC mimic or inhibitor were co-cultured with BC cells. Effects on Nuclear factor-κb (NF-κb) pathway were investigated by NF-κb nuclear translocation assay and western blot analysis. RNA pull-down assay was performed to identify 3′tRF-Ala-AGC interacting proteins.ResultsA 3′tRF fragment of 3′tRF-AlaAGC was screened, which is significantly overexpressed in BC specimens and Adriamycin-resistant cells. 3′tRF-AlaAGC could promote cell malignant activity and facilitate M2 polarization of macrophages in vitro and in vivo. Higher expression of M2 macrophages were more likely to have lymph node metastasis and deeper invasion in BC patients. Mechanistically, 3′tRF-AlaAGC binds Type 1-associated death domain protein (TRADD) in BC cells, and suppression of TRADD partially abolished the enhanced effect of 3′tRF-AlaAGC mimic on phenotype of M2. The NF-κb signaling pathway was activated in BC cells co-cultured with M2 macrophages transfected with 3′tRF-AlaAGC mimic.Conclusions3′tRF-AlaAGC might modulate macrophage polarization via binding to TRADD and increase the effect of M2 on promoting the chemoresistance in BC cells through NF-κb signaling pathway.

USP53 Affects the Proliferation and Apoptosis of Breast Cancer Cells by Regulating the Ubiquitination Level of ZMYND11

Breast cancer is the most common female malignancy worldwide. Ubiquitin-specific peptidase 53 (USP53) has been shown to exert cancer-suppressing functions in several solid tumors, but its role and the underlying mechanism in breast cancer has not been clearly elucidated. Therefore, we have carried out a series of detailed studies on this matter at the levels of bioinformatics, clinical tissue, cell function and animal model. We found that USP53 expression was downregulated in breast cancer specimens and was negatively correlated with the clinical stages. Gain- and loss-of-function experiments demonstrated USP53 inhibited proliferation, clonogenesis, cell cycle and xenograft growth, as well as induced apoptosis and mitochondrial damage of breast cancer cells. Co-immunoprecipitation data suggested that USP53 interacted with zinc finger MYND-type containing 11 (ZMYND11), and catalyzed its deubiquitination and stabilization. The 33–50 amino acid Cys-box domain was key for USP53 enzyme activity, but not essential for its binding with ZMYND11. The rescue experiments revealed that the anti-tumor role of USP53 in breast cancer cells was at least partially mediated by ZMYND11. Both USP53 and ZMYND11 were prognostic protective factors for breast cancer. USP53-ZMYND11 axis may be a good potential biomarker or therapeutic target for breast cancer, which can provide novel insights into the diagnosis, treatment and prognosis.

The misunderstanding of the R Classification—a survey amongst medical specialties treating breast cancer

Frequent discussions in the tumour board about the Residual tumour (R) Classification of the UICC’s “TNM Classification of Malignant Tumours”, especially in the case of breast surgery specimens, raised the question about differing interpretations amongst different medical specialties. Thus, we designed a survey about the R Classification with a special focus on breast cancer specimens. An online survey was conducted, where a web link to the survey was distributed via email to various medical professional societies dealing with breast cancer in Austria and Germany with the request to distribute the link to their members. The study population consisted of physicians of all educational levels of different medical professions, who deal with breast carcinomas in their daily routine. Two hundred two participants, of which 160 (79.2%) have more than 10 years’ professional experience, took part in the survey; 88 (43.6%) were surgeons/gynaecologists, 80 (39.6%) pathologists, 19 (9.4%) radiation oncologists/ therapists, 8 (4.0%) radiologists, and 7 (3.5%) oncologists. We show that the R Classification is not completely mastered by anyone and that there are significant differences in the interpretation of the R Classification between different medical specialties. For better differentiation between the residual tumour (R Classification) of the TNM and a pure resection margin assessment, we suggest the use of a Resection margin (Rm) Classification to avoid further misunderstandings. To assist better multidisciplinary cooperation and to ensure better patient care all medical disciplines should be educated about the actual meaning and correct application of the R Classification.

TSP50 facilitates breast cancer stem cell-like properties maintenance and epithelial-mesenchymal transition via PI3K p110α mediated activation of AKT signaling pathway

BackgroundStudies have confirmed that epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC)-like properties are conducive to cancer metastasis. In recent years, testes-specific protease 50 (TSP50) has been identified as a prognostic factor and is involved in tumorigenesis regulation. However, the role and molecular mechanisms of TSP50 in EMT and CSC-like properties maintenance remain unclear.MethodsThe expression and prognostic value of TSP50 in breast cancer were excavated from public databases and explored using bioinformatics analysis. Then the expression of TSP50 and related genes was further validated by quantitative RT-PCR (qRT-PCR), Western blot, and immunohistochemistry (IHC). In order to investigate the function of TSP50 in breast cancer, loss- and gain-of-function experiments were conducted, both in vitro and in vivo. Furthermore, immunofluorescence (IF) and immunoprecipitation (IP) assays were performed to explore the potential molecular mechanisms of TSP50. Finally, the correlation between the expression of TSP50 and related genes in breast cancer tissue microarray and clinicopathological characteristics was analyzed by IHC.ResultsTSP50 was negatively correlated with the prognosis of patients with breast cancer. TSP50 promoted CSC-like traits and EMT in both breast cancer cells and mouse xenograft tumor tissues. Additionally, inhibition of PI3K/AKT partly reversed TSP50-induced activation of CSC-like properties, EMT and tumorigenesis. Mechanistically, TSP50 and PI3K p85α regulatory subunit could competitively interact with the PI3K p110α catalytic subunit to promote p110α enzymatic activity, thereby activating the PI3K/AKT signaling pathway for CSC-like phenotypes maintenance and EMT promotion. Moreover, IHC analysis of human breast cancer specimens revealed that TSP50 expression was positively correlated with p-AKT and ALDH1 protein levels. Notably, breast cancer clinicopathological characteristics, such as patient survival time, tumor size, Ki67, pathologic stage, N stage, estrogen receptor (ER) and progesterone receptor (PR) levels, correlated well with TSP50/p-AKT/ALDH1 expression status.ConclusionThe effects of TSP50 on EMT and CSC-like properties promotion were verified to be dependent on PI3K p110α. Together, our study revealed a novel mechanism by which TSP50 facilitates the progression of breast cancer, which can provide new insights into TSP50-based breast cancer treatment strategies.

Functional ex vivo DNA fibre assay to measure replication dynamics in breast cancer tissue.

Replication stress (RS) is a key trait of cancer cells, and a potential actionable target in cancer treatment. Accurate methods to measure RS in tumour samples are currently lacking. DNA fibre analysis has been used as a common technique to measure RS in cell lines. Here, we investigated DNA fibre analysis on fresh breast cancer specimens and correlated DNA replication kinetics to known RS markers and genomic alterations. Fresh, treatment-naïve primary breast cancer samples (n = 74) were subjected to ex vivo DNA fibre analysis to measure DNA replication kinetics. Tumour cell proliferation was confirmed by EdU incorporation and cytokeratin AE1/AE3 (CK) staining. The RS markers phospho-S33-RPA and γH2AX and the RS-inducing proto-oncogenes Cyclin E1 and c-Myc were analysed by immunohistochemistry. Copy number variations (CNVs) were assessed from genome-wide single nucleotide polymorphism (SNP) arrays. We found that the majority of proliferating (EdU-positive) cells in each sample were CK-positive and therefore considered to be tumour cells. DNA fibre lengths varied largely in most tumour samples. The median DNA fibre length showed a significant inverse correlation with pRPA expression (r = -0.29, p = 0.033) but was not correlated with Cyclin E1 or c-Myc expression and global CNVs in this study. Nuclear Cyclin E1 expression showed a positive correlation with pRPA levels (r = 0.481, p < 0.0001), while cytoplasmic Cyclin E1 expression exhibited an inverse association with pRPA expression (r = -0.353, p = 0.002) and a positive association with global CNVs (r = 0.318, p = 0.016). In conclusion, DNA fibre analysis performed with fresh primary breast cancer samples is feasible. Fibre lengths were associated with pRPA expression. Cyclin E1 expression was associated with pRPA and the percentage of CNVs. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

CDCA5 accelerates progression of breast cancer by promoting the binding of E2F1 and FOXM1

BackgroundBreast cancer is one of the most common malignant tumors in women. Cell division cycle associated 5 (CDCA5), a master regulator of sister chromatid cohesion, was reported to be upregulated in several types of cancer. Here, the function and regulation mechanism of CDCA5 in breast cancer were explored.MethodsCDCA5 expression was identified through immunohistochemistry staining in breast cancer specimens. The correlation between CDCA5 expression with clinicopathological features and prognosis of breast cancer patients was analyzed using a tissue microarray. CDCA5 function in breast cancer was explored in CDCA5-overexpressed/knockdown cells and mice models. Co-IP, ChIP and dual-luciferase reporter assay assays were performed to clarify underlying molecular mechanisms.ResultsWe found that CDCA5 was expressed at a higher level in breast cancer tissues and cell lines, and overexpression of CDCA5 was significantly associated with poor prognosis of patients with breast cancer. Moreover, CDCA5 knockdown significantly suppressed the proliferation and migration, while promoted apoptosis in vitro. Mechanistically, we revealed that CDCA5 played an important role in promoting the binding of E2F transcription factor 1 (E2F1) to the forkhead box M1 (FOXM1) promoter. Furthermore, the data of in vitro and in vivo revealed that depletion of FOXM1 alleviated the effect of CDCA5 overexpression on breast cancer. Additionally, we revealed that the Wnt/β-catenin signaling pathway was required for CDCA5 induced progression of breast cancer.ConclusionsWe suggested that CDCA5 promoted progression of breast cancer via CDCA5/FOXM1/Wnt axis, CDCA5 might serve as a novel therapeutic target for breast cancer treatment.

A study of the assessment of pathological response to neoadjuvant chemotherapy in patients with breast cancer

Abstract: BACKGROUND: Neoadjuvant chemotherapy therapy ( NACT) has emerged as an integrated therapeutic approach to treat locally advanced breast cancer in order to down stage the tumour and to assess tumour response to chemotherapy. The aims of this study were to estimate the incidence of complete and partial pathological response in patients undergoing NACT for breast cancer and to evaluate the clinico pathological parameters associated with pathological response. MATERIALS AND METHODS: The study included breast cancer specimens from patients who had received NACT. The pathological response to NACT was assessed by the AJCC protocol and the morphological features associated with NACT were recorded. The clinicopathological parameters associated with complete pathological response were also studied. The association between the clinicopathological paramaters and pathological response was studies using Chi square and Fisher exact tests. RESULTS: There were a total of 50 women who underwent surgery for breast cancer following NACT. The mean age was 56.5 years. The majority (59.1%) belonged to stage IIIB followed by Stage III A ( 30.6%). 73.4% of cases showed clinical partial response, 14.2 % showed no response and 12.2 percent showed Pathological Complete Response (pCR). ER and PR negative and Her 2 positive status were the parameters significantly associated with pCR. CONCLUSIONS: Pathological evaluation is the most reliable method to assess response to NACT in breast cancer patients which may further influence management decisions. This study showed a much lower frequency of pCR when compared to other Indian studies. It is recommended that all patients who are treated with NACT undergo prior core needle biopsy with hormone receptor and Her 2 testing as they may have a bearing on pCR.

Identification of GNB1 as a downstream effector of the circRNA-0133711/miR-145-5p axis involved in breast cancer proliferation and metastasis

Abstract Objectives Despite the involvement of the G protein beta-1 (GNB1) protein in various cancer types, its relationship to breast tumours is presently uncertain. This research focused on the expression of GNB1 in breast cancer and its possible biological ramifications in an effort to explain this confusion. Methods The expression levels of GNB1 in adjacent normal tissues and breast cancer were compared. We next constructed GNB1-overexpressed or -knockdown MDA-MB-231 cell lines in order to clarify GNB1’s function in breast cancer. We used colony-formation assays, CCK-8 assays, xenograft models, and transwell migration/invasion assays to evaluate the effect of GNB1 on tumorigenicity, migration, and invasion. Moreover, we used western blot analysis to investigate the significance of FAK/mTOR signalling in GNB1-regulated tumour stimulatory effects in breast cancer. Finally, we investigated the upstream regulatory signaling of GNB1 using luciferase reporter and functional repair assays. Results When comparing human breast cancer specimens to specimens of normal tissue, we discovered that GNB1 was noticeably overexpressed. This phenotype was also found to be substantially associated with unfavourable clinical outcomes. Functional research findings indicate that elevated expression of GNB1 stimulated the proliferation and metastasis of breast cancer cells. Additionally, we discovered that GNB1 activated the FAK/mTOR signalling cascade by directly inducing the phosphorylation of the FAK protein through specific contacts. According to the results of the RNA pull-down assays and dual-luciferase reporter, we concluded that circRNA-0133711 functions as a competitive endogenous RNA (ceRNA) that sequesters miR-145-5p and thereby relieves its repressive effect on GNB1 expression. Conclusions Collectively, our research findings elucidate the hitherto unexplored important role of the circRNA-0133711/miR-145-5p/GNB1 axis in the formation of breast cancer, and provide a new biomarker for clinical diagnosis and treatment of breast cancer.

Human-derived Tumor-On-Chip model to study the heterogeneity of breast cancer tissue

One of the leading causes that complicate the treatment of some malignancies, including breast cancer, is tumor heterogeneity. In addition to inter-heterogeneity and intra-heterogeneity of tumors that reflect the differences between cancer cell characteristics, heterogeneity in the tumor microenvironment plays a critical role in tumor progression and could be considered an overlooked and a proper target for the effective selection of therapeutic approaches. Due to the difficulty of completely capturing tumor heterogeneity in conventional detection methods, Tumor-on-Chip (TOC) devices with culturing patient-derived spheroids could be an appropriate alternative. In this research, human-derived spheroids from breast cancer individuals were cultured for 6 days in microfluidic devices. To compare TOC data with conventional detection methods, immunohistochemistry (IHC) and ITRAQ data were employed, and various protein expressions were validated using the transcriptomic databases. The behavior of the spheroids in the collagen matrix and the cell viability were monitored over 6 days of culture. IHC and immunocytochemistry (ICC) results revealed that inter and intra-heterogeneity of tumor spheroids are associated with HER2/ER expression. HER2 expression levels revealed a more important biomarker associated with invasion in the 3D culturing of spheroids. The expression levels of CD163 (as a marker for Ma2 macrophages) and CD44 (a marker for cancer stem cells (CSCs)) were also evaluated. Interestingly, the levels of M2a macrophages and CSCs were higher in triple-negative specimens and samples that showed higher migration and invasion. Cell density and extracellular matrix (ECM) stiffness were also important factors affecting the migration and invasion of the spheroids through the matrix. Among these, rigid ECM revealed a more crucial role than cell density. To sum up, these research findings demonstrated that human-derived spheroids from breast cancer specimens in microfluidic devices provide a dynamic condition for predicting tumor heterogeneity in patients, which can help move the field forward for better and more accurate therapeutic strategies.

Suppressing mitochondrial inner membrane protein (IMMT) inhibits the proliferation of breast cancer cells through mitochondrial remodeling and metabolic regulation

Metabolic reprogramming is widely recognized as a hallmark of malignant tumors, and the targeting of metabolism has emerged as an appealing approach for cancer treatment. Mitochondria, as pivotal organelles, play a crucial role in the metabolic regulation of tumor cells, and their morphological and functional alterations are intricately linked to the biological characteristics of tumors. As a key regulatory subunit of mitochondria, mitochondrial inner membrane protein (IMMT), plays a vital role in degenerative diseases, but its role in tumor is almost unknown. The objective of this research was to investigate the roles that IMMT play in the development and progression of breast cancer (BC), as well as to elucidate the underlying biological mechanisms that drive these effects. In this study, it was confirmed that the expression of IMMT in BC tissues was significantly higher than that in normal tissues. The analysis of The Cancer Genome Atlas (TCGA) database revealed that IMMT can serve as an independent prognostic factor for BC patients. Additionally, verification in clinical specimens of BC demonstrated a positive association between high IMMT expression and larger tumor size (> 2 cm), Ki-67 expression (> 15%), and HER-2 status. Furthermore, in vitro experiments have substantiated that the suppression of IMMT expression resulted in a reduction in cell proliferation and alterations in mitochondrial cristae, concomitant with the liberation of cytochrome c, but it did not elicit mitochondrial apoptosis. Through Gene Set Enrichment Analysis (GSEA) analysis, we have predicted the associated metabolic genes and discovered that IMMT potentially modulates the advancement of BC through its interaction with 16 metabolic-related genes, and the changes in glycolysis related pathways have been validated in BC cell lines after IMMT inhibition. Consequently, this investigation furnishes compelling evidence supporting the classification of IMMT as prognostic marker in BC, and underscoring its prospective utility as a novel target for metabolic therapy.