Breast Cancer Tissue Sections Research Articles

A Dual-Modal, Label-Free Raman Imaging Method for Rapid Virtual Staining of Large-Area Breast Cancer Tissue Sections.

As one of the most common cancers, accurate, rapid, and simple histopathological diagnosis is very important for breast cancer. Raman imaging is a powerful technique for label-free analysis of tissue composition and histopathology, but it suffers from slow speed when applied to large-area tissue sections. In this study, we propose a dual-modal Raman imaging method that combines Raman mapping data with microscopy bright-field images to achieve virtual staining of breast cancer tissue sections. We validate our method on various breast tissue sections with different morphologies and biomarker expressions and compare it with the golden standard of histopathological methods. The results demonstrate that our method can effectively distinguish various types and components of tissues, and provide staining images comparable to stained tissue sections. Moreover, our method can improve imaging speed by up to 65 times compared to general spontaneous Raman imaging methods. It is simple, fast, and suitable for clinical applications.

Clinical and Analytical Validation of Two Methods for Ki-67 Scoring in Formalin Fixed and Paraffin Embedded Tissue Sections of Early Breast Cancer.

Proliferation determined by Ki-67 immunohistochemistry has been proposed as a useful prognostic and predictive marker in breast cancer. However, the clinical validity of Ki-67 is questionable. In this study, Ki-67 was retrospectively evaluated by three pathologists using two methods: a visual assessment of the entire slide and a quantitative assessment of the tumour margin in 411 early-stage breast cancer patients with a median follow-up of 26.8 years. We found excellent agreement between the three pathologists for both methods. The risk of recurrence for Ki-67 was time-dependent, as the high proliferation group (Ki-67 ≥ 30%) had a higher risk of recurrence initially, but after 4.5 years the risk was higher in the low proliferation group. In estrogen receptor (ER)-positive patients, the intermediate Ki-67 group initially followed the high Ki-67 group, but eventually followed the low Ki-67 group. ER-positive pN0-1 patients with intermediate Ki-67 treated with endocrine therapy alone had a similar outcome to patients treated with chemotherapy. A cut-off value of 20% appeared to be most appropriate for distinguishing between the high and low Ki-67 groups. To summarize, a simple visual whole slide Ki-67 assessment turned out to be a reliable method for clinical decision-making in early breast cancer patients. We confirmed Ki-67 as an important prognostic and predictive biomarker.

Immunohistochemical Observation and Correlation of Epidermal Growth Factor Receptor Downstream Signaling Proteins with Plasma Medium-Chain Fatty Acids Levels in Breast Cancer

Abstract Aim: The aim of this study was to correlate the expression of epidermal growth factor receptor (EGFR) downstream signaling molecules with plasma medium-chain fatty acids in breast cancer (BC). Materials and Methods: One hundred and sixteen plasma samples were collected from BC and control subjects. mRNA was extracted and gene expression of mRNA was analyzed by reverse transcriptase-quantitative polymerase chain reaction. Plasma levels of phosphorylated EGFR (p-EGFR), p-Akt, and phosphorylated mammalian target of rapamycin (p-mTOR) were measured using an ELISA kit. Thirty BC tissue sections were used to analyze the expression of EGFR/p-EGFR, Akt/p-Akt, and mammalian target of rapamycin (mTOR)/p-mTOR by immunohistochemistry. Results: The relative mRNA expression levels of EGFR, Akt, and mTOR were high in BC compared to the control subjects, whereas plasma levels of phosphorylated EGFR downstream signaling molecules were upregulated in BC patients, which was statistically significant. The expression of p-EGFR was high in both the cytoplasm and the nuclei, and also, the higher the expression of the downstream molecules in BC tissue sections. Conclusion: In the present investigation, there was a noteworthy inverse relationship between plasma levels of caprylic and lauric acid and the expression of EGFR downstream signaling molecules in BC. These findings suggest that EGFR signaling in BC may be efficiently repressed by increasing the amount of caprylic and lauric acid supplements taken through diet.

OR22-05 Progesterone Receptor-Grb2 Interaction Is Associated With Better Outcomes In Breast Cancer

Abstract Disclosure: N. Wittayavimol: None. E. Iwabuchi: None. P. Pateetin: None. Y. Miki: None. Y. Onodera: None. H. Sasano: None. V. Boonyaratanakornkit: None. The presence of progesterone receptor (PR) is usually associated with better clinical outcomes for several endocrine-related cancers. However, the molecular mechanism of how PR mediates growth inhibition remains unclear. In addition to its role as a nuclear transcription, PR also possesses an extranuclear function, mediated mainly through PR polyproline domain (PPD) interaction with the SH3 domains of cytoplasmic signaling molecules. Our previous study suggested a possible role of PR-PPD in mediating SH3-domain interaction. We showed that PR-PPD and SH3 domain interaction inhibited EGF-mediated signaling and decreased lung cancer cell proliferation. An essential adapter protein that helps transduce growth factor signaling is Grb2 which has an SH2 domain flanked by two SH3 domains. In this study, we hypothesized that PR interferes with cytoplasmic signaling through interaction between PR-PPD and Grb2-SH3. We used several protein-protein interaction assays in vitro and in cells to demonstrate PR-Grb2 interaction. GST-pulldown analysis showed that PR-PPD interacts specifically with SH3 domains of Grb2. No interaction was detected when key prolines in the PR-PPD to alanines. Immunofluorescence staining showed colocalization of PR and Grb2 in both the nucleus and cytoplasm of BT474 breast cancer cells. Using Bimolecular Fluorescence Complementation (BiFC) analysis, we showed that PR and Grb2 interacted in breast cancer cells, specifically through the Grb2-SH3 domain. No BiFC signal was detected when Grb2-SH3 domains were deleted. Furthermore, we used Proximity Ligation Assay (PLA) analysis to examine protein-protein interaction of a distance less than 40nm in cells. We found PR-Grb2 interaction in PR-positive breast cancer tissue sections. No PLA signal was detected in PR-negative breast cancer cells. Importantly, we examined 43 PR-positive breast cancer specimens and showed that PR-Grb2 interaction was associated with low histological stage and negatively correlated with lymph node invasion and metastasis of breast cancer. Our results provide a molecular mechanism for how PR can inhibit growth factor signaling by interfering with Grb2 signaling and a basis for developing novel therapeutic strategies for future cancer treatment. Presentation: Saturday, June 17, 2023

MALDI-IHC-Guided In-Depth Spatial Proteomics: Targeted and Untargeted MSI Combined.

Recently, a novel technology was published, utilizing the strengths of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) and immunohistochemistry (IHC), achieving highly multiplexed, targeted imaging of biomolecules in tissue. This new technique, called MALDI-IHC, opened up workflows to target molecules of interest using MALDI-MSI that are usually targeted by standard IHC. In this paper, the utility of targeted MALDI-IHC and its complementarity with untargeted on-tissue bottom-up spatial proteomics is explored using breast cancer tissue. Furthermore, the MALDI-2 effect was investigated and demonstrated to improve MALDI-IHC. Formalin-fixed paraffin-embedded (FFPE) human breast cancer tissue sections were stained for multiplex MALDI-IHC with six photocleavable mass-tagged (PC-MT) antibodies constituting a breast cancer antibody panel (CD20, actin-αSM, HER2, CD68, vimentin, and panCK). K-means spatial clusters were created based on the MALDI-IHC images and cut out using laser-capture microdissection (LMD) for further untargeted LC-MS-based bottom-up proteomics analyses. Numerous peptides could be tentatively assigned to multiple proteins, of which three proteins were also part of the antibody panel (vimentin, keratins, and actin). Post-ionization with MALDI-2 showed an increased intensity of the PC-MTs and suggests options for the development of new mass-tags. Although the on-tissue digestion covered a wider range of proteins, the MALDI-IHC allowed for easy and straightforward identification of proteins that were not detected in untargeted approaches. The combination of the multiplexed MALDI-IHC with image-guided proteomics showed great potential to further investigate diseases by providing complementary information from the same tissue section and without the need for customized instrumentation.

The characteristics and clinical significance of atypical mitosis in breast cancer

AbstractAtypical 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.

Dual-miRNA-Propelled Three-Dimensional DNA Walker for Highly Specific and Rapid Discrimination of Breast Cancer Cell Subtypes in Clinical Tissue Samples

Dual-miRNA-Propelled Three-Dimensional DNA Walker for Highly Specific and Rapid Discrimination of Breast Cancer Cell Subtypes in Clinical Tissue Samples

Efficient prediction of a spatial transcriptomics profile better characterizes breast cancer tissue sections without costly experimentation

Spatial transcriptomics is an emerging technology requiring costly reagents and considerable skills, limiting the identification of transcriptional markers related to histology. Here, we show that predicted spatial gene-expression in unmeasured regions and tissues can enhance biologists’ histological interpretations. We developed the Deep learning model for Spatial gene Clusters and Expression, DeepSpaCE, and confirmed its performance using the spatial-transcriptome profiles and immunohistochemistry images of consecutive human breast cancer tissue sections. For example, the predicted expression patterns of SPARC, an invasion marker, highlighted a small tumor-invasion region difficult to identify using raw spatial transcriptome data alone because of a lack of measurements. We further developed semi-supervised DeepSpaCE using unlabeled histology images and increased the imputation accuracy of consecutive sections, enhancing applicability for a small sample size. Our method enables users to derive hidden histological characters via spatial transcriptome and gene annotations, leading to accelerated biological discoveries without additional experiments.

High nerve density in breast cancer is associated with poor patient outcome.

Active crosstalk between the nervous system and breast cancer cells has been experimentally demonstrated in vitro and in animal models. However, low frequencies of peripheral nerve presence in human breast cancers reported in previous studies (~30% of cases) potentially negate a major role of the nervous system in breast cancer development and progression. This study aimed to clarify the incidence of nerves within human breast cancers and to delineate associations with clinicopathological features. Immunohistochemical staining was conducted in formalin‐fixed paraffin‐embedded breast cancer tissue sections using antibodies against the pan‐neuronal markers protein gene product 9.5 and growth‐associated protein 43, and the sympathetic nerve‐specific marker tyrosine hydroxylase. Nerve trunks and isolated nerve fibers were quantitated. The chi‐squared test was used to determine the associations between nerve counts and clinicopathological parameters. The log‐rank test was used to compare differences in patient progression‐free survival (PFS) and overall survival (OS). The overall frequency of peripheral nerves in breast cancers was 85%, a markedly higher proportion than reported previously. Of note, most nerves present in breast cancers were of the sympathetic origin. While high density of nerve trunks or isolated nerve fibers was associated with poor PFS and OS of patients, high nerve trunk density appeared also to predict poor patient PFS independently of lymph node metastasis. Innervation of breast cancers is a common event correlated with poor patient outcomes. These findings support the notion that the nervous system plays an active role in breast cancer pathogenesis.

Abstract P1-05-02: Intratumor molecular tumor heterogeneity in low ER-expressing primary breast tumors

Abstract Background: Change in estrogen receptor (ER) status between primary breast cancer and distant recurrence are seen in the clinic in up to 15-20% of patients. This is difficult to reconcile with the current understanding of breast cancer molecular subtypes, the large-scale molecular differences between ER-positive (ER+) and ER-negative (ER-) cancers suggest different cellular origins, luminal versus basal breast epithelium, respectively. A potential explanation for ER status switch is the presence of mixed molecular subtypes at diagnosis. ER+ cancers with less than 100% positivity may be composed by both luminal and basal-like cancer cells, and a recurrence might arise from one subtype that survived adjuvant therapy. The goal of this study was to test if molecular subtype heterogeneity exists at diagnosis in less than 100% ER+ breast cancer using the NanoString GeoMx™ platform. Methods: GeoMx™ is a highly multiplexed assay that quantifies RNA expression from spatially discrete regions of interest (ROIs) within formalin fixed paraffin embedded (FFPE) tissue sections. We identified 4 cancers with 30-40% ER-positivity on routine immunohistochemistry (IHC) staining (clone SP1). Eight ROIs per case were selected to represent both ER-high and -low regions of the section using ER IHC (clone 1D5). Invasive tumor cells were defined as PanCK positive cells. 1,825 mRNA species were measured separately in each ROI. mRNA expression results (unique molecular index [UMI] counts) were quantile normalized and differential expression analyses for all genes, ESR1, ER-regulated gene set, and PAM50 gene set were performed between ROIs within the same cancer. We also assessed PAM50 subtypes of each cases on bulk RNA using NanoString BC360 panel. Results: Three of the 4 cases had heterogenous ER IHC (1D5) staining; the fourth case was uniformly ER-negative during ROI selection. For this last case, ROIs were selected based on PanCK staining from different locations in the cancer. Bulk PAM50 subtyping indicated 1 basal-like, 2 HER2-enriched (HER2-E), and 1 luminal (lum)A cancer. We also attempted ROI-level subtyping; however, Pearson correlations to the nearest centroid were low (<0.5) for all ROIs, possibly due to low expression of PAM50 genes or to the exploratory methodology used. There was substantial within cancer, between ROI, expression heterogeneity for many genes including ESR1 and sensitivity to endocrine therapy (SET) index genes. Conclusions: ER- and ER+ cells are intermixed which makes defining low and high ER regions challenging. We observed region to region variation in ESR1 and SET index gene expression. ROI-level PAM50 subtyping was unstable. The novel NanoString GeoMx™ DSP technology can be used to study intratumor molecular heterogeneity in regions of interest on FFPE breast cancer tissue sections. We found substantial region-to-region gene expression differences within tissue sections. Case ID1234ER IHC SP130%40%40%30%ER IHC 1D50%40%40%30%Bulk PAM50 SubtypeBasalHER2-ELumAHER2-EESR1 UMI average (range across ROIs)28.45 (22.4-34.6)64.9 (18.8-173)179.9 (114-245)34.8 (25.1-45.4)SET Index (genes positively correlated with ESR1) UMI average (range across ROIs)41.5 (39.1-44.8)120.1 (86.9-182.9)94.9 (72.9-118.4)143.5 (124.9-150.3)SET Index (genes negatively correlated with ESR1) UMI average (range across ROIs)57.6 (45-75.3)43.4 (38.8-52.6)33.7 (31.8-36.7)44.8 (41.4-50.6) Citation Format: Julia Foldi, Emily Reisenbichler, Liuliu Pan, Krsitina Sorg, Sarah E. Church, Lajos Pusztai. Intratumor molecular tumor heterogeneity in low ER-expressing primary breast tumors [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-05-02.

Role of Ag NORs in improve diagnosis of breast cancer with different grades and subtypes’ among Sudanese women

This study aimed to evaluate the role of Ag NORs in improves diagnosis of Breast cancer with different subtypes’ among Sudanese Patients.This study includes tissue sections of breast cancer diagnosed women, they were 30, ductal and lobular invasive carcinoma were 10 for each, while ductal and lobular in-situ carcinoma were 5 each. Found correlation between subtypes of breast cancer and Ag NOR , Invasive ductal carcinoma had more NOR while the lobular carcinoma in situ was less one , Stage III most frequency than the other stage.Silver staining were performed and Ag-NOR were detected in ductal and lobular invasive carcinoma more than ductal and lobular in-situ carcinoma, grade III has more frequency of Ag-NOR than other stages, and no correlation found between Ag-NOR and age group. In conclusion,The presence of mAgNOR was found among ductal and lobular invasive carcinomas more than ductal and lobular in-situ carcinoma and more often among patients sorted as grade III more than others.

Role of Ag NORs in Improve Diagnosis of Breast Cancer with Different grades and Subtypes’ among Sudanese women

This study aimed to evaluate the role of Ag NORs in improves diagnosis of Breast cancer with different subtypes’ among Sudanese Patients. This study include tissue sections of breast cancer diagnosed women, they were 30, ductal and lobular invasive carcinoma were 10 for each, while ductal and lobular in-situ carcinoma were 5 each. Found correlation between subtypes of breast cancer and Ag NOR , Invasive ductal carcinoma had more NOR while the lobular carcinoma in situ was less one , Stage III most frequency than the other stage. Silver staining were performed and Ag-NOR were detected in ductal and lobular invasive carcinoma more than ductal and lobular in-situ carcinoma, grade III has more frequency of Ag-NOR than other stages, and no correlation found between Ag-NOR and age group

Protein citrullination as a source of cancer neoantigens

BackgroundCitrulline post-translational modification of proteins is mediated by protein arginine deiminase (PADI) family members and has been associated with autoimmune diseases. The role of PADI-citrullinome in immune response in cancer...

Selection of aptamers against triple negative breast cancer cells using high throughput sequencing

Triple-negative breast cancer is the most aggressive subtype of invasive breast cancer with a poor prognosis and no approved targeted therapy. Hence, the identification of new and specific ligands is essential to develop novel targeted therapies. In this study, we aimed to identify new aptamers that bind to highly metastatic breast cancer MDA-MB-231 cells using the cell-SELEX technology aided by high throughput sequencing. After 8 cycles of selection, the aptamer pool was sequenced and the 25 most frequent sequences were aligned for homology within their variable core region, plotted according to their free energy and the key nucleotides possibly involved in the target binding site were analyzed. Two aptamer candidates, Apt1 and Apt2, binding specifically to the target cells with K_{d} values of 44.3 ± 13.3 nM and 17.7 ± 2.7 nM, respectively, were further validated. The binding analysis clearly showed their specificity to MDA-MB-231 cells and suggested the targeting of cell surface receptors. Additionally, Apt2 revealed no toxicity in vitro and showed potential translational application due to its affinity to breast cancer tissue sections. Overall, the results suggest that Apt2 is a promising candidate to be used in triple-negative breast cancer treatment and/or diagnosis.

Development and Validation of Measurement Traceability for In Situ Immunoassays.

Immunoassays for protein analytes measured in situ support a $2 billion laboratory testing industry that suffers from significant interlaboratory disparities, affecting patient treatment. The root cause is that immunohistochemical testing lacks the generally accepted tools for analytic standardization, including reference standards and traceable units of measure. Until now, the creation of these tools has represented an insoluble technical hurdle. We address the need with a new concept in metrology-that is, linked traceability. Rather than calculating analyte concentration directly, which has proven too variable, we calculate concentration by measuring an attached fluorescein, traceable to NIST Standard Reference Material 1934, a fluorescein standard. For validation, newly developed estrogen receptor (ER) calibrators were deployed in tandem with an array of 80 breast cancer tissue sections in a national external quality assessment program. Laboratory performance was assessed using both the ER standards and the tissue array. Similar to previous studies, the tissue array revealed substantial discrepancies in ER test results among the participating laboratories. The new ER calibrators revealed a broad range of analytic sensitivity, with the lower limits of detection ranging from 7310 to 74 790 molecules of ER. The data demonstrate, for the first time, that the variable test results correlate with analytic sensitivity, which can now be measured quantitatively. The reference standard enables precise interlaboratory alignment of immunohistochemistry test sensitivity for measuring cellular proteins in situ. The introduction of a reference standard and traceable units of measure for protein expression marks an important milestone.

A novel aptamer-based histochemistry assay for specific diagnosis of clinical breast cancer tissues

Pathological detection using immunohistochemistry (IHC) has become an indispensable process in the diagnosis confirmation of various cancers. However, the production of monoclonal antibodies is always very complex, expensive and time-consuming, and the batch differences are significant due to the corporeity and health statuses of animals may be different. In this work, an aptamer-based histochemistry (aptahistochemistry) assay was developed using a DNA aptamer for specific diagnosis of clinical breast cancer tissue sections. This aptahistochemistry assay can specifically distinguish Luminal A breast cancer molecular subtype from Luminal B (HER2+), HER2-enriched, and triple-negative breast cancer molecular subtypes, as well as para-carcinoma tissue, mastitis tissue and normal breast tissue. The accuracy of this aptahistochemistry assay for the diagnosis of Luminal A breast cancer was as high as 80%, which showed a great potential for clinical pathological diagnosis applications.

10P Targeting Angiomotin-p130 reverse resistance to antiangiogenic therapies in breast cancer by inhibition of vasculogenic mimicry

Breast cancer (BC) was shown to relapse faster and displayed therapeutic resistance to antiangiogenic therapies (AATs) through an alternative tumor cell-driven mechanism of neovascularization called vascular mimicry (VM). This study aims to ascertain the correlation between Angiomotin-p130 expressing and the formation of VM in antiangiogenic therapy. Human BC tissue sections and tissue array were used to ascertain the clinical relevance of Angiomotin-p130 expressing in tumor cells and the formation of VM. We utilized MCF7 and MD-MBA-231 human tumor cells to observe VM in an orthotopic BC model and bevacizumab (anti-VEGF) was used to identify the resistance mechanism of antiangiogenic therapies in breast cancer. Later, tube formation assay was used to explore the efficacy of bevacizumab and anlotinib (anti-VEGFR,PDGFR,FGFR and c-Kit) in the inhibition of VM. According to human datasets and histological analysis Angiomotin-p130 was identified as a bonafide candidate to regulation VM formation through in vitro and in vivo experiments. Stable overexpression of Angiomotin-p130 in tumor cells led to decreased tumor growth as well as incomplete VM structures in the animal models. We identified highly downregulated Angiomotin-p130 in human breast tumor cells and AAT-treated orthotopic mammary tumors. AAT-treated (bevacizumab and anlotinib) tumors displayed a higher number of Angiomotin-p130-negative tumor cells than endothelial-like phenotypes. Tube formation assay showed that the bevacizumab and anlotinib had no significant difference in inhibition on VM, though anlotinib inhibited the proliferation of tumor cells. The present study suggests that tumor cell autonomous Angiomotin-p130 pathway may play an important role in AAT-mediated resistance and VM formation in BC. Therefore, targeting Angiomotin-p130 can be combined with standard antiangiogenic therapies to improve the therapeutic outcomes in clinical trials.

Extracellular Neuroglobin as a Stress-Induced Factor Activating Pre-Adaptation Mechanisms against Oxidative Stress and Chemotherapy-Induced Cell Death in Breast Cancer.

Components of tumor microenvironment, including tumor and/or stromal cells-derived factors, exert a critical role in breast cancer (BC) progression. Here we evaluated the possible role of neuroglobin (NGB), a monomeric globin that acts as a compensatory protein against oxidative and apoptotic processes, as part of BC microenvironment. The extracellular NGB levels were evaluated by immunofluorescence of BC tissue sections and by Western blot of the culture media of BC cell lines. Moreover, reactive oxygen species (ROS) generation, cell apoptosis, and cell migration were evaluated in different BC cells and non-tumorigenic epithelial mammary cells treated with BC cells (i.e., Michigan Cancer Foundation-7, MCF-7) conditioned culture media and extracellular NGB. Results demonstrate that NGB is a component of BC microenvironment. NGB is released in tumor microenvironment by BC cells only under oxidative stress conditions where it can act as autocrine/paracrine factor able to communicate cell resilience against oxidative stress and chemotherapeutic treatment.

Identification and transfer of spatial transcriptomics signatures for cancer diagnosis

BackgroundDistinguishing ductal carcinoma in situ (DCIS) from invasive ductal carcinoma (IDC) regions in clinical biopsies constitutes a diagnostic challenge. Spatial transcriptomics (ST) is an in situ capturing method, which allows quantification and visualization of transcriptomes in individual tissue sections. In the past, studies have shown that breast cancer samples can be used to study their transcriptomes with spatial resolution in individual tissue sections. Previously, supervised machine learning methods were used in clinical studies to predict the clinical outcomes for cancer types.MethodsWe used four publicly available ST breast cancer datasets from breast tissue sections annotated by pathologists as non-malignant, DCIS, or IDC. We trained and tested a machine learning method (support vector machine) based on the expert annotation as well as based on automatic selection of cell types by their transcriptome profiles.ResultsWe identified expression signatures for expert annotated regions (non-malignant, DCIS, and IDC) and build machine learning models. Classification results for 798 expression signature transcripts showed high coincidence with the expert pathologist annotation for DCIS (100%) and IDC (96%). Extending our analysis to include all 25,179 expressed transcripts resulted in an accuracy of 99% for DCIS and 98% for IDC. Further, classification based on an automatically identified expression signature covering all ST spots of tissue sections resulted in prediction accuracy of 95% for DCIS and 91% for IDC.ConclusionsThis concept study suggest that the ST signatures learned from expert selected breast cancer tissue sections can be used to identify breast cancer regions in whole tissue sections including regions not trained on. Furthermore, the identified expression signatures can classify cancer regions in tissue sections not used for training with high accuracy. Expert-generated but even automatically generated cancer signatures from ST data might be able to classify breast cancer regions and provide clinical decision support for pathologists in the future.

Recombinant Dual-target MDM2/MDMX Inhibitor Reverses Doxorubicin Resistance through Activation of the TAB1/TAK1/p38 MAPK Pathway in Wild-type p53 Multidrug-resistant Breast Cancer Cells.

Chemotherapy resistance represents a major obstacle for the treatment of patients with breast cancer (BC) and greatly restricts the therapeutic effect of the first-line chemotherapeutic agent doxorubicin (DOX). The present study aimed to investigate the feasibility of the recombinant dual-target murine double minute 2 (MDM2) and murine double minute X (MDMX) inhibitor in reversing the DOX resistance of BC. Both DOX-resistant human breast carcinoma cell lines exhibited a multidrug resistance (MDR) phenotype. The ability of the dual-target MDM2/MDMX inhibitor in reversing doxorubicin resistance was subsequently verified, (9.15 and 13.92 - fold reversal indexes) respectively. We observed that the MDM2/MDMX inhibitor in combination with DOX could suppress proliferation, promote cell cycle arrest and induce apoptosis. In addition, it was capable of reducing rhodamine123 efflux in DOX-resistance BC cell lines and further played a key role in BC nude mice model. The groups that were treated with the combination of the drugs had decreased P-glycoprotein/multidrug resistance-associated protein/cdc 2/Bcl-2 expression and increased CyclinB1/Bax expression. These effects were caused due to activation of the transforming growth factor β-activated kinase 1 (TAK1)-binding protein 1 (TAB1)/TAK1/p38 mitogen-activated protein kinase (MAPK) signaling pathway, as shown by small interfering RNA (siRNA) silencing and immumohistochemical staining of BC tissue sections. Furthermore, high MDM2/MDMX expression was positively associated with weak TAB1 expression in BC patients. Therefore, the recombinant dual-target MDM2/MDMX inhibitor could reverse doxorubicin resistance via the activation of the TAB1/TAK1/p38 MAPK pathway in wild-type p53 multidrug-resistant BC.