Primary Breast Cancer Biopsies Research Articles

Unexpected Expression and Function of FcεRI in Immortalized Breast Cancer Cells: A Cautionary Null Study.

The high-affinity IgE receptor, FcεRI, is typically associated with type 2 effectors such as mast cells (MC). The relatively unique expression profile of FcεRI and accumulating evidence from pre-clinical and clinical settings, such as MC interactions with tumors, have led us to study MCs as a potential therapeutic target in breast cancer. Our work identified MCs interacting with tumor cells at primary sites using the 4T1 (BALB/c) adenocarcinoma model in vivo. However, this analysis was complicated by a surprising finding that the tumor cells intrinsically and strongly expressed FcεRI. We further studied the expression and function of FcεRI in breast cancer cells in vitro. The 4T1 cells expressed FcεRI to a level similar to mouse bone marrow-derived MC (BMMC). Additionally, two established breast cancer cultures derived from human T-47D cells, one estrogen-dependent (E3) and the other estrogen-withdrawn (EWD8), also expressed FcεRI with EWD8 cells showing the greatest abundance. Functional analyses indicated that IgE-mediated antigen stimulation did not elicit classic Ca2+ flux in breast cancer cells as seen in the respective species' MCs; however, FcεRI crosslinking could stimulate IL-6 production from the T-47D derivatives. Preliminary analysis of primary breast cancer biopsy datasets using R2: Genomics Analysis and Visualization Platform was discordant with our in vivo model and in vitro observations. Indeed, FcεRI mRNA abundance declined in metastatic breast cancers compared to non-cancerous breast tissue. Altogether, we report a previously unidentified and immunologically substantive difference between breast cancer models and human primary tumors. Investigators pursuing FcεRI-relevant therapeutics in this context should be aware of this translational barrier.

Proof-of-concept study linking ex vivo sensitivity testing to neoadjuvant anthracycline-based chemotherapy response in breast cancer patients

We developed a functional ex vivo anthracycline-based sensitivity test. Surgical resection material of primary breast cancer (BC) was used to determine criteria for the ex vivo sensitivity assay based on morphology, proliferation and apoptosis. Subsequently, a proof-of-concept study was performed correlating results of this assay on primary BC biopsies with in vivo response after treatment with anthracycline-containing neoadjuvant chemotherapy (NAC). Cut off values for the ex vivo anthracycline-based sensitivity test were established based on analysis of 21 primary breast tumor samples obtained after surgery. In the proof-of-concept study based on a new set of tumor biopsies, 41 patients were included. Eight biopsies did not contain tumor cells and three patients could not be biopsied for various reasons. In the remaining 30 biopsies, the success rate of the ex vivo test was 77% (23/30); six out of seven failed tests were due to excessive apoptosis, our pre-specified test criteria. Of the 23 patients with a successful ex vivo test result, three patients did not undergo NAC after the biopsy. Here we report the ex vivo anthracycline-based sensitivity assay is feasible on biopsy material and shows 75% concordance between ex vivo outcomes and in vivo MRI response. Unfortunately, the percentage of unsuccessful tests is rather high. This study provides the foundation for further development of ex vivo sensitivity assays.

Abstract 3445: Immune cells infiltration and activation are higher in breast cancers resistant to antiestrogen therapy

Abstract We investigated the tumor microenvironment (TME) in estrogen receptor positive (ER+) primary breast cancers from stage I-III patients treated with estrogen deprivation (ED, induced with letrozole) for 10-21 days. We used primary breast cancer biopsies, collected from 198 patients, to assess tumor-infiltrating lymphocytes (TILs), transcriptomic profiles, and TME composition. Diagnostic biopsy and surgical on-treatment tumor specimens were collected; Ki67, ERα, PR, and HER2 expression were measured using automated quantitative analysis (AQUA). Tumors were categorized as estrogen deprivation-sensitive (ED-S) or -resistant (ED-R) based on the natural log (ln) of the Ki67 score (ln ≤1.0 or ≤2.7% Ki67+ cells vs. ln ≥2.0 or ≥7.4%, respectively) in the on-treatment (surgical) biopsy. Scoring of stromal (s)TILs, using hematoxylin and eosin staining of on-treatment specimens, revealed that sTILs were elevated in ED-R compared to ED-S tumors (p<0.0001). We next constructed tissue microarrays (TMAs) of on-treatment tumor sections and performed cyclic immunofluorescence (CyCIF) with 38 antibodies, including markers of hormone receptors, immune cells, signaling pathways, proliferation, DAPI (nuclei), and others. We then segmented single cells from each tumor core and designated them as immune, cancer, or stromal cells based on expression of a set of markers. Briefly, cells that stained strongly for CD45 (leukocyte marker), and/or CD4 (T lymphocyte marker), or CD68 (macrophage marker) were considered immune cells. CD45/CD4/CD68-negative cells were categorized as tumor, if E-cadherin and/or cytokeratin-positive, or stromal cells, if -negative. To investigate the TME composition, we next assessed cell specific spatial enrichment by quantifying the expression of immune markers in the area immediately adjacent to each tumor cell. Relative to ED-S, ED-R samples exhibited higher CD45+ cells (p<0.0001) and higher CD8+ T cells (p=0.0329), while CD4+ T cells showed no difference. Conversely, immune-suppressive T-reg (CD4+FOXP3+) cells were enriched in the ED-S samples (p=0.0004). In addition, PD1, a marker for T cell exhaustion, was higher in the ED-S group (p=0.0004), as were CD68+ cells (p<0.0001). To confirm and expand upon these findings, RNA-sequencing was performed on the on-treatment tumor sections. Gene set enrichment analysis of hallmark gene signatures revealed that immune-related gene sets, such as “IFNα response”, “IFNɣ response”, “allograft rejection”, and “inflammatory response” were enriched in ED-R vs. ED-S tumors. In summary, our study shows that activated anti-tumor immune cells are enriched in the TME of ER+ breast tumors resistant to estrogen deprivation, whereas ED-sensitive tumors show a more immunosuppressed or immune cold milieu. These data suggest a potential causal link between antiestrogen treatment and modulation of antitumor immunity in ER+ breast cancers. Citation Format: Fabiana Napolitano, Dhivya R. Sudhan, Yunguan Wang, Paula I. González-Ericsson, Luigi Formisano, Kyung-Min Lee, Chang-Ching Lin, María Rosario Chica-Parrado, Hongli Ma, Nathaniel Evans, Alberto Servetto, Saurabh Mendiratta, Spencer Barnes, Yisheng V. Fang, Justin M. Balko, Gordon B. Mills, Marilyne Labrie, Ariella B. Hanker, Carlos L. Arteaga. Immune cells infiltration and activation are higher in breast cancers resistant to antiestrogen therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3445.

Abstract P1-06-07: Enhanced differentiation of clinical behavior by breast carcinomas utilizing quantification of estrogen and progestin receptor proteins and gene expression subsets

Abstract Background: IHC analyses of clinical biomarkers for breast cancer management yield semi-quantitative results potentially complicated by variation in methods and interpretation. We are mining a comprehensive database established over four decades that contains deidentified results of quantified protein and RNA biomarkers with clinical outcomes to detect candidates for companion diagnostics and molecular targets. Assays were performed in a Reference Laboratory holding both CLIA and Commonwealth of Kentucky licenses. Methods: This retrospective investigation utilized ER/PR protein levels, expressed as fmol/mg cytosol protein, that were quantified for each of 2339 carcinoma biopsies with FDA-approved kits and cut-off values by radio-ligand binding assay (LBA, NEN/DuPont) and/or enzyme immunoassay (EIA, Abbott Labs). Of these, 454 specimens had both LBA and EIA results recorded. Biomarker results and de-identified clinical outcomes were examined using REMARK criteria. Gene expression, quantified biomarkers, features of primary breast cancers and clinical patient outcomes were analyzed by univariable and multivariable Cox regressions, Fisher’s Exact Test, Kaplan Meier plots and with R software v4.0.0. Microarray results of ~22,000 genes were obtained from RNA of LCM-procured breast carcinoma cells of 247 primary breast biopsies and expression levels of 85 cancer-associated genes were determined by RTqPCR of 278 specimens after RNA extraction and purification with established procedures. Results: Quantified levels of both ER and PR protein were expressed to a greater extent in primary carcinomas from post-menopausal patients compared to cancers from premenopausal women regardless of assay type (p = 0.005). ESR1 expression by microarray was only greater in LCM-procured carcinoma cells of post-menopausal patients while both ESR1 and PGR relative expression levels measured by qPCR were greater in tissue biopsies of post-menopausal women (p = 0.05). Box plots of untransformed EIA/LBA results of either ER (n=454) or PR (n=452) using specimens on which both quantifying assays were performed revealed a significant positive ER bias by EIA (median ratio=1.54) compared to that of PR (median ration=1.13). Transformed data of ratios confirmed this observation. Kaplan Meier plots of patient outcomes with ER or PR or both assessed by each assay type suggested LBA was a slightly better predictor. Microarray analyses of LCM-procured cells revealed 2994 genes distinguishing ER+ from ER- breast cancers of which ~200 genes were identified that differentiated 4 distinct molecular subtypes (2 ER+ and 2 ER-) exhibiting different outcomes upon Kaplan-Meier analyses. Expression levels of the 85 genes (including ERBB2), validated by qPCR with RNA from biopsy tissue sections, were evaluated relative to quantified levels of either ER or PR protein or both in primary breast cancer biopsies. Collectively, use of quantified ER/PR protein or ESR1/PGR expression with clinical outcomes enhanced identification of small molecular signatures and drug development candidates. Conclusions: Measurements of ER, PR and HER2 proteins with tests that quantify their levels as well as by microarray and qPCR assessment of ESR1, PGR and ERBB2 relative gene expression were correlated with prediction of clinical outcomes of breast carcinomas exhibiting various features (e.g., pathology classification, TNBC). Results reinforced importance of assessing levels of the clinically relevant biomarkers in a quantified fashion to enhance their use in breast cancer management and prediction of risk of recurrence. These investigations also revealed numerous over-expressed genes in carcinomas with poor clinical outcomes suggesting candidates for development of novel therapeutics. Citation Format: James L Wittliff, Michael W. Daniels. Enhanced differentiation of clinical behavior by breast carcinomas utilizing quantification of estrogen and progestin receptor proteins and gene expression subsets [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 P1-06-07.

Human Plasma-Derived 3D Cultures Model Breast Cancer Treatment Responses and Predict Clinically Effective Drug Treatment Concentrations.

Lack of efficacy and a low overall success rate of phase I-II clinical trials are the most common failures when it comes to advancing cancer treatment. Current drug sensitivity screenings present several challenges including differences in cell growth rates, the inconsistent use of drug metrics, and the lack of translatability. Here, we present a patient-derived 3D culture model to overcome these limitations in breast cancer (BCa). The human plasma-derived 3D culture model (HuP3D) utilizes patient plasma as the matrix, where BCa cell lines and primary BCa biopsies were grown and screened for drug treatments. Several drug metrics were evaluated from relative cell count and growth rate curves. Correlations between HuP3D metrics, established preclinical models, and clinical effective concentrations in patients were determined. HuP3D efficiently supported the growth and expansion of BCa cell lines and primary breast cancer tumors as both organoids and single cells. Significant and strong correlations between clinical effective concentrations in patients were found for eight out of ten metrics for HuP3D, while a very poor positive correlation and a moderate correlation was found for 2D models and other 3D models, respectively. HuP3D is a feasible and efficacious platform for supporting the growth and expansion of BCa, allowing high-throughput drug screening and predicting clinically effective therapies better than current preclinical models.

Wire guided localisation for targeted axillary node dissection is accurate in axillary staging in node positive breast cancer following neoadjuvant chemotherapy

AimThis study investigated whether wire localisation of the histologically proven positive, clipped axillary lymph node (ALN) with subsequent targeted axillary dissection (TAD) following neoadjuvant chemotherapy (NACT) improves axillary staging in breast cancer. Materials and methodsWe performed a retrospective review of patients with primary breast cancer and core biopsy proven metastatic ALNs, that had an excellent nodal radiological response following NACT, treated at our centre between January 2016 and December 2018. The initial cohort of patients (Group 1) underwent sentinel lymph node biopsy (SLNB), with a minimum of three nodes were sampled. The subsequent cohort (Group 2) had a marker clip inserted in the metastatic ALN prior to NACT. This cohort underwent wire guided excision of the clipped node in addition to SLNB, with a minimum of three nodes sampled. ResultsA total of 47 patients were identified. Group 1 comprised 22 patients with a sentinel lymph node (SLN) identification rate (IR) of 95%. 25 patients (Group 2) underwent wire guided clip location and the SLN IR was 100% with a 92% clipped node IR. Evidence of pathological complete response (pCR) in the clipped node was associated with pCR in other nodes. ConclusionTargeted axillary dissection is a feasible technique following excellent response to NACT in selected patients with limited volume ALN metastasis, at diagnosis. The identification of the positive ALN during surgery is vital and the IR can be improved by clipping the node prior to NACT and wire guided localisation at the time of surgery.

Heterogeneity in signaling pathway activity within primary breast cancer and between primary and metastases.

589 Background: Treatment with targeted drugs aims to block tumor driving signaling pathway(s). Drug choice is often based on a single preoperative primary breast cancer biopsy. It is important that biopsied cancer tissue is representative for the primary tumor (PT) or metastases to treat. Little is known about pathway heterogeneity within the PT, and between PT and metastatic tumors. A novel analysis method was developed to identify and quantify activity of signal transduction pathways in cancer tissue, based on Bayesian models that infer a pathway activity score from transcription factor target gene mRNA levels (Cancer Res 2014;74:2936-45). Methods: Pathway analysis, originally developed for AffymetrixU133Plus2.0, was adapted to RT-qPCR for use on formalin fixed paraffin embedded tissue. Estrogen (ER) and androgen (AR) receptor, PI3K, Hedgehog (HH), TGFβ and Wnt pathway activities were analyzed. Samples were from multiple locations (“quadrants”) in 15 luminal A, 9 luminal B, and 8 ER-negative primary breast cancers; from subdivided quadrant samples (4 “subquadrants”) of respectively 9, 4, and 4 PTs; and from 13 distant and 24 lymph node (LN) metastases of respectively 9 and 7 matched luminal PTs. Analysis of pathway activity score (PAS) variance was performed with linear mixed models with subgroup-dependent standard deviations. Results: In primary breast cancer intra-tumor PAS variance was not larger at macroscale (“quadrant”) than at microscale (“subquadrant”). For ER, AR, HH, and Wnt pathways, PAS variation was higher between distant metastases and PT than within the PT (p < 0.0002). For HH and Wnt pathways, PAS variation was higher between LN metastases than within the PT (p < 0.002). Correlation between primary and metastatic pathway activities ranged from -0.34 for ER to 0.47/0.50 for TGFβ/HH pathways. Conclusions: A single location tissue sample was representative for the whole primary tumor with respect to signaling pathway activity, suggesting one biopsy as generally sufficient for (neo)adjuvant therapy choice. Pathway activities varied between primary cancer and metastases, indicating the necessity of metastatic sample analysis (biopsies or liquid biopsy) to improve therapy choice.

The Role of Proliferation in Determining Response to Neoadjuvant Chemotherapy in Breast Cancer: A Gene Expression-Based Meta-Analysis.

To provide further insight into the role of proliferation and other cellular processes in chemosensitivity and resistance, we evaluated the association of a diverse set of gene expression signatures with response to neoadjuvant chemotherapy (NAC) in breast cancer. Expression data from primary breast cancer biopsies for 1,419 patients in 17 studies prior to NAC were identified and aggregated using common normalization procedures. Clinicopathologic characteristics, including response to NAC, were collected. Scores for 125 previously published breast cancer-related gene expression signatures were calculated for each tumor. Within each receptor-based subgroup or PAM50 subtype, breast tumors with high proliferation signature scores were significantly more likely to achieve pathologic complete response to NAC. To distinguish "proliferation-associated" from "proliferation-independent" signatures, we used correlation and linear modeling approaches. Most signatures associated with response to NAC were proliferation associated: 90.5% (38/42) in ER+/HER2- and 63.3% (38/60) in triple-negative breast cancer (TNBC). Proliferation-independent signatures predictive of response to NAC in ER+/HER2- breast cancer were related to immune activity, while those in TNBC comprised a diverse set of signatures, including immune, DNA damage, signaling pathways (PI3K, AKT, Ras, and EGFR), and "stemness" phenotypes. Proliferation differences account for the vast majority of predictive capacity of gene expression signatures in neoadjuvant chemosensitivity for ER+/HER2- breast cancers and, to a lesser extent, TNBCs. Immune activation signatures are proliferation-independent predictors of pathologic complete response in ER+/HER2- breast cancers. In TNBCs, significant proliferation-independent signatures include gene sets that represent a diverse set of cellular processes. Clin Cancer Res; 22(24); 6039-50. ©2016 AACR.

Platelets surrounding primary tumor cells are related to chemoresistance.

Platelets are crucial components of the tumor microenvironment that function to promote tumor progression and metastasis. In the circulation, the interaction between tumor cells and platelets increases invasiveness, protects tumor cells from shear stress and immune surveillance, and facilitates tumor cell extravasation to distant sites. However, the role and presence of platelets in the primary tumor have not been fully determined. Here, we investigated the presence of platelets around breast cancer primary tumor cells and the associations between these cells. We further investigated the associations among platelets, tumor cells, chemoresistance, and epithelial-mesenchymal transition(EMT). We retrospectively analyzed data from 74patients with human epidermal growth factor receptor2(HER2)‑negative breast cancer who underwent biopsies before treatment and subsequent neo-adjuvant chemotherapy. In biopsy specimens, we evaluated the expression of platelet-specific markers and EMT markers using immunohistochemistry. The associations among the expression of platelet‑specific markers in biopsy specimens, EMT, response to neo‑adjuvant chemotherapy, and survival were analyzed. The presence of platelets was observed in 44 out of 74 (59%) primary breast cancer biopsy specimens. Platelet‑positive tumor cells showed EMT‑like morphological changes and EMT marker expression. Primary tumor cells associated with platelets were less responsive to neo‑adjuvant chemotherapy (pCR rate: 10vs.50%, respectively; p=0.0001). Platelets were an independent predictor of the response to chemotherapy upon multivariable analysis (p<0.0001). In conclusion, there was a significant association between platelets surrounding primary tumor cells in the biopsy specimens and the chemotherapeutic response in breast cancer. Platelets surrounding primary tumor cells may represent novel predictors of chemotherapeutic responses.

Fulvestrant response prediction from transcriptome data obtained from primary breast cancer biopsies.

e12056Background: Fulvestrant is a highly selective estrogen receptor (ER) antagonist used in the treatment of postmenopausal patients with ER-positive advanced breast cancer. To identify patients ...

Abstract 2009: Flow cytometric analysis, sorting and molecular analysis of dissociated cells from human solid tumors derived from PDX mouse models

Abstract Functional and genetic heterogeneity in tumor tissue has been a well described phenomenon for many decades but only recently emerged as a potentially crucial contributor to cancer development and progression. The correlation between cellular heterogeneity and aggressiveness, metastatic potential and drug susceptibility of a cancerous lesion have led to models in which the existence of multiple clonal cell lineages is a central feature enabling a neoplastic lesion to overcome selective pressures caused by the surrounding tissues’ defensive capabilities as well as therapeutic interventions. In addition, the role of the tumor microenvironment as an integral part of tumorigenesis was recognized and infiltrating leukocytes or tumor associated fibroblasts are no longer viewed as mere contaminants of a solid tumor biopsy. The emerging picture is compared to macroscopic ecosystems and a detailed understanding of the interactions between numerous cell subgroups seems necessary for the complete understanding of cancer pathogenesis. Scarcity of appropriate tools and model systems are an obstacle to the investigation of this heterogeneity at a molecular level but advances over the last few years have led to a significant acceleration in this field. More sensitive and far cheaper methods for collection of genomic and transcriptomic data have revealed a complex picture of the evolution of individual solid tumors. To turn this deeper understanding of tumorigenesis into improved clinical outcomes, routine methods are required to separate complex tumors into subpopulations. This stratification will provide a more comprehensive characterization of the tumor and enable more detailed prediction of disease progression and resistance development. We have developed an integrated workflow for dissociation and flow cytometric analysis and sorting for multiple downstream analysis modalities. Using patient derived xenograft (PDX) mouse models derived from primary human breast cancer biopsies we have demonstrated the ability to identify distinct immunophenotypes for each model and use this analysis to isolate distinct subpopulations. Our successful optimization of a variety of well characterized surface markers (e.g. CD 24, 44, 133, 184, 326 (EpCAM), and CD45) provides a basis for effective fingerprinting of cancer cells from a variety of sources. In an effort to demonstrate the potential of FACS sorting of solid tumor derived cell populations we have interrogated sorted fractions by NGS as well as RT-PCR array analysis and show distinct genotypic as well as gene expression signatures for each subgroup. The evidence provided by our data suggests that the single cell focused approach flow cytometry has traditionally enabled in hematological cancers is accessible for solid tumors as well and may unlock valuable biological insights. Citation Format: Rainer Blaesius, Friedrich Hahn, Eileen Snowden, Warren Porter, Mitchell Ferguson, Frances Tong, Stewart Jurgensen, Chang Chen, Daphne Clancy, Jamal Sirriyah, John Alianti, Perry Haaland, Shannon Dillmore, Jeff Baker, Aaron Middlebrook, Joyce Ruitenberg, Maria Suni, Smita Ghanekar. Flow cytometric analysis, sorting and molecular analysis of dissociated cells from human solid tumors derived from PDX mouse models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2009. doi:10.1158/1538-7445.AM2015-2009

Abstract P1-07-10: Immunohistochemical (IHC) marker discordance between primary breast cancer biopsy and recurrent cancer: Would IHC testing of the surgical breast or lymph node have altered treatment?

Abstract Objective: Based on emerging data on tumor heterogeneity and the evolutionary branching of tumor cells, tumor cells in the lymph node may represent more virulent clones with the inherent capability of metastasis. IHC discordance from original cancer diagnosis to recurrence is documented to occur in up to 20% of cases, raising the question if characterization of these likely more virulent cells would more accurately guide treatment and predict prognosis. Our pilot study sought to determine if crucial clinical information is gained by IHC testing of the surgical breast or lymph node specimens at the time of initial surgery. Methods: Using the cancer registry and oncology records, all invasive breast cancers diagnosed after 2001 with subsequent recurrence were identified. Cases missing all IHC data were disqualified. We then evaluated ER and HER2 of the primary cancer biopsy and recurrence biopsy to identify discordances. Those with discordances who had surgical breast and lymph node specimens available were accessed, tested, and evaluated by our breast cancer pathologist. Results: A total of 128 recurrence cases with partial or complete primary and recurrence IHC data were identified. Of the 95 initially ER positive cases with recurrence IHC available, 13/95 had discordant, or ER negative, recurrence. Additionally, 5/27 initially ER negative tumors, 3/14 initially HER2 positive tumors, and 6/69 initially HER2 negative tumors had discordant recurrence results. In 128 cases, 27 cases were identified to have ER or HER2 discordance from primary biopsy diagnosis to recurrence. Of all cases with original surgical breast or positive lymph node specimen available, 9 markers on 7 patients were performed for our pilot study. One of seven surgical breast specimens and one of two lymph node specimens were concordant with the recurrence, but not the initial biopsy. The tested surgical breast was ER positive, while the surgical lymph node was HER2 positive, concordant to their recurrences, but discordant with initial biopsy. Breast BiopsyRecurrence ConcordantRecurrence DiscordantER Positive98/12782/9513/95 (14%)ER Negative29/12724/275/27 (19%) Total ER Discordance 18/122 (15%)HER2 Positive19/11911/143/14 (22%)HER2 Negative100/11963/696/69 (9%) Total HER2 Discordance 9/83 (11%) Conclusion: Tumor discordance of the original cancer biopsy and recurrence is not uncommon. Our pilot study demonstrated that ER and HER2 discordance occurred in 15% and 11% of cases, respectively. Though our pilot study was limited by small sample size, we found that IHC testing of the surgical breast and lymph node specimen may provide additional clinical information and affect management. Of the two cases that had a positive lymph node available, one was HER2 positive and concordant with the recurrence. Of the seven breast specimens tested, one was ER positive and concordant with the recurrence. Had IHC testing been performed at that time of surgery, adjuvant treatment management would have been altered. Further testing of our IHC discordant recurrence patient population will be pursued to investigate the potential benefits of surgical breast and lymph node IHC testing. Citation Format: Michele M Gage, Martin Rosman, Charles Mylander, Crystal Tran, Rubie S Jackson, Lorraine Tafra. Immunohistochemical (IHC) marker discordance between primary breast cancer biopsy and recurrent cancer: Would IHC testing of the surgical breast or lymph node have altered treatment? [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-10.

Abstract P6-03-11: Erk5 as a therapeutic target in triple negative breast cancer (TNBC)

Abstract TNBC is a subtype of breast cancer known for its aggressive behavior and poor prognosis. TNBC accounts for 17-24% of all invasive breast cancers and is a subtype that lacks the expression of hormone receptors; oestrogen [ER], progesterone [PR]) and epidermal growth factor receptor 2 (HER2) receptors. Currently there has been a shift from using empirically derived agents that inhibit tumor cell growth and/or survival, to molecular therapies that target specific molecules that regulate these and other important biological processes. Despite the success of some new-targeted therapies to treat breast cancer, the outlook for the majority of patients with TNBC remains poor. Therefore we have utilized the KinexTM antibody array to resolve potential new targetable "nodes". We have screened 43 primary breast cancer biopsies (16 TNBC, 16 ER/PR positive and 11 HER2-positive) and 16 breast cancer cell lines for protein/phosphoprotein levels. Extracellular-signal-regulated kinase 5 (Erk5) is a member of the MAPK family. Erk5 has a large, unique C-terminal-half not found in other MAPK family members and because of this unique 400-amino acid extension, ERK5 is also called big MAP kinase 1 (BMK1). By extensive in silico analyses, we identified that the Erk5 metagene is prognostic and identifies TNBC/BLBC with poor prognosis. Our kinome study of primary breast cancer has further confirmed that the MEK5-Erk5 pathway is upregulated in the TNBC subgroup compared to the other TNBC subgroups and that pharmacological inhibition of Erk5 is therapeutic against primary MDA-MB-231 TNBC xenografts (1). Our preliminary data has shown that Erk5 is overexpressed in the TNBC subtype at the protein level. We have also shown that silencing Erk5 by siRNA or shRNA inhibits migration of TNBC cell lines in vitro. Expression of Erk5 (total and phosphoproteins) will be investigated in primary breast tumours and metastases samples from patient by immunohistochemistry. We will also overexpress Erk5 in non-metastatic breast cancer cell lines to study the effect of Erk5 overexpression on metastases. In an elegant study, site directed mutagenesis was used to modify Erk5 and show that phosphorylations of the C-terminal domain of kinase activated Erk5 is required for the transcriptional activity of Erk5. Using in vitro and in vivo models we will determine if the transcriptional activity of activated Erk5 is a major player to drive metastatic phenotype. Moreover, whether kinase-dependent or transcription-dependent activity of Erk5 on several pathways is sufficient to drive metastasis will also be determined. Our results strongly suggest the MEK5-Erk5 pathway as a prognostic and therapeutic pathway in a poor-prognosis subset of TNBC/BCLC. Characterization of this pathway in clinical cohorts particularly in TNBC tumors, mechanistic understanding of its contribution to metastasis and therapeutic targeting in vivo would help develop a personalised approach to identify and treat TNBC tumors and metastases with activated Erk5 pathway. 1. Al-Ejeh F, Miranda M et al. Kinome profiling reveals breast cancer heterogeneity and identifies targeted therapeutic opportunities for triple negative breast cancer. Oncotarget. March 19, 2014. Citation Format: Mariska Sauzine Miranda, Fares Al Ejeh, Wei Shi, Peter Simpson, Sunil Lakhani, Kum Kum Khanna. Erk5 as a therapeutic target in triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-03-11.

Nuclear CSPP1 expression defined subtypes of basal-like breast cancer

Background:The multi-exon CSPP1 gene, encoding for centrosome and microtubule-associated proteins involved in ciliogenesis and cell division, is a candidate oncogene in luminal breast cancer but expression of CSPP1 proteins remained unexplored.Methods:CSPP1 gene and protein expression was examined in normal mammary tissue, human breast cancer cell lines, and primary breast cancer biopsies from two patient cohorts. Cell type and epitope-dependent subcellular-specific CSPP1 staining pattern in normal mammary gland epithelium and cancer biopsies were correlated to molecular and clinical parameters.Results:A novel, nuclear localised CSPP1 isoform was exclusively detected in luminal epithelial cells, whereas cytoplasmic CSPP-L was generally expressed in normal mammary epithelium. Luminal cell-related nuclear CSPP1 expression was preserved in type-matched cell lines and carcinomas, and correlated to gene copy number and mRNA expression. In contrast, basal-like carcinomas displayed generally lower CSPP1 mRNA expression. Yet, a subgroup of basal-like breast carcinomas depicted nuclear CSPP1 expression, displayed luminal traits, and differed from nuclear CSPP1 devoid counterparts in expression of eight genes. Eight-gene signature defined groups of basal-like tumours from an independent cohort showed significant differences in survival.Conclusions:Differential expression of a nuclear CSPP1 isoform identified biologically and clinically distinct subgroups of basal-like breast carcinoma.

Abstract A198: Flow cytometric analysis and sorting of dissociated cells from human solid tumors derived from PDX mouse models.

Abstract Functional and genetic heterogeneity in tumor tissue was first observed over 50 years ago. Today, tumor heterogeneity is frequently evoked in describing the pathway from pre-cancerous lesions to aggressive, metastatic cancer. During this progression, multiple clonal lineages are thought to arise, leading to subpopulations of the tumor showing different metastatic profiles and susceptibility to anti-cancer therapy. In addition, the role of the tumor microenvironment became recognized and infiltrating leukocytes or tumor associated fibroblasts are no longer viewed as mere contaminants of a solid tumor biopsy. The emerging picture is increasingly compared to macroscopic ecosystems and a detailed understanding of the interactions between numerous cell subgroups seems necessary for the complete understanding of cancer pathogenesis. Scarcity of appropriate tools and model systems are an obstacle to the investigation of this heterogeneity at a molecular level but advances over the last few years have led to a significant acceleration in this field. More sensitive and far cheaper methods for collection of genomic and transcriptomic data have revealed a complex picture of the evolution of individual solid tumors. To turn this deeper understanding of tumorigenesis into improved clinical outcomes, routine methods are required to separate complex tumors into subpopulations. This stratification will provide a more comprehensive characterization of the tumor and enable more detailed prediction of disease progression and resistance development. We have developed dissociation methods for solid tumor tissue which allows flow cytometric analysis as well as sorting to provide cells for multiple downstream analysis modalities. Using patient derived xenograft (PDX) mouse models derived from primary human breast, colorectal and lung cancer biopsies we have demonstrated efficient dissociation, surface marker analysis and nucleic acid purification from sorted populations. Conditions have been optimized for a range of relevant surface markers (e.g. CD 24, 44, 133, 184, 326 (EpCAM), and CD45) which are suitable to identify cells predicted to have stem cell, endothelial, epithelial or immune cell functions, respectively. Through sequencing of subpopulations identified by their phenotype we have demonstrated the compatibility of our workflow with downstream analysis methods such as Next Generation Sequencing (NGS). Our RNA stability measurements suggest that gene expression analysis is equally feasible. Our data provide a standardized basis for in depth investigation of subpopulations of cells from solid tumors with various molecular techniques. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A198. Citation Format: Rainer Blaesius, Friedrich Hahn, Eileen Snowden, Warren Porter, Mitchell Ferguson, Tina Marmura, Frances Tong, Shannon Dillmore, Aaron Middlebrook, Joyce Ruitenberg, Maria Suni, Smita Ghanekar. Flow cytometric analysis and sorting of dissociated cells from human solid tumors derived from PDX mouse models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A198.

TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer

After an initial response to chemotherapy, many patients with triple-negative breast cancer (TNBC) have recurrence of drug-resistant metastatic disease. Studies with TNBC cells suggest that chemotherapy-resistant populations of cancer stem-like cells (CSCs) with self-renewing and tumor-initiating capacities are responsible for these relapses. TGF-β has been shown to increase stem-like properties in human breast cancer cells. We analyzed RNA expression in matched pairs of primary breast cancer biopsies before and after chemotherapy. Biopsies after chemotherapy displayed increased RNA transcripts of genes associated with CSCs and TGF-β signaling. In TNBC cell lines and mouse xenografts, the chemotherapeutic drug paclitaxel increased autocrine TGF-β signaling and IL-8 expression and enriched for CSCs, as indicated by mammosphere formation and CSC markers. The TGF-β type I receptor kinase inhibitor LY2157299, a neutralizing TGF-β type II receptor antibody, and SMAD4 siRNA all blocked paclitaxel-induced IL8 transcription and CSC expansion. Moreover, treatment of TNBC xenografts with LY2157299 prevented reestablishment of tumors after paclitaxel treatment. These data suggest that chemotherapy-induced TGF-β signaling enhances tumor recurrence through IL-8-dependent expansion of CSCs and that TGF-β pathway inhibitors prevent the development of drug-resistant CSCs. These findings support testing a combination of TGF-β inhibitors and anticancer chemotherapy in patients with TNBC.

Abstract P6-10-06: Rational combination therapy against triple-negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with higher incidence of recurrence, more distant metastasis, and poorer survival. This subtype is also characterized by complex genomes where little of their genomes remain at normal copy number but without high, focal copy number amplifications. At the transciptome level, the majority of TNBC (∼75%) are classified as basal-like breast cancer (BLBC) according to the five intrinsic subtypes. Despite considerable genomic and gene expression characterization of TNBC, proteomic and phospho-proteomic investigations of this disease are limited with no available targeted therapies in clinical use. Methods &amp; Results: We used the Kinex™ antibody array (http://www.kinexus.ca/) to interrogate protein/phosphoproteins levels in 43 primary breast cancer biopsies (16 TNBC, 16 ER/PR positive and 11 HER2-positive) and 16 breast cancer cell lines. Unsupervised hierarchical clustering of protein/phosphoprotein levels revealed two subgroups of TNBC in comparison to other subtypes. Western blotting and Proteome Profiler™ Arrays (R&amp;D Systems) were used to validate deregulated proteins/phosphoproteins in TNBC. Pathway analysis revealed that one subgroup of TNBC exploits overlapping and cross-talking networks for survival. These signaling networks are downstream from elevated activation of EGFR, integrins and Insulin-like growth factor 1 receptor (IGF1R). Targeted molecular inhibitors of activated kinases in these pathways showed specificity against basal-like/TNBC cell lines compared to other subtypes in vitro. These activated kinases/networks represent druggable targets for the treatment of TNBC but may be limited by compensatory effect of the complex cross-talking signaling networks. To overcome compensatory downstream signaling that would limit the inhibition of a given pathway; we developed EGFR-targeted radioimmunotherapy (RIT) strategy to systemically deliver cytotoxic loads of beta particles (177Lu) that would kill targeted cells and surrounding cells by crossfire effect. The combination of EGFR-directed RIT with chemotherapy and PARP inhibition successfully treated orthotopic and metastatic TNBC models established from cell lines and patient-derived xenografts. The superior efficacy of this triple-agent combination therapy is explained by enhanced DNA damage and reduced DNA repair response, higher apoptotic cell death and the elimination of putative breast cancer stem cells. Conclusion: Proteomic analysis of TNBC provides a powerful tool to elucidate druggable signaling networks with therapeutic potential. TNBC utilizes complex interacting signaling networks and rational combination therapies are required for effective therapy. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-10-06.

Serine Protease PRSS23 Is Upregulated by Estrogen Receptor α and Associated with Proliferation of Breast Cancer Cells

Serine protease PRSS23 is a newly discovered protein that has been associated with tumor progression in various types of cancers. Interestingly, PRSS23 is coexpressed with estrogen receptor α (ERα), which is a prominent biomarker and therapeutic target for human breast cancer. Estrogen signaling through ERα is also known to affect cell proliferation, apoptosis, and survival, which promotes tumorigenesis by regulating the production of numerous downstream effector proteins.In the present study, we aimed to clarify the correlation between and functional implication of ERα and PRSS23 in breast cancer. Analysis of published breast cancer microarray datasets revealed that the gene expression correlation between ERα and PRSS23 is highly significant among all ERα-associated proteases in breast cancer. We then assessed PRSS23 expression in 56 primary breast cancer biopsies and 8 cancer cell lines. The results further confirmed the coexpression of PRSS23 and ERα and provided clinicopathological significance. In vitro assays in MCF-7 breast cancer cells demonstrated that PRSS23 expression is induced by 17β-estradiol-activated ERα through an interaction with an upstream promoter region of PRSS23 gene. In addition, PRSS23 knockdown may suppress estrogen-driven cell proliferation of MCF-7 cells.Our findings imply that PRSS23 might be a critical component of estrogen-mediated cell proliferation of ERα-positive breast cancer cells. In conclusion, the present study highlights the potential for PRSS23 to be a novel therapeutic target in breast cancer research.

STAT1-deficient mice spontaneously develop estrogen receptor α-positive luminal mammary carcinomas.

IntroductionAlthough breast cancers expressing estrogen receptor-α (ERα) and progesterone receptors (PR) are the most common form of mammary malignancy in humans, it has been difficult to develop a suitable mouse model showing similar steroid hormone responsiveness. STAT transcription factors play critical roles in mammary gland tumorigenesis, but the precise role of STAT1 remains unclear. Herein, we show that a subset of human breast cancers display reduced STAT1 expression and that mice lacking STAT1 surprisingly develop ERα+/PR+ mammary tumors.MethodsWe used a combination of approaches, including histological examination, gene targeted mice, gene expression analysis, tumor transplantaion, and immunophenotyping, to pursue this study.ResultsForty-five percent (37/83) of human ERα+ and 22% (17/78) of ERα- breast cancers display undetectable or low levels of STAT1 expression in neoplastic cells. In contrast, STAT1 expression is elevated in epithelial cells of normal breast tissues adjacent to the malignant lesions, suggesting that STAT1 is selectively downregulated in the tumor cells during tumor progression. Interestingly, the expression levels of STAT1 in the tumor-infiltrating stromal cells remain elevated, indicating that single-cell resolution analysis of STAT1 level in primary breast cancer biopsies is necessary for accurate assessment. Female mice lacking functional STAT1 spontaneously develop mammary adenocarcinomas that comprise > 90% ERα+/PR+ tumor cells, and depend on estrogen for tumor engraftment and progression. Phenotypic marker analyses demonstrate that STAT1-/- mammary tumors arise from luminal epithelial cells, but not myoepithelial cells. In addition, the molecular signature of the STAT1-/- mammary tumors overlaps closely to that of human luminal breast cancers. Finally, introduction of wildtype STAT1, but not a STAT1 mutant lacking the critical Tyr701 residue, into STAT1-/- mammary tumor cells results in apoptosis, demonstrating that the tumor suppressor function of STAT1 is cell-autonomous and requires its transcriptional activity.ConclusionsOur findings demonstrate that STAT1 suppresses mammary tumor formation and its expression is frequently lost during breast cancer progression. Spontaneous mammary tumors that develop in STAT1-/- mice closely recapitulate the progression, ovarian hormone responsiveness, and molecular characteristics of human luminal breast cancer, the most common subtype of human breast neoplasms, and thus represent a valuable platform for testing novel treatments and detection modalities.

A Divergent Substrate-Binding Loop within the Pro-oncogenic Protein Anterior Gradient-2 Forms a Docking Site for Reptin

Anterior gradient-2 (AGR2) functions in a range of biological systems, including goblet cell formation, limb regeneration, inhibition of p53, and metastasis. There are no well-validated binding proteins for AGR2 protein despite the wealth of data implicating an important cellular function in vertebrates. The yeast two-hybrid system was used to isolate the ATP binding protein Reptin as an AGR2-interacting protein. AGR2 formed a stable complex in human cell lysates with Reptin, thus validating Reptin as an AGR2 binding protein in cells. Reptin was also shown to be overproduced in a panel of primary breast cancer biopsy specimens, relative to normal adjacent tissue from the same patient, suggesting a role in cancer growth in vivo. Mutations were made at the two ATP binding motifs in Reptin to evaluate the effects of ATP on Reptin–AGR2 complex stability. Loss-of-ATP binding mutations at the Walker A motif (K83A) or gain-of-ATP binding mutations at the Walker B motif (D299N) resulted in Reptin mutants with altered oligomerization, thermostability, and AGR2 binding properties. These data indicate that the two ATP binding motifs of Reptin play a role in regulating the stability of the AGR2–Reptin complex. The minimal region of AGR2 interacting with Reptin was localized using overlapping peptide libraries derived from the AGR2 protein sequence. The Reptin docking site was mapped to a divergent octapeptide loop in the AGR2 superfamily between amino acids 104 and 111. Mutations at codon Y104 or F111 in full-length AGR2 destabilized the binding of Reptin. These data highlight the existence of a protein docking motif on AGR2 and an ATP-regulated peptide-binding activity for Reptin. This knowledge has implications for isolating other AGR2-interacting proteins, for developing assays to isolate small molecules that target the Reptin ATP binding site , and for measuring the effects of the Reptin–AGR2 complex in cancer cell growth.