Breast Tumor Tissues Research Articles

Abstract 4163: Clinical proteomics for patient stratification - a GCLP quantitative mass spectrometry workflow for multiplexed measurement of protein biomarkers in FFPE tissues

Abstract a) The advent of precision oncology has led a shift towards biomarker-driven clinical trial designs and molecular profiling of individual patients. Identification of patients with the target biomarker profile may be useful in guiding patient selection as an enrichment strategy for clinical trials. Targeted multiple reaction monitoring mass spectrometry (MRM-MS) analysis for multiplexed quantitation of biomarker proteins in FFPE tissue provides direct, more accurate and precise quantification over current ‘gold standard' immunohistochemistry (IHC) methods. However, MRM-MS has not yet been broadly applied to clinical trials. In this study, we demonstrate the systematic development, optimization and analytical validation of quantitative, multiplexed MRM-MS assays for robust biomarker quantification in clinical FFPE tissues, including sample analysis under GCLP. Results from an MRM panel targeting 8 clinically relevant biomarker proteins will also be shown, including the measured HER2 levels in FFPE breast tumors classified by IHC as 0, 1+, 2+ or 3+. b) MRM-MS biomarker panels were developed and optimized for multiplexed quantitation of ≤12 proteins, in which unique peptides derived from each target protein were monitored as a surrogate measure of protein levels. Tumor regions from FFPE tissue sections were dissected using laser capture or macrodissection, solubilized, digested with trypsin to generate peptides for analysis, spiked with fixed levels of stable isotope labeled (SIL) peptide standards, and analyzed by MRM-MS. Analytical validation was performed per NCI CPTAC guidelines, including response curves, assay repeatability, selectivity, stability, and reproducibility of endogenous detection. Clinical performance was assessed using commercially sourced FFPE-tumor tissues, including a cohort of breast tumor tissues with a wide range of HER2 expression. c) Assay performance results were protein/peptide dependent, with sensitivity in the low pg/μg total protein range. For HER2, assay linearity was demonstrated over 2.5 to 3 orders of magnitude, with a precision and accuracy of <15% over 3 independent runs. In sample analysis, the MRM-MS was sufficiently sensitive to detect HER2 in 1 μg total protein from FFPE breast tumor classified by IHC as negative (0). d) GCLP-compliant quantitative multiplexed large-scale clinical analysis of protein biomarkers by MRM-MS in FFPE tissue is feasible and enables precise and accurate quantitation of proteins when IHC methods are unsuitable or unavailable. Data can be used for patient stratification, optimization of treatment outcomes, drug resistance prediction, and to support clinical development of novel therapies. Citation Format: Maxim Isabelle, Michael Schirm, Gwenael Pottiez, Rudolf Guilbaud, Lorella Di Donato. Clinical proteomics for patient stratification - a GCLP quantitative mass spectrometry workflow for multiplexed measurement of protein biomarkers in FFPE tissues [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4163.

Eight immune-related genes predict survival outcomes and immune characteristics in breast cancer.

Advancements in immunotherapy have improved our understanding of the immune characteristics of breast cancer. Here, we analyzed gene expression profiles and clinical data obtained from The Cancer Genome Atlas database to identify genes that were differentially expressed between breast tumor tissues and normal breast tissues. Comparisons with the Immunology Database and Analysis Portal (ImmPort) indicated that many of the identified differentially expressed genes were immune-related. Risk scores calculated based on an eight-gene signature constructed from these immune-related genes predicted both overall survival and relapse-free survival outcomes in breast cancer patients. The predictive value of the eight-gene signature was validated in different breast cancer subtypes using external datasets. Associations between risk score and breast cancer immune characteristics were also identified; in vitro experiments using breast cancer cell lines confirmed those associations. Thus, the novel eight-gene signature described here accurately predicts breast cancer survival outcomes as well as immune checkpoint expression and immune cell infiltration processes.

MiR-204/COX5A axis contributes to invasion and chemotherapy resistance in estrogen receptor-positive breast cancers.

Breast cancer is the most common cancer among women worldwide, with 70% being estrogen receptor-positive (ER+). Although ER-targeted treatment is effective in treating ER+breast cancer, chemoresistance and metastasis still prevail. Outcome-predictable biomarkers can help improve patient prognosis. Through the analysis of the Array Express database, The Cancer Genome Atlas-Breast Cancer datasets, and breast tumor tissue array results, we found that cytochrome c oxidase subunit 5a (COX5A) was related to poor prognosis of ER+breast cancer. Further studies revealed that COX5A was positively associated with metastasis and chemoresistance in ER+breast cancer. In vitro experiments showed that knockdown of COX5A was accompanied by a decrease in ERα expression, cell cycle arrest, and epithelial-mesenchymal transition blockade, resulting in an inhibition of proliferation and invasion. Knockdown of COX5A enhanced the chemosensitivity of breast cancer cells by decreasing adenosine triphosphate and increasing reactive oxygen species levels. We report that miR-204 can target and inhibit the expression of COX5A, thus, reversing the functions of COX5A in ER+breast cancer cells. We found that COX5A may serve as a prognostic biomarker in ER+breast cancer.

Rapid Microfluidic Formation of Uniform Patient-Derived Breast Tumor Spheroids.

Breast cancer is a highly complex, heterogeneous, and multifactorial disease that poses challenges for rapid and efficient treatment and development of personalized therapy. Here, we describe a rapid and reliable method to generate three-dimensional (3D) tumor spheroids in vitro that recapitulate an individual patient's tumor for testing treatments. By employing droplet microfluidics and scaffold materials, tumor cells were encapsulated into a large number of Matrigel-in-oil droplets with precise control over cell numbers and components per droplet. After removal of the oil, large numbers of uniform tumor spheroids were formed within a few hours via Matrigel-supported cell self-assembly. Our microfluidic technique produces uniform-sized tumor spheroids in less than 1 day. This method was used to reproducibly and rapidly generate uniform-sized tumor spheroids derived from patients' breast tumor tissues. As a proof-of-concept application, this method was used to quickly evaluate cancer treatments. We demonstrated that our microfluidic patient-derived tumor cultures not only preserve the genetic characteristics of the original tumor tissue but also provide heterogeneous responses to targeted therapies within 2 days. We believe this method will enable a timely and reliable 3D in vitro culture model, which may be applicable to personalized treatment prediction, drug discovery, and toxicity testing.

Evaluation of EphB4 as Target for Image-Guided Surgery of Breast Cancer.

Background: Targeted image-guided surgery is based on the detection of tumor cells after administration of a radio-active or fluorescent tracer. Hence, enhanced binding of a tracer to tumor tissue compared to healthy tissue is crucial. Various tumor antigens have been evaluated as possible targets for image-guided surgery of breast cancer, with mixed results. Methods: In this study we have evaluated tyrosine kinase receptor EphB4, a member from the Eph tyrosine kinase receptor family, as a possible target for image-guided surgery of breast cancers. Two independent tissue micro arrays, consisting of matched sets of tumor and normal breast tissue, were stained for EphB4 by immunohistochemistry. The intensity of staining and the percentage of stained cells were scored by two independent investigators. Results: Immunohistochemical staining for EphB4 shows that breast cancer cells display enhanced membranous expression compared to adjacent normal breast tissue. The enhanced tumor staining is not associated with clinical variables like age of the patient or stage or subtype of the tumor, including Her2-status. Conclusion: These data suggest that EphB4 is a promising candidate for targeted image-guided surgery of breast cancer, especially for Her2 negative cases.

RETRACTED ARTICLE: Ameliorated monopole antenna with perforated ground for breast tumor detection

This letter confer an ameliorated monopole antenna for locating the tumor in breast tissue. The posited UWB antenna with a size of 35 × 30 mm provides good impedance matching, gain and radiation characteristics over the wide range of 3.1 GHz to 12.8 GHz. Homogeneous breast model was enlarged to analyze the area as well as size of the tumor in breast, The absorbed power and the SAR for average mass of 10 g of breast tissue at different frequency, different size of the tumor and different distance among the antenna and the breast model were analyzed. In order to locate the tumor, the coordinates of the maximum SAR value are discerned. The antenna and breast phantom were designed using CST studio simulator.

MiR‑28‑5p inhibits the migration of breastcancer by regulating WSB2.

MicroRNAs (miRNAs or miRs) play an important role in the tumorigenesis and progression of breast cancer. However, the function of miR-28-5p in breast cancer migration has yet to be determined. In the present study, Human MicroRNA Expression Database (HMED) analysis revealed that the expression level of miR-28-5p was significantly lower in breast cancer tissue than in normal breast tissue. Kaplan -Meier plotter (KMPLOT) analysis revealed that the low expression level of miR-28-5p was associated with a poor survival in breast cancer. In addition, reverse transcription-quantitative PCR (RT-qPCR) revealed that the expression of miR-28-5p was significantly lower in breast cancer cell lines compared with that in human mammary epithelial cells (HMECs). Moreover, transfection with miR-28-5p mimics suppressed the migration of MCF-7 cells, whereas an miR-28-5p inhibitor exerted the opposite effect. Gene chip assay identified 648 differentially expressed genes (DEGs) in cells overexpressing miR-28-5p. The DEGs are enriched in the 'focal adhesion' and 'pathway in cancer' pathways. The expression levels of Ras-related protein Rap-1b (RAP1B), WD repeat and SOCS box containing 2 (WSB2) and vascular endothelial growth factor A (VEGFA) were confirmed by RT-qPCR. Furthermore, transfection with miR-28-5p mimics decreased WSB2 expression, whereas the miR-28-5p inhibitor increased the expression of WSB2, at both the transcriptional and translational levels. miR-28-5p targets the 3′UTR of WSB2, and the binding site is conserved in multiple species, with a consensus motif of 5′-AGCUCCUU-3′. Moreover, WSB2 overexpression promoted the migration of MCF-7 cells which had been inhibited by miR-28-5p. UALCAN analysis revealed that WSB2 was significantly upregulated in primary breast tumor tissue, and a high expression level of WSB2 was associated with a poor survival in breast cancer. Furthermore, immunohistochemistry revealed that the expression of WSB2 was markedly higher in breast cancer tissue compared with that in adjacent normal breast tissue. Taken together, the findings of the present study demonstrate that miR-28-5p inhibits the migration of breast cancer cells by regulating WSB2 expression, and the miR-28-5p/WSB2 axis may be a novel therapeutic target in breast cancer.

MAM domain containing 2 is a potential breast cancer biomarker that exhibits tumour-suppressive activity.

ObjectivesThe aim of this study was to discover new potential biomarkers of breast cancer and investigate their cellular functions.Materials and methodsWe analysed the gene expression profiles of matched pairs of breast tumour and normal tissues from 24 breast cancer patients. Tetracycline‐inducible MAMDC2 expression system was established and used to evaluate cell proliferation in vitro and in vivo. MAMDC2‐mediated signalling was determined using immunoblot analysis.ResultsWe identified MAMDC2 as a down‐regulated gene showing significant prognostic capability. Overexpression of MAMDC2 or treatment with MAMDC2‐containing culture medium significantly inhibited the cell proliferation of T‐47D cells. Furthermore, MAMDC2 expression reduced in vivo growth of T‐47D xenograft tumours. MAMDC2 may exert its growth‐inhibitory functions by attenuating the MAPK signalling pathway.ConclusionWe report that MAMDC2 has a tumour‐suppressive role and, as a secretory protein, it might be useful as a biomarker for breast cancer treatment.

CCL2 and IL18 expressions may associate with the anti-proliferative effect of noncontact electro capacitive cancer therapy in vivo

Background: Noncontact Electro Capacitive Cancer Therapy (ECCT) is a novel treatment modality in cancer. Chemokine (C-C motif) ligand 2 (CCL2) has a major role in the outgrowth of metastatic breast cancer. Interleukin 18 (IL18) plays a role in macrophage alteration, which leads to excessive angiogenesis. This study aims to elaborate on the association of CCL2, IL18, IL23α, and TNF-α (tumor necrosis factor-alpha) expression with the anti-proliferative effect of ECCT in rat breast tumor tissue. Methods: Low intensity (18 Vpp) and intermediate frequency (150 kHz) alternating current-electric field (AC-EF) between two capacitive electrodes were exposed as external EF to a rat cage. Twenty-four rats were divided into four groups of six replicates. Breast tumor tissues were collected from 7, 12-dimethylbenz[a]anthracene (DMBA)-induced rats. Two groups were non DMBA-induced rats without ECCT exposure (NINT) and with (NIT). The other two groups were DMBA-induced rats without ECCT exposure (INT) and with (IT). Mammary glands and breast tumor tissues were collected from each group and preserved. Hematoxylin-eosin and immunohistochemistry staining were performed on paraffin sections of tissues using anti-PCNA, anti-ErbB2, anti-Caspase3, and anti-CD68. CCL2, IL18, IL23α, and TNF-α mRNA relative expressions were analyzed using qRT-PCR. Results: ECCT exposure may cause the reduction of PCNA protein expression as well as ErbB2 on breast tumor tissues, but it causes the increase of Caspase3 and macrophage CD68 protein. In rat breast tumor tissues of IT groups, the mRNA expression of CCL2 and IL18 are significantly down-regulated, in contrast with the up-regulated expression of these cytokines in tumor tissues of the INT group. IL23α and TNF- α expression remained similar in both groups. Conclusion: CCL2 and IL18 expressions have an association with the inhibition of breast tumor cell proliferation affected by ECCT exposure.

Establishment and validation of a novel autophagy-related gene signature for patients with breast cancer.

There exists considerable evidence conforming that autophagy may play an important role in the biological process of breast cancer. This study aimed to construct and evaluate a novel autophagy-related gene signature as a potential prognostic factor and therapeutic target in breast cancer patients based on high-throughput sequencing datasets. Autophagy-related genes obtained from the Human Autophagy Database and high-sequencing data obtained from The Cancer Genome Atlas (TCGA) were analyzed to identify differential expressed genes (DEGs) between tumor and normal tissues. Then GO and KEGG analysis were performed to explore potential biological and pathological functions of DEGs. Autophagy-related prognostic genes were identified by univariate COX regression analysis. Subsequently stepwise model selection using the Alkaike information criterion (AIC) and multivariate COX regression model was performed to construct autophagy-related gene signature. Then patients were divided into high- and low-risk groups based on the risk score identified by the autophagy-related gene signature. Multivariate COX regression model and stratification analysis were used to specify the prognostic value of this gene signature in whole cohort and various subgroups. T-test and ANOVA analysis were used to compare the expression differences of continuous variables (5 prognostic genes and risk score) in binary and multiple category groups respectively. Kaplan-Meier analysis, log-rank tests and the area under receiver operating characteristic (ROC) curve (AUC) were conducted to validate the accuracy and precise of the autophagy-related gene signature based on GSE20685 and GSE21653 datasets. We profiled autophagy-related DEGs in normal and breast tumor tissues. GO and KEGG analysis indicated that autophagy-related DEGs might participate in breast cancer occurrence, development and drug resistance. Then we identified five autophagy-related genes (EIF4EBP1, ATG4A, BAG1, MAP1LC3A and SERPINA1) that had significantly prognostic values for breast cancer. Autophagy-related gene signature was constructed and patients were divided into high- and low- risk groups based on their risk score. Patients in the high-risk group tended to have shorter overall survival (OS) and relapse-free survival (RFS) times than those in the low-risk group (OS: HR=1.620, 95%CIs: 1.345-1.950; P<0.001; RFS: HR=1.487, 95%CIs: 1.248-1.771, P<0.001). Autophagy-related gene signature had significant prognostic value in stratified subgroups especially in advanced breast cancer subgroups (T3-4; N2-3; stage III-IV). Its prognostic value was further confirmed in two GEO validation datasets (GSE20685: P=6.795e-03; GSE21653: P=1.383e-03). Finally, association analysis between clinicopathological factors and gene signature showed the risk score was higher in patients with ER/PR negative, higher clinical stage or T stage (P<0.01). We established and confirmed a novel autophagy-related gene signature for patients with breast cancer that had independent survival prognostic value especially in advanced breast cancer subgroups. Our research might promote the molecular mechanism study of autophagy-related genes in breast cancer.

CCL2 and IL18 expressions may associate with the anti-proliferative effect of noncontact electro capacitive cancer therapy in vivo

Background: Noncontact Electro Capacitive Cancer Therapy (ECCT) is a novel treatment modality in cancer. Chemokine (C-C motif) ligand 2 (CCL2) has a major role in the outgrowth of metastatic breast cancer. Interleukin 18 (IL18) plays a role in macrophage alteration, which leads to excessive angiogenesis. This study aims to elaborate on the association of CCL2, IL18, IL23α, and TNF-α (tumor necrosis factor-alpha) expression with the anti-proliferative effect of ECCT in rat breast tumor tissue. Methods: Low intensity (18 Vpp) and intermediate frequency (150 kHz) alternating current-electric field (AC-EF) between two capacitive electrodes were exposed as external EF to a rat cage. Twenty-four rats were divided into four groups of six replicates. Breast tumor tissues were collected from 7, 12-dimethylbenz[a]anthracene (DMBA)-induced rats. Two groups were non DMBA-induced rats without ECCT exposure (NINT) and with (NIT). The other two groups were DMBA-induced rats without ECCT exposure (INT) and with (IT). Mammary glands and breast tumor tissues were collected from each group and preserved. Hematoxylin-eosin and immunohistochemistry staining were performed on paraffin sections of tissues using anti-PCNA, anti-ErbB2, anti-Caspase3, and anti-CD68. CCL2, IL18, IL23α, and TNF-α mRNA relative expressions were analyzed using qRT-PCR. Results: ECCT exposure may cause the reduction of PCNA protein expression as well as ErbB2 on breast tumor tissues, but it causes the increase of Caspase3 and macrophage CD68 protein. In rat breast tumor tissues of IT groups, the mRNA expression of CCL2 and IL18 are significantly down-regulated, in contrast with the up-regulated expression of these cytokines in tumor tissues of the INT group. IL23α and TNF- α expression remained similar in both groups. Conclusion: CCL2 and IL18 expressions have an association with the inhibition of breast tumor cell proliferation affected by ECCT exposure

Cellular heterogeneity map of diverse immune and stromal phenotypes within breast tumor microenvironment.

BackgroundCellular heterogeneity within the tumor microenvironment is essential to tumorigenesis and tumor development. A high-resolution global view of the tumor-infiltrating immune and stromal cells in breast tumors is needed.MethodsxCell was used to create a cellular heterogeneity map of 64 cell types in 1,092 breast tumor and adjacent normal tissues. xCell digitally dissects tissue cellular heterogeneity based on gene expression. Integrated statistical analyses were then performed.ResultsThere were noticeable differences between the cell fractions in tumor tissues and normal tissues. Tumors displayed higher proportions of immune cells, including CD4+ Tem, CD8+ naïve T cells, and CD8+ Tcm compared with normal tissues. Immune inhibitory receptors (PD1, CTLA4, LAG3 and TIM3) were co-expressed on certain subtypes of T cells in breast tumors, and PD1 and CTLA4 were both positively correlated with CD8+ Tcm and CD8+ T cells. 28 cell types were significantly associated with overall survival in univariate analysis. CD4+ Tem, CD8+ Tcm, CD8+ T-cells, CD8+ naive T-cells, and B cells were positive prognostic factors but CD4+ naive T-cells were negative prognostic factors for breast cancer patients. TDRD6 and TTK are promising T cell and B cell targets for tumor vaccines. Endothelial cells and fibroblasts were significantly less prevalent in tumor tissues; astrocytes and mesangial cells were negatively correlated with the T stage. Mesangial cells and keratinocytes were found to be favorable prognostic factors and myocytes were negative prognostic factors. Five cell types were found to be independent prognostic factors and we used these to create a reliable prognostic model for breast cancer patients. Cellular heterogeneity was discovered among different breast cancer subtypes by Her2, ER, and PR status. Tri-negative patients had the highest fraction of immune cells while luminal type patients had the lowest. The various cells may have diverse or opposing roles in the prognosis of breast cancer patients.ConclusionsWe created a uniquecellular map for the diverse heterogeneity of immune and stromal phenotypes within the breast tumor microenvironment. This map may lead to potential therapeutic targets and biomarkers with prognostic utility.

In-Silico study of microwave ablation applicators of different size for breast cancer treatment

Breast cancer is one of the most common types of cancer in females around the world, surgery is the preferred treatment for this disease in early stages, however there are some emergent treatment options with less clinical and cosmetically repercussions, being one of these the microwave ablation. Microwave ablation is a minimally invasive technique used to treat tumors in different organs of the human body, mainly in liver, lung and kidney. It consist on the destruction of the tumor by rising the temperature of the cells until necrosis is achieve. In breast tumors, this technique has been rarely used in human patients. Depending on the type of tissue, an applicator must be made to be coupled to ensure that energy used is not reflected, so the standing wave ratio is closest to 1. In order to determine an appropriate design for the applicators that could be used for the treatment of breast cancer, an in-silico study of microwave ablation of breast cancer was made to show the difference between micro-coaxial applicators constructed with two different types of coaxial cable (UT-47 and UT-85) utilizing the same design in three possible scenarios breast tissue, tumor tissue and tumor surrounded by breast tissue. The simulation was realized using COMSOL Multiphysics commercial software, due to its capabilities to simulate the electromagnetics and thermals effects. A slot type applicator was selected because of its ease of manufacture and the available literature on it. The results shows a better coupling for the applicator constructed with the UT-47 cable, especially in the tumor phantom surrounded by breast phantom test.

K-means clustering of coherent Raman spectra from extracellular vesicles visualized by label-free multiphoton imaging.

Extracellular vesicles (EVs) have emerged as potential biomarkers in cancer research and for clinical diagnosis. Little is known, however, about their spatial distributions in tissue and the different subpopulations that may exist. Here we report the use of label-free nonlinear optical imaging techniques to provide spatially resolved chemical information of EVs within untreated tissues. A multimodal nonlinear optical imaging system incorporating multiphoton autofluorescence and hyperspectral coherent anti-Stokes Raman scattering (CARS) imaging was built to visualize the spatial tissue distribution and probe the spectra of EVs. K-means clustering is performed on the CARS spectra from EVs in rat mammary tissues and human breast tumor tissue to reveal both the spatial distribution of EV clusters and their different chemical signatures. Correlations are identified between EV clusters and metabolic information.

Development of an HPLC-MS/MS Method for the Determination of Silybin in Human Plasma, Urine and Breast Tissue.

Silybin is a flavonolignan extracted from Silybum marianum with chemopreventive activity against various cancers, including breast. This study was designed to develop an HPLC-MS/MS method for the determination of silybin in human plasma, urine and breast tissue in early breast cancer patients undergoing Siliphos® supplementation, an oral silybin-phosphatidylcholine complex. The determination of silybin was carried out by liquid–liquid extraction with methyl-tert-butyl ether (MTBE); total silybin concentration was determined by treating the samples with β–glucuronidase, while for the determination of free silybin, the hydrolytic step was omitted. Naringenin and naproxen were selected as internal standards. The detection of the analyte was carried out by mass spectrometry and by chromatography. The HPLC-MS/MS method was evaluated in terms of selectivity, linearity, limit of quantification, precision and accuracy, and carryover. The method proved to be selective, linear, precise and accurate for the determination of silybin. To the best of our knowledge, this presents the first analytical method with the capacity to quantify the major bioactive components of milk thistle in three different biological matrices with a lower limit of quantification of 0.5 ng/mL for plasma. Silybin phosphatidylcholine, taken orally, can deliver high blood concentrations of silybin, which selectively accumulates in breast tumor tissue.

Analysis of Spatial Distribution and Prognostic Value of Different Pan Cytokeratin Immunostaining Intensities in Breast Tumor Tissue Sections.

Cancer risk prognosis could improve patient survival through early personalized treatment decisions. This is the first systematic analysis of the spatial and prognostic distribution of different pan cytokeratin immunostaining intensities in breast tumors. The prognostic model included 102 breast carcinoma patients, with distant metastasis occurrence as the endpoint. We segmented the full intensity range (0–255) of pan cytokeratin digitized immunostaining into seven discrete narrow grey level ranges: 0–130, 130–160, 160–180, 180–200, 200–220, 220–240, and 240–255. These images were subsequently examined by 33 major (GLCM), fractal and first-order statistics computational analysis features. Interestingly, while moderate intensities were strongly associated with metastasis outcome, high intensities of pan cytokeratin immunostaining provided no prognostic value even after an exhaustive computational analysis. The intense pan cytokeratin immunostaining was also relatively rare, suggesting the low differentiation state of epithelial cells. The observed variability in immunostaining intensities highlighted the intratumoral heterogeneity of the malignant cells and its association with a poor disease outcome. The prognostic importance of the moderate intensity range established by complex computational morphology analyses was supported by simple measurements of its immunostaining area which was associated with favorable disease outcome. This study reveals intratumoral heterogeneity of the pan cytokeratin immunostaining together with the prognostic evaluation and spatial distribution of its discrete intensities.

Association of circulating Progesterone Receptor Membrane Component-1 (PGRMC1) with PGRMC1 expression in breast tumour tissue and with clinical breast tumour characteristics

ObjectivesProgesterone receptor membrane component-1 (PGRMC1) in breast cancer tissue has been suggested to predict a worse prognosis. The aim of this study was to assess for the first time whether PGRMC1 expressed in cancer tissue is associated with PGRMC1 blood concentrations and whether both are correlated with clinical tumour characteristics known to predict a worse outcome. MethodsIn total, 201 patients with invasive breast cancer and 65 with benign breast disease (control group) were recruited. PGRMC1 blood concentrations were measured by a recently developed ELISA, PGRMC1 in breast cancer tissue was assessed by immunohistochemistry, and the correlation between the two was calculated. Receiver-operating characteristic (ROC) curve analysis was used to assess area under the curve (AUC). Furthermore, PGRMC1 was correlated with tumour characteristics such as tumour diameter, tumour grade and metastatic status, and with known blood tumour markers. ResultsAUC for the breast cancer group was 0.713, which was significantly higher than in the control group (p < 0.01). Blood PGRMC1 concentrations had a strong (positive) correlation with tissue PGRMC1 expression (p < 0.01) but were not associated with serum tumour markers CEA, CA125, CA153 and TPS. Tissue PGRMC1, ER and cancer stage were positively associated with blood PGRMC1 (p < 0.05). ConclusionsAs PGRMC1 expression levels in cancer tissue were significantly correlated with PGRMC1 in blood, and because concentrations in blood were also positively associated with breast tumour characteristics known to predict a worse prognosis, PGRMC1 may be valuable as a new tumour marker and may be superior to known tumour markers such as CEA, CA125, CA153 and TPS.

Age-associated genes in human mammary gland drive human breast cancer progression

BackgroundAging is a comorbidity of breast cancer suggesting that aging-associated transcriptome changes may promote breast cancer progression. However, the mechanism underlying the age effect on breast cancer remains poorly understood.MethodWe analyzed transcriptomics of the matched normal breast tissues from the 82 breast cancer patients in The Cancer Genome Atlas (TCGA) dataset with linear regression for genes with age-associated expression that are not associated with menopause. We also analyzed differentially expressed genes between the paired tumor and non-tumor breast tissues in TCGA for the identification of age and breast cancer (ABC)-associated genes. A few of these genes were selected for further investigation of their malignancy-regulating activities with in vitro and in vivo assays.ResultsWe identified 148 upregulated and 189 downregulated genes during aging. Overlapping of tumor-associated genes between normal and tumor tissues with age-dependent genes resulted in 14 upregulated and 24 downregulated genes that were both age and breast cancer associated. These genes are predictive in relapse-free survival, indicative of their potential tumor promoting or suppressive functions, respectively. Knockdown of two upregulated genes (DYNLT3 and P4HA3) or overexpression of the downregulated ALX4 significantly reduced breast cancer cell proliferation, migration, and clonogenicity. Moreover, knockdown of P4HA3 reduced growth and metastasis whereas overexpression of ALX4 inhibited the growth of xenografted breast cancer cells in mice.ConclusionOur study suggests that transcriptome alterations during aging may contribute to breast tumorigenesis. DYNLT3, P4HA3, and ALX4 play significant roles in breast cancer progression.

Clinicopathological factors associated with tumor-infiltrating lymphocyte reactivity in breast cancer.

The clinical significance of adoptive tumor-infiltrating lymphocyte (TIL) therapy has been demonstrated in many clinical trials. We analyzed the in vitro reactivity of cultured TILs against autologous breast cancer cells. TILs and cancer cells were cultured from 31 breast tumor tissues. Reactivity of TILs against cancer cells was determined by measuring secreted interferon-gamma. Expression levels of epithelial markers, major histocompatibility complex molecules, and programmed death-ligand 1 (PD-L1) in cancer cells, and T cell markers (memory, T cell activation and exhaustion, and regulatory T cell markers) in expanded TILs were analyzed and compared between the reactive and non-reactive groups. In seven cases, TILs showed reactivity to autologous cancer cells. Six of these cases were associated with triple-negative breast cancer (TNBC). All reactive TNBCs were derived from surgical specimens after neoadjuvant chemotherapy (NAC). Higher expression of Ki67 in tumor tissues and lower expression of PD-L1 in cultured cancer cells were associated with reactivity. Proliferation of reactive TILs was high. High proportions of T cells and PD-1+CD4+ and PD1+CD8+ T cells were associated with reactivity in TNBC cases, while other activation or exhaustion markers were not. TILs from approximately half the TNBC cases with NAC showed reactivity against autologous cancer cells. The proportion of PD-1+ T cells was higher in the reactive group. Adoptive TIL therapy combined with PD-1 inhibitors might be promising for TNBC patients with residual tumors after NAC.

Abstract B056: Tumor-associated ACKR1/DARC is correlated with a unique signature of proinflammatory chemokines and TILs in African-American women with breast cancer

Abstract Part of the delicate interplay of interactions between tumor cells, immune cells, and other cell types cells contribute to the complexities of the tumor microenvironment (TME). Specific interactions between chemokines and their receptors can influence the migration of immune cells, thereby directing tumor immune responses. In general immune response, the Atypical Chemokine Receptor 1 (ACKR1/DARC) modulates chemokine levels through the sequestration of proinflammatory chemokines in circulation and transcytosis across endothelial and epithelial tissue. This can affect not only the concentration of immune cells brought to the TME, but also the profile of immune cell types, given that ACKR1 is a promiscuous binder of both chemokine classes (CXCL and CCL). ACKR1 also serves as an ancestral informative marker. The gene harbors a mutation (“Duffy-null”) in its promoter/5′ UTR region that abolishes its normal erythrocytic expression, a status that only exists in populations of sub-Saharan African descent, conferring an evolutionary advantage against malaria. This polymorphism is carried by 60-80% of African-Americans, and based on our findings, it can influence chemokine and immune cell migration in the context of tumorigenesis. Our study reveals the potential effect of this mutation in women with breast cancer (BC) by showing correlations between circulating chemokine concentrations, measured through Luminex multiplexing immunoassays, and relative levels of ACKR1 and tumor-infiltrating lymphocytes (TIL), scored through immunohistochemistry. Our initial investigation of epithelial expression of ACKR1 on breast tumors revealed supporting data of our hypothesis that differential expression of ACKR1 on breast tumor tissue is correlated with a distinct signature of TILs and associated proinflammatory chemokines. Specifically, lower levels of tumor-associated ACKR1 were seen in African-Americans, in addition to a less robust signature of TILs, including macrophages and T-cells. Genotyping for the Duffy-null mutation also showed correlations with proinflammatory chemokines, including CCL2 and CXCL8. Our data suggest that ACKR1 levels in circulation and tissues can indirectly influence the immune cell profile in African-American women with BC, potentially leading to changes in tumor aggressiveness and response to treatment. Citation Format: Brittany D. Jenkins, Talina Fleifel, Rachel N. Martini, Haythem Ali, Lisa A. Newman, Melissa B. Davis. Tumor-associated ACKR1/DARC is correlated with a unique signature of proinflammatory chemokines and TILs in African-American women with breast cancer [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B056.