Predictive Value In Breast Cancer Research Articles

A panoptic segmentation dataset and deep-learning approach for explainable scoring of tumor-infiltrating lymphocytes

Tumor-Infiltrating Lymphocytes (TILs) have strong prognostic and predictive value in breast cancer, but their visual assessment is subjective. To improve reproducibility, the International Immuno-oncology Working Group recently released recommendations for the computational assessment of TILs that build on visual scoring guidelines. However, existing resources do not adequately address these recommendations due to the lack of annotation datasets that enable joint, panoptic segmentation of tissue regions and cells. Moreover, existing deep-learning methods focus entirely on either tissue segmentation or cell nuclei detection, which complicates the process of TILs assessment by necessitating the use of multiple models and reconciling inconsistent predictions. We introduce PanopTILs, a region and cell-level annotation dataset containing 814,886 nuclei from 151 patients, openly accessible at: sites.google.com/view/panoptils. Using PanopTILs we developed MuTILs, a neural network optimized for assessing TILs in accordance with clinical recommendations. MuTILs is a concept bottleneck model designed to be interpretable and to encourage sensible predictions at multiple resolutions. Using a rigorous internal-external cross-validation procedure, MuTILs achieves an AUROC of 0.93 for lymphocyte detection and a DICE coefficient of 0.81 for tumor-associated stroma segmentation. Our computational score closely matched visual scores from 2 pathologists (Spearman R = 0.58–0.61, p < 0.001). Moreover, computational TILs scores had a higher prognostic value than visual scores, independent of TNM stage and patient age. In conclusion, we introduce a comprehensive open data resource and a modeling approach for detailed mapping of the breast tumor microenvironment.

Pre-treatment peripheral blood immunophenotyping and response to neoadjuvant chemotherapy in operable breast cancer

BackgroundTumor immune infiltration and peripheral blood immune signatures have prognostic and predictive value in breast cancer. Whether distinct peripheral blood immune phenotypes are associated with response to neoadjuvant chemotherapy (NAC) remains understudied.MethodsPeripheral blood mononuclear cells from 126 breast cancer patients enrolled in a prospective clinical trial (NCT02022202) were analyzed using Cytometry by time-of-flight with a panel of 29 immune cell surface protein markers. Kruskal–Wallis tests or Wilcoxon rank-sum tests were used to evaluate differences in immune cell subpopulations according to breast cancer subtype and response to NAC.ResultsThere were 122 evaluable samples: 47 (38.5%) from patients with hormone receptor-positive, 39 (32%) triple-negative (TNBC), and 36 (29.5%) HER2-positive breast cancer. The relative abundances of pre-treatment peripheral blood T, B, myeloid, NK, and unclassified cells did not differ according to breast cancer subtype. In TNBC, higher pre-treatment myeloid cells were associated with lower pathologic complete response (pCR) rates. In hormone receptor-positive breast cancer, lower pre-treatment CD8 + naïve and CD4 + effector memory cells re-expressing CD45RA (TEMRA) T cells were associated with more extensive residual disease after NAC. In HER2 + breast cancer, the peripheral blood immune phenotype did not differ according to NAC response.ConclusionsPre-treatment peripheral blood immune cell populations (myeloid in TNBC; CD8 + naïve T cells and CD4 + TEMRA cells in luminal breast cancer) were associated with response to NAC in early-stage TNBC and hormone receptor-positive breast cancers, but not in HER2 + breast cancer.Trial registrationNCT02022202. Registered 20 December 2013.

Identification of Novel Anoikis-Related Gene Signatures to Predict the Prognosis, Immune Microenvironment, and Drug Sensitivity of Breast Cancer Patients.

Breast cancer is one of the most prevalent types of cancer and a leading cause of cancer-related death among females worldwide. Anoikis, a specific type of apoptosis that is triggered by the loss of anchoring between cells and the native extracellular matrix, plays a vital role in cancer invasion and metastasis. However, studies that focus on the prognostic values of anoikis-related genes (ARGs) in breast cancer are scarce. Gene expression data were obtained from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) databases. Five anoikis-related signatures (ARS) were selected from ARGs through univariate Cox regression analysis, LASSO regression analysis, and multivariate Cox regression analysis. Subsequently, an ARGs risk score model was established, and breast cancer patients were divided into high and low risk groups. The correlation between risk groups and overall survival (OS), tumor mutation burden (TMB), tumor microenvironment (TME), stemness, and drug sensitivity were analyzed. Moreover, RT-qPCR was performed to verify the gene expression levels of the five ARS in breast cancer tissues. Furthermore, a nomogram model was constructed based on ARGs risk score and clinicopathological factors. A novel ARGs risk score model was constructed based on five ARS (CEMIP, LAMB3, CD24, PTK6, and PLK1), and breast cancer patients were divided into high and low risk groups. Correlation analysis showed that the high and low risk groups had different OS, TMB, TME, stemness, and drug sensitivity. Both the ARGs risk score model and the nomogram showed promising prognosis predictive value in breast cancer. ARS could be used as promising biomarkers for breast cancer prognosis predication and treatment options selection.

Development and validation of Ran as a prognostic marker in stage I and stage II primary breast cancer

PurposeExisting prognostic biomarkers are inadequate for stratifying breast cancer patients with the highest risk of tumor progression at the time of diagnosis. Here, we demonstrate that the small GTPase Ran has predictive value for breast cancer (BC) patients as a whole, and for specific BC subtypes. Patients and methodsRan expression was quantified by immunohistochemistry in 263 patients with primary breast cancer diagnosed at the Breast Unit, Royal Liverpool Hospital. Additionally as an independent validation, we also analyzed the mRNA expressions of Ran, ER, PR, and Cerb-2, the triple-negative endocrine receptors, and their associations with patient survival in a combined patient cohorts of multiple public datasets (n = 1079). We analyzed the data with Spearman's rank correlation and Kaplan-Meier plots coupled with Wilcoxon-Gehan tests, respectively. All statistical tests were two-sided. ResultsRan nuclear, cytoplasmic, and total staining are substantially associated with poor survival, independent of conventional prognostic markers such as estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), and lymph node status. According to the datasets, Ran was significantly correlated with distant metastasis-free survival (DMFS) and relapse-free survival (RFS). ConclusionWe found that Ran expression is a unique predictive biomarker for patient survival, metastasis, and tumor relapse. This biomarker could be used for diagnostic purposes, using formalin-fixed, paraffin-embedded tumor biopsy samples from breast cancer patients in the early stages.

Increased expression of ECT2 predicts the poor prognosis of breast cancer patients

Breast cancer is the most common malignancy and the second leading cause of cancer-related death in women. Recent studies have indicated that aberrant activation of Rho GTPases relates to the malignant properties of breast cancer cells. As the guanine nucleotide exchange factor of Rho GTPases, the role of ECT2 (epithelial cell transforming 2) in breast cancer is still unclear. Tissue microarrays and multiple public databases were utilized to investigate the relationship between ECT2 level and clinical-pathological features of breast cancer patients. Kaplan Meier-plotter online tool and tissue microarray with survival information were used to investigate the predictive value for breast cancer. Here, we found increased ECT2 level was highly associated with advanced TNM stage, poor differentiation, and loss of hormone receptors of breast cancer. Gene expression profile showed that ECT2 level was closely correlated to cell-proliferation-associated pathways. Integration analysis using public databases and tissue microarray indicated that high ECT2 was an adverse prognostic factor for breast cancer patients. We believe the ECT2 level might be a valuable complement for commercially available predictors such as the 21 genes test. Furthermore, ECT2 would be a novel target for drug development for breast cancer.

Comparison of BIRADS lexicon to breast biopsy findings in low resource countries

Breast cancer is the most common malignancy in women worldwide and early detection is of utmost importance. In developed countries, mandatory mammographic screening programs help in early detection, whereas, in developing countries cancer is often detected at an advanced stage. The BIRADS guidelines permit a standard approach and follow up for breast lesions. Many newer imaging modalities are being available for better diagnosis. Breast lesions have a varied spectrum and the gold standard for diagnosis of breast cancer is based on histopathological examination of tissue. At times, even on trucut biopsy, it is difficult to categorize the lesion as the tissue studied is limited and some evolving lesions may have overlapping features. As there are limitations to both radiologic and pathologic approaches, the general and accepted way is to combine both modalities to arrive at a diagnosis. The aim: The aim of the study was to find out how well the BIRADS radiological findings correlate with histopathological findings on breast biopsies. Materials and methods: A MEDLINE search for articles published in English language, with key words as breast biopsy histopathology and BIRADS was done for the years between 1985 and 2021. In addition, other cross-referenced articles were also searched for relevant data. Results: There is good correlation between BIRADS category 1, 2 and 5 with the findings on core needle biopsy in breast lumps i.e., good correlation is seen at the end of spectrum of breast lesions in totally benign and unequivocally malignant lesions. But this correlation is lacking in the middle of the spectrum i.e., in borderline/intermediate category of BIRADS. Conclusion: The non-suspicious (BIRADS 1/ 2) and highly suspicious (BIRADS 4C/5) compare very well with the histopathologic findings. It is the grey zone i.e., BIRADS 3/4A which has a wide and variable predictive value for breast cancer when compared with histopathology and imaging study alone is insufficient and mandates histopathology in all such cases

Identification of FOXM1 and CXCR4 as key genes in breast cancer prevention and prognosis after intermittent energy restriction through bioinformatics and functional analyses

We explored potential biomarkers and molecular mechanisms regarding breast cancer (BC) risk reduction after intermittent energy restriction (IER) and further explored the association between IER and BC prognosis. First, we identified differentially expressed genes (DEGs) in breast tissues before and after IER intervention by analyzing the expression profile from gene expression summary (GEO). Then, enrichment analysis was used to identify important pathways of DEGs and hub genes were selected from protein-protein interaction (PPI) network. After that, gene expression profiling interactive analysis (GEPIA), receiver operating characteristic (ROC), and Kaplan–Meier (KM) plotter were used to explore the preventive and prognostic value of hub genes. A total of 331 up-regulated genes and 126 down-regulated genes were identified and 6 genes were selected as hub genes for further study. After analyzing by GEPIA and KM, it was found that FOXM1 and CXCR4 were highly expressed in BC tissues and associated with the worse prognosis. FOXM1 and CXCR4 were down-regulated after IER intervention, which meant that FOXM1 and CXCR4 might be the most important key genes for reducing the risk and improving prognosis of BC after IER intervention. ROC curve indicated that FOXM1 and CXCR4 also had the predictive value for BC. Our study contributed to a better understanding of the specific mechanisms in protective effects of IER on BC and provided a new approach to improve the prognosis of BC, which might provide partial guidance for the subsequent development of more effective treatments and prevention strategies.

Abstract P4-05-10: Composite score combining multi-gene testing with liquid biopsy may have stronger prognostic value in HR+/HER2- breast cancer

Abstract Background: For patients with early-stage, hormone receptor-positive (HR+) and HER2-negative breast cancer, multigene testing (MGT) is commonly used to personalize treatment. Published data suggests that the 70-gene MammaPrint (MP) assay, while used less commonly, may be superior to the 21-gene OncotypeDX panel at identifying high-risk patients requiring chemotherapy adjunct to endocrine therapy, versus endocrine therapy alone. We previously showed that the presence of circulating tumor cells (CTCs) has prognostic and predictive value in breast cancer. We assess the prognostic value of composite scoring combining liquid biopsy data with MGT. Methods: This retrospective analysis of the National Cancer Database explores the prognostic value of ODX, MP and liquid biopsy identifying CTCs in patients diagnosed between 2004-2017 with early-stage (AJCC clinical stage I-II), HR+/HER- breast cancer. We examined the prognostic value of the following models: 1) ODX alone, 2) MP alone, and 3) ODX-liquid biopsy composite score. The ODX-liquid biopsy composite score was created to be a binary risk stratification as follows: when circulating tumor cells were present, intermediate-risk as per ODX was upstaged to high-risk, but when absent, intermediate-risk was down-staged to low-risk. We compared the effect size of hazard ratios generated by each model’s inclusion in Cox regressions for overall survival that controlled for age and race. We also evaluated the prognostic value of each model through generated Harrell’s C-indices (i.e. area under ROC curve), with a C-index closer to 1 indicating superiority of a model in differentiating high- from low-risk groups. Results: Among early-stage, HR+/HER2- patients (n=841,716), n=271,416 (32.2%) had documented ODX data, n=12,417 (1.5%) had MP data, and n=1,141 (0.14%) had both ODX and CTC data. Based on liquid biopsy results, n=63 patients were upstaged from intermediate- to high-risk in the partial composite ODX-CTC model (20.5% of all intermediate-risk), while the rest (79.5%) were down-staged. A comparison of effect sizes for HRs corroborates preliminary data in the literature that MP alone (HR 2.49, 95% CI 1.94-3.19) may be superior to ODX alone (HR 2.23, 95% CI 2.07-2.39) at prognosticating overall survival. Additionally, we show that a binary composite score of ODX with liquid biopsy may better identify patients with a higher risk of mortality (HR 3.46, 95% CI 1.63-7.31). Comparison of C-indices was not possible for MP scoring or ODX-liquid biopsy scoring, likely due to an inadequate sample size leading to non-significance. Conclusion: Composite scoring based on ODX and liquid biopsy using CTCs may be superior to ODX/MP alone to differentiate high-risk from low-risk prognosis in patients with early-stage, HR+/HER2- BC. If confirmed in larger studies, this composite scoring would be useful in practice to more accurately identify a higher risk patient population to target for additional treatment and close monitoring. Table 1. Summary of prognostic parameters (HR and C-index) of 3 scoring systems.Prognostic ModelCOX RegressionROC AnalysisHR* (95% CI)C-index (95% CI)p-value1. ODX scoring alone (n=182,861)2.23 (2.07-2.39)0.566 (0.558-0.573)&amp;lt;0.0012. MP scoring alone (n=8,643)2.49 (1.94-3.19)0.527 (0.492-0.562)0.1213. ODX-liquid biopsy composite scoring (n=685) 3.46 (1.63-7.31)0.497 (0.391-0.602)0.952*HRs comparing high-risk to low-risk stratifications, calculated from COX regression for overall survival after controlling for age (5-70) and race (White). Citation Format: Nadeem Bilani, Marita Yaghi, Diana Saravia, Iktej Jabbal, Maroun Bou Zerdan, Leah Elson, Hong Liang, Zeina Nahleh. Composite score combining multi-gene testing with liquid biopsy may have stronger prognostic value in HR+/HER2- breast cancer [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 P4-05-10.

Development of an Inflammation-Related lncRNA-miRNA-mRNA Network Based on Competing Endogenous RNA in Breast Cancer at Single-Cell Resolution.

The role and mechanism of inflammation in breast cancer is unclear. This study aims to probe the relationship between inflammation and long non-coding RNAs (lncRNAs) and to stablish an inflammation-related competing endogenous RNA (ceRNA) network in breast cancer. Inflammation-related lncRNAs and target genes were screened based on the data from four single-cell RNA sequencing (scRNA-seq) studies and miRNAs were bioinformatically predicted according to ceRNA hypothesis. A series of in silico analyses were performed to construct an inflammation-related ceRNA network in breast cancer. Consequently, a total of seven inflammation-related lncRNAs were selected, after which LRRC75A-AS1 was identified as the most potential lncRNA in view of its expression and prognostic predictive value in breast cancer. Finally, an inflammation-related ceRNA network in breast cancer at the single cell level was established based on lncRNA LRRC75A-AS1, miR-3127-5p, miR-2114-3p, RPL36 and RPL27A mRNAs. Collectively, the lncRNA LRRC75A-AS1 and the LRRC75A-AS1-based on ceRNA network may exert crucial roles in modulating inflammation response during the initiation and progression of breast cancer.

Analysis of Ki-67 expression in women with breast cancer: Comparative evaluation of two different methodologies by immunophenotyping

The Ki-67 antigen is a nuclear protein with proven prognostic value in different neoplasms and recognizes the predictive value in breast cancer (BC). No consensus exists on the ideal cutoff point. In this study, Ki-67 expression was evaluated in samples of BC by flow cytometry (FC) and compared with immunohistochemical (IHC) examination. For this, the BC tissue samples were sectioned, macerated, filtered, and marked with anti-Ki-67 FITC and anti-CD45 V500 antibodies. We selected the neoplastic cells according to CD45 expression and size and internal complexity (FSC × SSC) using the Infinicity 1.7 software. Lymphocytes were negative control. We compared the results with IHC analyses carried out in parallel and independently. The expression of Ki-67 was evaluated in both methodologies through Bland–Altman analysis. Among the 44 samples analyzed, only three showed bias higher than the established confidence interval (mean bias 2.1%, p = 0.62), with no significant difference for the perfect mean bias (0%). Therefore, one can state that FC provides results equivalent to IHC analysis and possibly analyzes more cells simultaneously. The results obtained in this study show the absence of observational bias through software analysis in a larger number of tumor cell populations. We can conclude that FC may be a promising alternative method for investigating Ki-67 in solid tumours.

Urinary estrogen metabolites and breast cancer risk in Chinese population.

BackgroundIn China, the association between estrogen metabolism and breast cancer risk and the differences in metabolic pattern between breast cancer patients and controls are poorly understood.MethodsA total of 84 patients with invasive breast cancer and 47 controls with benign breast diseases were included in this study. Estrogen metabolites from their morning urine were determined by HPLC-MS/MS and evaluated in both groups, and the predictive value of each estrogen metabolite in the malignant group according to their menstrual status was analyzed.ResultsUrinary concentration of estrogen metabolites 2-hydroxyestrone (2-OHE1), 2-hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2), 4-methoxyestrone (4-MeOE1), and 16α-hydroxyestrone were lower in postmenopausal patients with breast cancer, compared with benign controls. In logistic regression model, breast cancer risk increased with the decline in the levels of 4-OHE2 and 4-MeOE1. In premenopausal patients, a difference in the level of 2-OHE2 was observed between both groups, and 2-OHE2 was found to have predictive value for breast cancer. Additionally, urinary 2-OHE2 level in premenopausal hormone receptor positive (HR+) patients was considerably higher compared with hormone receptor negative patients.ConclusionsWe found that lower urinary levels of 4-OHE2 and 4-MeOE1 had predictive value for breast cancer, and higher 2-OHE1 were associated with HR+ breast cancer in premenopausal women.

Independent Clinical Validation of the Automated Ki67 Scoring Guideline from the International Ki67 in Breast Cancer Working Group.

Ki67 is an important biomarker with prognostic and potential predictive value in breast cancer. However, the lack of standardization hinders its clinical applicability. In this study, we aimed to investigate the reproducibility among pathologists following the guidelines of the International Ki67 in Breast Cancer Working Group (IKWG) for Ki67 scoring and to evaluate the prognostic potential of this platform in an independent cohort. Four algorithms were independently built by four pathologists based on our study cohort using an open-source digital image analysis (DIA) platform (QuPath) following the detailed guideline of the IKWG. The algorithms were applied on an ER+ breast cancer study cohort of 157 patients with 15 years of follow-up. The reference Ki67 score was obtained by a DIA algorithm trained on a subset of the study cohort. Intraclass correlation coefficient (ICC) was used to measure reproducibility. High interobserver reliability was reached with an ICC of 0.938 (CI: 0.920–0.952) among the algorithms and the reference standard. Comparing each machine-read score against relapse-free survival, the hazard ratios were similar (2.593–4.165) and showed independent prognostic potential (p ≤ 0.018, for all comparisons). In conclusion, we demonstrate high reproducibility and independent prognostic potential using the IKWG DIA instructions to score Ki67 in breast cancer. A prospective study is needed to assess the clinical utility of the IKWG DIA Ki67 instructions.

Abnormal Long Non-Coding RNAs Expression Patterns Have the Potential Ability for Predicting Survival and Treatment Response in Breast Cancer.

Abnormal long non-coding RNAs (lncRNAs) expression has been documented to have oncogene or tumor suppressor functions in the development and progression of cancer, emerging as promising independent biomarkers for molecular cancer stratification and patients’ prognosis. Examining the relationship between lncRNAs and the survival rates in malignancies creates new scenarios for precision medicine and targeted therapy. Breast cancer (BRCA) is a heterogeneous malignancy. Despite advances in its molecular classification, there are still gaps to explain in its multifaceted presentations and a substantial lack of biomarkers that can better predict patients’ prognosis in response to different therapeutic strategies. Here, we performed a re-analysis of gene expression data generated using cDNA microarrays in a previous study of our group, aiming to identify differentially expressed lncRNAs (DELncRNAs) with a potential predictive value for response to treatment with taxanes in breast cancer patients. Results revealed 157 DELncRNAs (90 up- and 67 down-regulated). We validated these new biomarkers as having prognostic and predictive value for breast cancer using in silico analysis in public databases. Data from TCGA showed that compared to normal tissue, MIAT was up-regulated, while KCNQ1OT1, LOC100270804, and FLJ10038 were down-regulated in breast tumor tissues. KCNQ1OT1, LOC100270804, and FLJ10038 median levels were found to be significantly higher in the luminal subtype. The ROC plotter platform results showed that reduced expression of these three DElncRNAs was associated with breast cancer patients who did not respond to taxane treatment. Kaplan–Meier survival analysis revealed that a lower expression of the selected lncRNAs was significantly associated with worse relapse-free survival (RFS) in breast cancer patients. Further validation of the expression of these DELncRNAs might be helpful to better tailor breast cancer prognosis and treatment.

Composite scoring combining OncotypeDX risk with circulating tumor cell status improves prognostication.

e12577 Background: Genomic assays, such as the 21-gene OncotypeDX (ODX) and the 70-gene MammaPrint (MP) panels, can be used to personalize treatment for women with early-stage, hormone receptor-positive (HR+), HER2-negative (HER2-) breast cancer (BC). We previously showed that 1) circulating tumor cell (CTC) status has prognostic and predictive value in BC, and that 2) there is no clear association between ODX risk and CTCs. We, hereby, assess the prognostic value of composite scoring combining ODX and CTC data. Methods: This analysis of the 2004-2017 National Cancer Database registry includes patients with early-stage (AJCC I-II), HR+/HER- BC, as well as ODX, MP, and CTC data. A composite model was created with ODX and CTC data: low-risk escalated to intermediate-risk, or intermediate- to high-risk, if positive for CTCs. The association between a) ODX, b) ODX-CTC composite, or c) MP and overall survival (OS) was examined using Cox regressions, controlling for age (50-70); and stratifying by race (White vs. Black) and tumor histology (ductal vs. lobular). Prognostic value was evaluated using Harrell’s C-index (i.e. area under ROC curve) for each model. Results: N = 841,716 patients with early-stage, HR+/HER2- BC were identified. N = 271,416 (32.2%) had ODX data, n = 12,417 (1.5%) had MP data, and n = 1,141 (0.14%) had both ODX and CTC data. Due to CTC-positivity, n = 46 patients were upstaged from low- to intermediate-risk (23.1% of all low-risk), while n = 63 patients were upstaged from intermediate- to high-risk (20.5% of all intermediate-risk) in the composite ODX-CTC model. All 3 prognostic scoring systems were associated (p &lt; 0.05) with OS as per Cox regressions. As indicated by a C-index closer to 1, MP appears superior to ODX alone at differentiating high- from low-risk based on OS (0.597 versus 0.565) patients. However, composite ODX-CTC scoring was superior to MP alone (0.673 versus 0.597). This model could not be validated in Black patients (versus White) or those with lobular histology (compared to ductal) due to limited ODX-CTC data in these groups. Conclusions: Composite ODX-CTC risk scoring appears superior to MP alone. If confirmed, this prognostic model would more accurately identify early-stage, HR+/HER2- patients requiring escalated systemic therapy including chemotherapy compared to low-risk BC. More data is needed in ethnic minorities and patients with atypical histology to validate these promising results.[Table: see text]

A simple digital image analysis system for automated Ki67 assessment in primary breast cancer.

Ki67 is a well-established immunohistochemical marker associated with cell proliferation that has prognostic and predictive value in breast cancer. Quantitative evaluation of Ki67 is traditionally performed by assessing stained tissue slides with light microscopy. Automated image analysis systems have become available and, if validated, could provide greater standardisation and improved precision of Ki67 scoring. Here, we aimed to evaluate the use of the Cognition Master Professional Suite (CogM) image analysis software, which is a simple system for scoring Ki67 in primary breast cancer samples. Sections from 94 core-cut biopsies, 20 excision specimens and 29 pairs of core-cut biopsies and excision specimens were stained for Ki67 with MIB1 antibody and the Dako EnVision FLEX Detection System. Stained slides were scanned to convert them to digital data. Computer-based Ki67 scoring was performed with CogM. Manual Ki67 scoring assessment was conducted on previously stained sections from the same biopsies with a clinically validated system that had been calibrated against the risk of recurrence. A high correlation between manual and digital scores was observed [rCores =0.92, 95% confidence interval (CI) 0.87-0.94, P<0.0001; rExcisions =0.95, 95% CI 0.86-0.98, P<0.0001] and there was no significant bias between them (P=0.45). There was also a high correlation of Ki67 scores between paired core-cut biopsies and excision specimens when CogM was used (r=0.78, 95% CI 0.59-0.89, P<0.0001). CogM image analysis allows for standardised automated Ki67 scoring that accurately replicates previously clinically validated and calibrated manual scores.

Tissue Plasminogen Activator Levels and Risk of Breast Cancer in a Case-Cohort Study on Italian Women: Results from the Moli-sani Study.

Elevated levels of key enzymes of the fibrinolytic system, such as tissue plasminogen activator (tPA), are reported as predictors of poor outcome in cancer patients. Limited information is available about their potential predictive value for breast cancer (BC) risk in the general population. We examined the association of tPA levels with BC risk in a case-cohort study including women from the prospective Moli-sani cohort. A sample of 710 women (mean age: 54.6 ± 12.1 years) was selected as a subcohort and compared with 84 BC cases, in a median follow-up of 4.2 years. Incident cases of BC were validated through medical records. tPA plasma levels were measured using an enzyme-linked immunosorbent assay kit. Hazard ratio (HR) and 95% confidence interval (CI), adjusted for relevant covariates, were estimated by a Cox regression model using the Prentice method. Compared with the lowest quartile (<4.9 ng/mL), women in the highest quartile of tPA (>11.2 ng/mL) had increased risk of BC (HRIVvsI: 2.20, 95% CI: 1.13-4.28) after adjusted for age, smoking, education, menopause, and residence. Further adjustment for biochemical markers did not modify this association. The risk of BC increased by 34% for each increase in 1 standard deviation of log-transformed tPA levels (p = 0.046). Elevated levels of tPA were associated mainly with estrogen-receptor-positive BC (2.08, 95% CI: 1.18-3.66). Higher levels of tPA, reported to predict cardiovascular risk, are a potential biomarker for BC risk, supporting the hypothesis of a "common soil" linking the pathogenic mechanisms of hormone-dependent tumors and cardiovascular disease.

Liquid biopsy of the immune environment: Evaluation of peripheral blood mononuclear cells (PBMCs) with CyTOF and response to trastuzumab (T)-based neoadjuvant chemotherapy (NAC) in HER2+ breast cancer (BC).

592 Background: Immune responses in the tumor microenvironment have prognostic and predictive value in BC. However, the potential of immune responses observed in peripheral blood as biomarkers in BC remains unclear. We have shown that a higher frequency of circulating monocytes and a lower frequency of antigen-experienced memory CD8+ T cells are associated with response to NAC in triple negative BC (Leon-Ferre et al SABCS 2019). Here, we used cytometry by time-of-flight (CyTOF) to evaluate associations between circulating immune cells, clinical features and response to T-based NAC in HER2+ BC. Methods: PBMC suspensions from 36 pts with stage I-III HER2+ BC were prospectively collected prior to initiation of T-based NAC, stained with 29 metal-tagged antibodies optimized to identify major human immune cell subsets, and acquired in the Helios CyTOF instrument. Differential abundance analysis of immune cells by clinical characteristics and by NAC response was evaluated using Wilcoxon rank sum test. % of immune cell subsets is presented as % of all PBMCs. Results: Most pts presented with ER- tumors (56%), measuring &gt; 5cm (64%) and with nodal metastases (78%). After NAC, 16 pts (44%) achieved pathologic complete response (pCR). Analysis of preNAC PBMCs demonstrated a significantly higher number of B cells (8% vs 5%, p = 0.05) and effector memory CD8+ T cells (CD45RA-/CCR7-, 3 vs 1%, p = 0.02) in pts with pCR compared to those with residual disease. Of the B cell subsets, naïve B cells (CD24-/CD27-) were higher in pts who achieved pCR vs not (7% vs 4%, 0 = 0.04). Regarding clinical characteristics, cN+ pts at presentation exhibited a lower number of peripheral blood T cells compared to cN- pts (47% vs 63%, p = 0.03). Of the T cell subsets, overall CD4+ and naïve CD4+ T cells (CD45RA+/CCR7+) were lower in cN+ vs cN- pts (31% vs 45%, p = 0.05; and 11% vs 24%, p = 0.04). We also observed differences in CD56+/CD16- NK cells by ER status (ER- 1% vs ER+ 3%, p = 0.01), and a moderate negative correlation between age and % circulating CD8+ T cells (rho -0.4669, p = 0.004). Conclusions: Distinct peripheral blood immune cell profiles are observed in HER2+ BC at diagnosis, and are associated with response to T-based NAC and initial clinical characteristics. Notably, pts who later achieved pCR had a relative abundance of B cells and effector memory CD8+ T cells at diagnosis. These data suggest that immune cell phenotyping in peripheral blood may have potential as a biomarker to predict response to NAC in BC.

Abstract P4-06-13: Programmed death-1 and programmed death-ligand 1 expression in sporadic breast cancer compared to BRCA germline mutation related breast cancer and male breast cancer

Abstract Introduction Programmed death 1 (PD-1) and its ligand PD-L1 seem to have a prognostic and predictive role in a variety of cancer types, especially the ones with a high mutational load and immunogenic profile. Despite that breast cancer is not considered as immunogenic, recent studies suggested that PD-1 and PD-L1 do have a prognostic and/or predictive value in breast cancer, which varies between molecular subtypes. However, the role of PD-1 and PD-L1 in BRCA germline mutation related breast cancer and male breast cancer has not yet been examined. We therefore evaluated the expression of PD-1 and PD-L1 in sporadic, BRCA related and male breast cancer. Methods Tissue microarrays were constructed of formalin-fixed paraffin-embedded resection material from 257 sporadic, 132 BRCA related and 175 male breast cancer patients and stained for PD-1 and PD-L1 by immunohistochemistry. Expression in BRCA related and male breast cancer was compared to the sporadic group and correlations with clinicopathological features were tested. Results PD-1 and/or PD-L1 expression was available for 245 sporadic, 145 male and 103 BRCA related breast cancers. PD-1 expression in the BRCA group was significantly higher than in the sporadic group (79.0% vs 64.1%), this in contradictory to the male group, where PD-1 was significantly less often expressed than in the sporadic group (46.9% vs. 64.1%). For PD-L1 expression no significant differences were seen between the groups. In subgroup analysis PD-1 expression was higher in grade 3 tumors compared to the lower grade tumors, which was significant in sporadic and male breast cancer. For PD-L1 a significantly higher expression was observed in grade 3 tumors in BRCA related breast cancer, while a significantly lower expression in grade 3 tumors was seen in the sporadic subgroup. No differences were observed looking at PD-(L)1 expression and molecular subtype. Subgroup analysis for PD-1 and PD-L1 expression and tumor grade Grade PD-1 negative, No. (%) PD-1 positive, No. (%) P-value PD-L1 negative, No. (%) PD-L1 positive, No. (%) P-value Sporadic116 (39.0)25 (61.0) 20 (52.6)18 (47.4) 237 (43.5)48 (56.5) 0.03740 (50.0)40 (50.0) 0.036 330 (26.5)83 (73.5) 68 (68.0)32 (32.0) BRCA1-1 (100) -1 (100) 28 (34.8)15 (65.2)0.18620 (87.0)3 (13.0) 0.011 313 (17.3)62 (82.7) 46 (61.3)29 (38.7) Male120 (64.5)11 (35.5) 20 (64.5)11 (35.5) 228 (58.3)20 (41.7) 0.03733 (66.0)17 (34.0)0.213 324 (39.3)37 (60.7) 31 (50.8)30 (49.2) Conclusion PD-1 expression is higher in BRCA related breast cancer compared to sporadic breast cancer, but lower in male breast cancer. Additionally, PD-1 expression is more frequent in grade 3 tumors. Patients with BRCA related breast cancer might therefore respond better to PD-1 inhibitors than patients with sporadic breast cancer, this in contrast to men with breast cancer. Citation Format: Manson QF, ter Hoeve ND, Moelans CB, van Diest PJ. Programmed death-1 and programmed death-ligand 1 expression in sporadic breast cancer compared to BRCA germline mutation related breast cancer and male breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-13.

PO-433 Prognostic relevance of PP1 in breast cancer

IntroductionProtein phosphatase 1 (PP1) is one of the major cellular serine/threonine protein phosphatases and it is implicated in the regulation of many cellular events including glycogen metabolism, neuronal signalling and protein synthesis. In addition, PP1 indirectly controls cell cycle progression by modulating the phosphorylation status of key cell-regulatory proteins, including retinoblastoma protein (pRb). In mammalian cells, three genes encode four isoforms of PP1 designated as PP1α, PP1β, PP1γ1 and PP1γ2. These isoforms are expressed in all tissues and cellular compartments except PP1γ2, which is found only in testes.Despite the numerous functions in which it is involved, PP1 itself does not exhibit substrate specificity. Indeed, specificity is achieved by its interaction with ~200 different regulatory proteins that associate with PP1 to form highly specific holoenzymes. These regulatory subunits bind to the catalytic subunit of PP1 (PP1C) generating structures and specific functions. Three different PP1C isoforms have been described, PPP1CA, PPP1CB and PPP1CC.As the role of PP1 in cell cycle regulation is crucial and its specificity depends on the binding of the regulatory protein to PP1C, we investigated the prognostic relevance of the different PP1 catalytic subunits in human tumours.Material and methodsTo evaluate the prognostic relevance of the three PP1 catalytic subunits in human tumours, we analysed correlations between grade, patient survival, tumour recurrence and PPP1CA/B/C gene expression in independent patient cohorts. To this end, we used different public transcriptomic databases of breast cancer from Oncomine (Compendia Biosciences, www.oncomine.org) and from R2: Genomics analysis and visualisation platform (http://r2.amc.nl/).Results and discussionsThe downregulation of two of the three catalytic subunits of PP1 (PPP1CA and C) is associated with worse prognosis in breast tumours, correlating with worse overall survival, a higher tumour grade and ER positive status.ConclusionThe catalytic subunit of PP1 seems to have a prognostic and predictive value for breast cancer. As several PP1 regulatory proteins have been described, identifying and understanding their role in PP1 regulation during cell cycle progression might be important to control cell growth. Therefore, PP1 regulatory proteins might be interesting as new therapeutic targets and/or biomarkers in cancer.

Decreased cellular and humoral immunity in patients with breast cancer.

4 Background: A growing body of evidence over the past few years suggests that the presence of immune elements within the tumor or in the tumor stroma has prognostic and predictive value in breast cancer. Immunotherapy is successfully used in many types of cancer, and modulation of immune system became standard part of the cancer patients treatment Responses in breast cancer have been more recently reported. However it has yet to be determined whether predictable biomarkers of response can be identified. Therefore in recent years, research focused on a precise description of the status and function of the immune system.The purpose of the study was to monitor immune responses in patients with breast cancer, particularly the examination of cellular (CD4, CD8, B cells) as well as humoral immunity (IgG, IgG1, IgG2, IgG3, IgG4. It appears that a factor contributing to the immunosupression may be a transforming factor-beta (TGF-beta).It is highly immunosuppressive factor that inhibits the natural and specific immunity against tumors. Methods: 50 patients included in the research project were implemented routine cancer treatment. Basic parameters (histological type and grade, the degree of expression of ER and PR, HER2, and the proliferative marker) were established. Patients were evaluated by a cancer clinical immunologist to exclude immune disorders, allergic or autoimmune origin. Anti-tumor cellular immunity (CD4, CD8, CD19) was measured by flow citometry, humoral immunity (IgG, IgG1, IgG2, IgG3, IgG4) was measured and TGF beta and VEGF production was monitored by ELISA. Results: In breast cancer patients mainly depression in cellular immunity was found. Immunglobuline plasma level was decreased as well (mainly IgG4 subtype). TGF beta as well as VEGF plasma level were increased. Most patients have shown clinical symptoms of immunodeficiency (frequent infections of respiratory or urinary tract, herpetic infections).Those patient could benefit from immunomodulation. Conclusions: The state of anticancer immunity could contribute to the selection of targeted immune therapy in breast cancer patients and to help to find optimal combination of immunotherapy. This project was supported by governmental grant AZV CR 15-28188A.