bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P1-05-05: Epithelial cell reprogramming assay reveals inter-individual heterogeneity in stem/basal and progenitor cell profile of the normal breast
Authors: Nakshatri H , Anjanappa M , Bhat-Nakshatri P
Published: August 2014

Objectives: The majority of our understanding of mammary epithelial cell hierarchy is from mouse models. In addition, due to limited replication potential, primary cells of either mouse or human origin have rarely been used for functional studies. In this study, we took advantage of the recently developed epithelial cell-reprogramming assay that utilizes irradiated fibroblasts and ROCK inhibitor to characterize breast epithelial cells from individuals with varying degree of breast cancer risk.

Methods: Primary breast tissues were collected at the time of surgery and cultured either immediately or at convenient time after freezing in liquid nitrogen with ROCK inhibitor. Flow cytometry sorting was employed to fractionate and propagate stem-like/basal (CD49f+/EpCAM-), luminal progenitor (CD49f+/EpCAM+), and mature (CD49f-/EpCAM+) cell enriched fractions. Similar fractionation/characterization was performed using CD133, CD10, CD271, EGFR and ALDEFLUOR.

Results: Inter-individual variability was observed in proportion of CD49f+/EpCAM+, CD49-/EpCAM+ and CD133+ cells. Epithelial cells from a patient considered high risk based on the history of hyperplasia as well as cells from BRCA1 and BRCA2 patients displayed unique CD49f and EpCAM staining pattern compared to cells isolated from adjoining “normal” tissue of two cancer patients. In addition, CD133+ population was higher in BRCA1 and BRCA2 mutant patients. Morphologically, cells isolated from BRCA1 patient displayed higher levels of cells with mesenchymal phenotype. Upon short-term culturing of cells under regular 2D culture without reprogramming conditions, cell surface marker profile remained unchanged suggesting that most of the cell surface marker profile is cell intrinsic. CD49f+/EpCAM-, CD49f+/EpCAMhigh, CD49f+/EpCAMmedium, CD49f-/EpCAM+, CD133+/EpCAM+, and CD10+/EpCAM+ cells were amicable for further propagation in culture. CD49f+/EpCAMmedium cells cultured for two weeks remained CD49f+/EpCAMmedium. Additional marker profiling including cancer stem cell markers is underway to further refine stem, progenitor, and mature cells.

Conclusion: Ability to propagate distinct subpopulation of normal cells from patients with different breast cancer risk would allow investigation of the impact of cell type origin of cancer on the course of the disease including response to therapy and the development of in vitro assay for breast cancer risk assessment.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-05-05.
Abstract P1-06-09: Engineered model matrix to mimic cancer stem cell microenvironment: Effect of integrin and heparin binding peptides
Authors: Jabbari E , Sarvestani SK
Published: August 2014

Introduction: It is critical to understand the mechanism of cancer stem cell (CSC) maintenance and its regulation by the microenvironment to prevent and treat cancer. Since cancer takes many years to grow, it is imperative to develop in vitro models to study the molecular basis of tumorigenesis and progression. In an effort to isolate and investigate the effect of individual physical, mechanical, and biological factors in the tumor microenvironment on the fate of cancer cells and maintenance of CSCs, we have developed novel inert synthetic hydroxy acid-chain-extended polyethylene glycol (PEGXA) hydrogels with defined physical, chemical, mechanical, and biological properties. The objective of this work was to investigate the effect of integrin-binding and heparin-binding peptides conjugated to a matrix with defined physiochemical properties and stiffness on tumorsphere formation and CSC maintenance.

Methods: Mouse 4T1, human MCF7, and human MDA-MB-231 breast cancer cells were encapsulated in PEGXA gels conjugated with integrin-binding and heparin-binding peptides. Peptides included laminin-derived IKVAV and YIGSR, collagen-IV derived TAGSCLRKFSTMY and GEFYFDLRLKGDKY, and fibronectin-derived WQPPRARI and RGD. Gels without peptide conjugation and those conjugated with laminin-derived CD44 binding RLVSYNGIGGLK peptide were used as positive (CSC forming) and negative controls, respectively. Tumorsphere size and density, and expression of CSC markers were determined with incubation time in tumor CSC culture medium. For in vivo, cell encapsulated gels were inoculated in syngeneic Balb/C mice and tumor formation was determined with time.

Results: All tumor cells types encapsulated in PEGXA gels without peptide formed tumorspheres and maintained CSCs. Gel stiffness had a bimodal effect on tumorsphere formation in the 1-50 kPa compressive modulus range. Tumor cells encapsulated in the gel with 5 kPa compressive modulus had the highest expression of CSC markers. Conjugation of CD44 binding peptide eliminated tumorsphere formation by encapsulated tumor cells. Interestingly, integrin-binding peptides (RGD, IKVAV and YIGSR) conjugated to the gel abolished tumorsphere formation by encapsulated cells whereas heparin-binding peptides (TAGSCLRKFSTMY, GEFYFDLRLKGDKY and WQPPRARI) improved rate of tumorsphere growth and the expression of CSC markers. It should be noted that peptides dissolved in the gel (not chemically attached to the gel) or gels conjugated with mutant peptides did not have a noticeable effect compared to the gels without peptide. Similar results were observed with the cell-encapsulated gels were implanted in Balb/C mice. The expression pattern of EMT markers suggested that enhanced EMT contributed to the increased tumorsphere formation in gels conjugated with heparin-binding peptides.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-06-09.
Abstract P1-06-11: Tumor-associated macrophages: CD206 expression and patient outcomes in locally advanced breast cancer
Authors: Reynolds JM , Hodges DB , Salam A , Shaw ET , Magoon V , Werlang-Perurena AM , Singh HB , Speights VO , Ruud CO , Bhat A
Published: August 2014

Introduction

The ability of a tumor to recur is related to the complex molecular biology of the tumor cells and the surrounding microenvironment. Tumor-associated macrophages (TAMs) account for up to 50% of the cell mass within a breast tumor, and the M2 macrophage phenotype has been reported to have pro-tumor activity. The mannose receptor, CD206, is a transmembrane receptor expressed predominantly in macrophages and shown in mouse studies to promote M2/TAM activity. This study evaluates CD206 expression in the tumor stroma with patient outcomes in locally advanced breast cancer.

Methods

The Scott & White tumor registry was queried for consecutive cases of women diagnosed with stage 3, HER-2 negative breast cancer between January 1999 and December 2006. To meet inclusion criteria, all biopsies must have been performed at S&W and patients must have had at least 5 years of follow up in our system documenting status of cancer (complete remission vs. metastatic recurrence). Archived formalin fixed and paraffin embedded tissue breast tissue from initial surgical biopsies was obtained from the Scott & White Pathology Department. Sections were cut at 4 microns thick and slides were stained for CD206. Two pathologists without knowledge of the patients’ clinical backgrounds reviewed each slide. The hot spot method was employed at 20x power to identify the 3 spots in tumor stroma that stained most heavily. The number of CD206+ cells in each of 3 hot spots was counted, and the mean number of CD206+ cells per hot spot reported by two independent pathologists was averaged for analysis.

Results

65 patients met inclusion criteria, of these 31 had a recurrence within 5 years of diagnosis. There was no correlation between likelihood of recurrence or overall survival and CD206 expression. In the 31 patients that had a recurrence, a high CD206 count of greater than or equal to 15 was associated with a shorter time to recurrence than a CD206 count of less than 15 (14.8 months vs. 22 months, respectively, p = 0.028). Mean age at diagnosis in the recurrence group was 59.6 vs. 60 years, and 15 (60%) vs. 2(33%) patients were ER positive in the low CD206 (<15) vs. high CD206 ( = >15) groups, respectively.

Conclusion

High expression of CD206 in the tumor stroma was associated with a shorter time to recurrence in patients with stage 3, HER-2 negative breast cancer; however, there were more ER negative tumors in the high CD 206 group. Our research supports in vitro data that high CD206 expression in macrophages may represent a more aggressive tumor phenotype. Future studies focusing on studying CD206 expression in HER-2 positive breast tumors along with histological stratification are needed to confirm this finding.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-06-11.
Abstract P1-07-12: Kaiso is a novel regulator of epithelial-to-mesenchymal transition in triple negative breast cancer cells
Authors: Bassey BI , Crawford C , Daniel JM
Published: August 2014

Introduction:

Breast cancer (BC) is the most frequent form of female cancer and a principal cause of female deaths worldwide. Most BC deaths are attributed to tumor metastasis to vital organs, and many studies have proposed a role for epithelial-to-mesenchymal transition (EMT) in BC metastasis especially the Triple Negative BC (TNBC) subtype. This theory is further strengthened by the observation that many triple-negative tumors display low E-cadherin expression, which is a hallmark of EMT. The TNBC subtype lack expression of the estrogen-receptor (ER), progesterone-receptor (PR) and human epidermal growth factor receptor-2, HER2. These tumors are highly aggressive with limited treatment options or targeted-therapies, and consequently TNBC patients have poor outcomes. Recently, increased nuclear expression of the transcription factor Kaiso was significantly correlated with high grade, BRCA1-related and basal/TNBCs. Kaiso is a novel transcription factor that we originally identified as a binding partner of p120-catenin (p120), a Src Kinase substrate and regulator of E-cadherin stability and turnover.

Rationale and Research Objective:

Though not much is known about the function of Kaiso in breast tumor progression and metastasis, Kaiso has been implicated in regulating E-cadherin expression, which raised the possibility that Kaiso might play a role in EMT. Thus we sought to elucidate the role of Kaiso in EMT and TNBC metastasis.

Results:

To achieve this goal, we created a stable Kaiso-depleted TNBC cell line (MDA-MB-231) to analyze the consequences of Kaiso-depletion on features attributed to metastasis, using immunoblot, wound healing and Matrigel invasion assays among other techniques. Interestingly, Kaiso-depletion led to reduced transcript and expression levels of the EMT markers Snail, Slug, ZEB1/2, TGFbRII, vimentin, and up-regulation of E-cadherin. Kaiso-depleted MDA-MB-231 cells also showed a gradual change in morphology from a mesenchymal to an epithelial phenotype referred to as mesenchymal to epithelial transition (MET). Loss of Kaiso further resulted in reduced cell motility and invasion. Ongoing studies seek to evaluate the effect of Kaiso-depletion on metastasis in vivo. Since there is evidence that the TGFβ signaling pathway drives EMT, and Kaiso-depletion resulted in reduced transcript levels of TGFβRII, future studies will determine if Kaiso plays a role in EMT/MET via promotion of the TGFβ signaling pathway.

Overall Significance:

Our study is the first to demonstrate a link between Kaiso and EMT/MET of TNBC cells and suggest that Kaiso may be a novel regulator of EMT in TNBC. Consequently, Kaiso might be useful as a therapeutic or prognostic tool in the treatment of TNBC.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-07-12.
Abstract P1-08-02: Chemosensitivity and endocrine sensitivity of MammaPrint and BluePrint in the prospective neo-adjuvant breast registry symphony trial (NBRST)
Authors: Whitworth P , Gittleman M , Akbari S , Stork-Sloots L , de Snoo F , Gibson J , Beitsch P
Published: August 2014

Background

Classification into molecular subtypes may be important for the selection of therapy for patients with early breast cancer. Previous analyses had shown that breast cancer subtypes have distinct clinical outcome (Sorlie, PNAS 2001; Esserman, BCRT 2011). The aim of the prospective NBRST study is to measure chemosensitivity as defined by pathological Complete Response (pCR), or endocrine sensitivity as defined by partial response (PR) and metastasis-free survival in molecular subgroups using MammaPrint and BluePrint.

Methods

The study includes women aged 18–90 with histologically proven breast cancer, who are scheduled to start neo-adjuvant chemotherapy (CT) or neo-adjuvant endocrine therapy (ET), and who provide written informed consent. Additional inclusion criteria include no excision biopsy or axillary dissection, no confirmed distant metastatic disease, and no prior therapy for breast cancer. Treatment is at the discretion of the physician adhering to NCCN approved regimens. pCR is defined as no invasive or noninvasive residual disease in the breast or axillary lymph nodes.

Results

191 Patients (median age 53, range 22-82), T1-4 N0-3, had definitive surgery and the overall pCR rate was 18%.

31 (16%) patients are classified as Luminal A-type (BluePrint Luminal/MammaPrint Low Risk) of whom 24 received neo-adjuvant CT; 1 patient (4%) had a pCR. While 7 patients received neo-adjuvant ET and 5 (71%) had a PR. 64 (34%) Patients are classified as Luminal B-type (BluePrint Luminal/MammaPrint High Risk). All but 1 patient received neo-adjuvant CT and 2 (3%) had a pCR. 30 (16%) Patients are classified as BluePrint HER2-type and all received neo-adjuvant CT and trastuzumab (except for 3 patients who did not receive trastuzumab); 11 (37%) had a pCR. 66 (31%) Patients are classified as BluePrint Basal-type and received neo-adjuvant CT; 19 (29%) had a pCR.

Comparison of IHC/FISH classification with molecular subtyping:

24/102 (24%) IHC/FISH ER+/Her2- patients are not classified as Luminal-type by BluePrint (4 Her2, 20 Basal). 22/48 (46%) IHC/FISH Her2+ patients are not Her2-type by BluePrint (4 Luminal A, 11 Luminal B, and 7 Basal). All IHC/FISH triple negative patients are BluePrint Basal-type.

Conclusions

Molecular subtyping using MammaPrint and BluePrint leads to a reclassification of 24% (46/191) of tumors. We observed differences in pCR to neo-adjuvant treatment in groups stratified by BluePrint and MammaPrint. Patients with Luminal A-type breast cancer have a high response to neo-adjuvant endocrine therapy (71% PR) and low pCR (4%) to neo-adjuvant CT. While patients with BluePrint HER2-type and Basal-type breast cancer have a high pCR rate to neo-adjuvant CT.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-02.
Abstract P1-08-03: Prediction of response to neoadjuvant chemotherapy in estrogen receptor positive (ER+) breast cancer by IHC4 or Ki67 alone
Authors: Sheri A , Smith IE , A'Hern R , Jones R , Parton M , Johnston SRD , Dowsett M
Published: August 2014

Aims To determine whether IHC4 score assessed on pre-treatment core biopsies (i) predicts response to neoadjuvant chemotherapy in ER positive breast cancer; (ii) provides more predictive information than Ki67 alone.

Background The IHC4 score, a composite of ER, PgR, Ki67 and HER2 scores provides prognostic information similar to the OncotypeDx 21-gene Recurrence Score in ER + primary breast cancer treated with endocrine therapy but it is unknown if it also predicts response to chemotherapy. Pathological complete response (pCR) following neoadjuvant chemotherapy is established as an intermediate marker of long-term outcome. More recently the residual cancer burden (RCB) has also been shown to be prognostic, with those patients with minimal residual disease (RCB1) following neoadjuvant chemotherapy having a similar prognosis to those with a pCR (RCB0). The development of these intermediate markers provides an opportunity to study the predictive role of pre-treatment biomarkers for benefit to a particular therapy.

Methods A total of 114 ER+ patients treated with neoadjuvant chemotherapy at the Royal Marsden Hospital between 2002-2010 were included in the study.

An assessment of the excision specimen was made for residual disease. IHC4 was determined on pre-treatment core biopsies, blinded to clinical outcome, by immunohistochemistry using quantitative scoring of ER (H-score), PgR (%) and Ki67 (%). Determination of HER2 status was made by immunohistochemistry and fluorescent in situ hybridization for 2+ cases. IHC4 and Ki67 scores were tested for their association with pCR rate and RCB score.

Results 19 (17%) of the 114 patients and 9 (10%) of the 90 HER2-ve cases showed a pCR. Ki67 and the IHC4 score were both positively associated with achievement of pCR (P<10-7 and P<10-9 respectively) and RCB0+1 (P<10-5 and P<10-9 respectively) following neoadjuvant chemotherapy in all patients. Rates of RCB0+1 were 45% and 66% in the highest quartiles of Ki67 and IHC4 scores respectively. In ER + HER2-ve cases pCR and RCB0+ 1 rates were 35% and 39%, respectively in the highest quartile of IHC4 and 30% and 39%, respectively in the highest quartile of Ki67 (Table 1). There were no pCRs in the lower half of IHC4 or Ki67 scores.

Conclusions A high IHC4 was strongly predictive of a pCR or near pCR in ER + breast cancers following neoadjuvant chemotherapy. Ki67 was an important component of this predictive ability.

Response according to IHC4 and Ki67 quartile in HER2 negative, ER+ breast cancersIntermediate endpointpCRpCRRCB0+RCB1RCB0+RCB1ScoreIHC4Ki67IHC4Ki67Quartile 10% (0/22)0% (0/22)5% (1/22)9% (2/22)Quartile 20% (0/23)0% (0/23)4% (1/23)0% (0/23)Quartile 35%(1/22)9% (2/23)9% (2/22)9% (2/23)Quartile 435% (8/23)30% (7/23)39% (9/23)39% (9/23)

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-03.
Abstract P1-08-07: High tumor CD68 mRNA content (intratumoral macrophages) predicts response to neoadjuvant chemotherapy
Authors: Wirtz RM , Aigner J , Marme F , Eidt S , Altevogt P , Sinn P , Schneeweiss A
Published: August 2014

Background

Immune cell infiltrates have been shown to play a prominent role with regard to prognosis of node negative breast cancer (Schmidt et al 2008). Moreover, it has been shown that B-cells and T-Cells are of particular importance for the response to chemotherapy (Schmidt et al. 2012). However, the role of macrophages remains unclear. It has been assumed, that tumor-associated macrophages play a role in promoting tumorigenesis. Still their role with regard to therapy prediction is unknown. Here we tested wether the infiltration of macrophages is associated with the response to neoadjuvant chemotherapy.

Materials and Methods

Pretreatment core cut biopsies from n = 100 patients with PBC treated within a randomized phase II trial (1) of anthracyline/taxane based NAC were examined. RNA from formalin-fixed, paraffin embedded (FFPE) routine biopsies were extracted using a bead-based extraction method (STRATIFYER XTRAKT kits). CD68 and ESR1, PGR, HER2, Ki67 as well as CALM2 as a house keeping gene were measured via a multiplex quantitative RT-PCR (RT-qPCR). Correlation analyses and partitioning tests were performed using the SAS JMP® 9.0.0 software.

Results

CD68 mRNA exhibited a normal data distribution (Median expression 40-DCT 34,66) in the core needle biopsies of advanced breast tumors. CD68 mRNA correlated strongly with Ki67 mRNA levels (Spearman r = 0,46; p<0,001), whereas there is virtually no correlation with other classical biomarkers such as ESR1, PGR and HER2. Partitioning analysis revealed that CD68 mRNA levels are superior to the previously and prospectively validated response markers ESR1 mRNA and HER2 mRNA. When using the median mRNA expression as an objective cut-off measurement no tumor having low CD68 mRNA expression did respond to chemotherapy, while 22% of the CD68 positive tumors responded by pathological complete response (ypT0ypN0). Based on molecular subtyping into Luminal, HER2 positive and Triple-Negative with predefined Cut-Off values for ESR1mRNA and HER2 mRNA levels, high CD68 were particularly informative within luminal tumors (22% pCR versus 0% pCR rate) and Triple negative tumors (38% pCR versus 0% pCR rate).

Conclusion

The presence of macrophages indicates tumors being sensitive to chemotherapy beyond the prospectively validated ESR1 and HER2 mRNA determinations (2), both in luminal and triple negative tumors. The mRNA expression levels of CD68 provide additional information beyond conventional subtyping and might therefore be useful for early assessment of non-response to chemotherapy und subsequent treatment planning.

1) Schneeweiss et al, Ann Oncol 2011

2) Denkert et al, Ann Oncol 2013.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-07.
Abstract P1-08-22: The impact of obesity on response to neoadjuvant chemotherapy in operable breast cancer patients
Authors: Lee HY , Jung SP , Kim S , Kim HY , Bae JW , Lee JB
Published: August 2014

Background

The association between obesity and response to neoadjuvant chemotherapy in breast cancer patients is not clear. We evaluated the impact of obesity on response to neoadjuvant chemotherapy in patients with operable breast cancer.

Methods

From May 2008 to December 2010, 104 patients were diagnosed with invasive breast cancer at Korea University Anam Hospital and received neoadjuvant chemotherapy before surgery. Patients were classified into those of normal (BMI of 18.5 to <25kg/m2), overweight (BMI of 25 to <30kg/m2), or Obese (BMI≥30kg/m2). The association between body mass index and pathologic response (pathologic complete response(pCR) and pathologic partial response(pPR)) to neoadjuvant chemotherapy was examined using logistic regression.

Results

Median age was 45 years. Mean BMI was 24.8 kg/m2; 53.8% had a normal BMI, 35.6% overweight, and 10.6% of patients was obese. BMI did not show a significant association with ER status, PR status, HER-2 status, lymph node involvement and neoadjuvant chemotherapy regimen. In univariate analysis, overweight and obese patients were significantly less likely to have a pCR compared with normal weight patients (odds ratio [OR] = 0.300; 95% CI, 0.115 to 0.784; p = 0.010). In multivariate analysis, ER negativity was significantly associated with a pCR and pPR to neoadjuvant chemotherapy (OR = 2.987; 95% CI, 1.128 to 7.907; p = 0.028), And there was significant difference in pCR for overweight and obese compared with normal weight patients (OR = 0.304;95% CI, 0.115 to 0.803; p = 0.016).

Conclusion

This study suggests that higher BMI should be considered to be a factor of worse response to neoadjuvant chemotherapy in patients with operable breast cancer.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-22.
Abstract P1-08-39: Tumor-infiltrating CD8+ lymphocytes as an independent predictive factor for pathologic complete response to primary systemic therapy in breast cancer
Authors: Seo AN , Lee HJ , Kim EJ , Kim HJ , Lee HE , Kim YJ , Kim JH , Park SY
Published: August 2014

Background: Tumor infiltrating lymphocytes (TILs), stem cell phenotype and epithelial-mesenchymal transition (EMT) are known to be associated with response to primary systemic therapy (PST) in breast cancer. However, these markers are strongly correlated with triple negative breast cancer, which shows high pathologic complete response (pCR) to PST. This study was conducted to evaluate clinical implication of these markers as predictive factor for pCR after PST in breast cancer and to find independent predictive factors, irrespective of breast cancer subtype.

Methods: The pre-chemotherapeutic biopsy specimens of 153 breast cancer patients who underwent surgical resection after anthracycline-or anthracycline/taxane-based PST were enrolled. TILs (CD4, CD8, and FOXP3), breast cancer stem cell phenotype (CD44+/CD24-, and ALDH1), expression of EMT markers [vimentin, smooth muscle actin (SMA), osteonectin, E-cadherin, and N-cadherin] were evaluated by immunohistochemistry and were correlated with pCR after PST.

Results: High infiltration of TILs (CD4+, CD8+, and FOXP3+), expression of stem cell markers (CD44+/CD24-, ALDH1+) and expression of EMT markers (expression of vimentin, SMA and osteonectin; loss of E-cadherin) were significantly associated with high histologic grade and triple negative breast cancer. Of them, high TILs (CD4+, CD8+, and FOXP3+) and expression of ALDH1, vimentin and osteonectin were associated with pCR. In multivariate analysis, CD8+ TILs and a conventional predictive marker, Ki67 were found to be independent predictors for pCR. In subgroup analyses, high CD8+ TILs was remained as a predictive factor for pCR, irrespective of PST regimen and tumor subtype.

Conclusion: CD8+ TILs could therefore be used as a reliable predictor of response to PST in breast cancer.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-39.
Abstract P1-09-14: Relationship between socioeconomic status, race, and breast cancer clinical stage at presentation
Authors: Wray CJ , Nguyen BC , Wiatrek RL , Robinson EK , Ko TC , Kao LS
Published: August 2014

Introduction: Racial and ethnic minority breast cancer patients present with later clinical stage at diagnosis, which may be due to lack of access to care due to poor socioeconomic status (SES). Our hypothesis was that the racial gap in clinical stage at breast cancer presentation would be eliminated after adjustment for SES.

Methods: Retrospective review was performed of breast cancer (excluding ductal carcinoma in situ) patients from 10 hospitals in Harris County Texas between 2002 and 2011. Demographic and clinical information was recorded. Clinical stage was calculated based on data from the institutional tumor registry and electronic medical record. Zip code-based SES data, including mean household income and percentage of households receiving social support, were downloaded from www.census.gov. A multivariate ordered logistic regression clustered by hospital was used to identify predictors of clinical stage.

Results: Of 3084 breast cancer patients, the mean age at diagnosis was 59 ± 14 years. The cohort included 1873 Caucasian (61%), 519 African-Americans (17%), 353 Hispanics (11%), and 339 Asian/others (11%). Age, race, mean household income, and percentage of household receiving social support were predictive of clinical stage at presentation (p<0.05).

Ordered Logistic RegressionVariableOdds RatioSEp-value95% CIMean Income0.998.2 e-060.0010.98 - 0.99% Social Support1.010.0020.0021.00 - 1.01Age0.980.0020.0410.97- 0.99Race    African-American4.581.220.0002.72 - 7.72Hispanic1.991.260.2730.58 - 6.89Asian/other1.450.760.4730.52 - 4.04Caucasian = referent value for Race, SE = standard error, CI = confidence interval

The largest racial gap was observed between African-Americans and Caucasians. African Americans from areas with a mean income up to $180K still had a lower predicted probability of presenting with clinical stage 1a disease than the Caucasians from the poorest mean income areas (<$5K). African Americans from the highest mean income areas (> $305K) still had a higher probability of presenting with clinical stage 4 disease as Caucasians from areas with a mean income as low as $100K.

Conclusion: Both African-American race and residence in a low SES zip code contribute independently to late clinical stage at presentation among breast cancer patients. Both genetic and social factors may play a role in this disparity. Addressing factors related to just race or low SES status alone is unlikely to improve outcomes for African-Americans from low SES zip codes.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-09-14.
Abstract P1-09-16: Factors affecting disparities in advanced stage breast cancer
Authors: Samiian L , Kraemer D , Butterfield R , Osian SR , Lukens-Bull K , Wood D
Published: August 2014

BACKGROUND:

African American women have a lower incidence of breast cancer than white American women. However, black women in the US are 40% more likely to die of breast cancer. Compared to white women, Black women in Florida have among the highest additional breast cancer deaths. Within Florida, Duval County has one of the highest rates of breast cancer. Furthermore, breast cancer mortality is significantly higher for black women (36.7 per 100,000) in Duval County, than for white women (24.0 per 100,000). Therefore, we evaluated breast cancer disparities in Duval County as a representative population to identify factors influencing breast cancer mortality.

METHODS:

Data on female breast cancer cases from 2004-2010 was extracted using the Florida Cancer Data System (FCDS). Classifications included health zones within Duval County, race, insurance, and age. Insurance categories included Medicare, Medicaid, Uninsured, and Tricare/commercial which were combined in one group. The primary outcome was late stage (Stage 4) at diagnosis versus other stages. Multivariable logistic regression was used to assess the association between late stage at diagnosis and insurance, race, age, and place of residence (health zone).

RESULTS:

Using the FCDS, 6,502 new breast cancers were identified in Duval County from 2004 – 2010. Of these women, 73% were white, 24% black, 5% Hispanic, 2% Asian, and 2% unknown/other. Health Zone 1 which represents the urban core has the largest population of Black women with breast cancer (76%), although the incidence of breast cancer was not higher in this group. Health Zone 1 has the highest volume of uninsured, and Medicaid breast cancer patients (10% and 8% vs. 4% average for all other zones), and the lowest number of breast cancer patients with Tricare/commercial insurance (33% vs. 56% average for all other zones). Black women overall had a higher percentage of advanced stage IV disease than any other race (8% vs. 4%, p< .001). Women in Health Zone 1 are more likely to present with stage IV breast cancer than any other health zone in Duval County (10% vs. 5%, p<0.01). Medicaid patients, followed by the uninsured had the highest rate of stage IV at diagnosis than those with commercial insurance (20% and 12% vs. 4%, p<.001). Using multivariable logistic regression analysis, only Medicaid insurance (OR = 5.3; 95% CI = (3.6, 7.9)) and living in the urban core (OR = 4.02; 95% CI = (1.9, 8.4)) was significantly associated with stage IV disease. After adjustment for insurance and place of residence, black race did not remain a significant factor (p = 0.1722).

CONCLUSION:

Living in the urban core, and lack of insurance or Medicaid funding was significantly associated with advanced stage at diagnosis in Duval County, Florida. These results suggest that socioeconomic factors are a major contributor to breast cancer mortality, irrespective of race.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-09-16.
Abstract P1-13-03: ER, PR, HER2, and Ki67 index and responsiveness to adjuvant tamoxifen in postmenopausal high-risk breast cancer patients enrolled in the DBCG 77C trial
Authors: Knoop AS , Lænkholm A-V , Jensen M-B , Nielsen KV , Andersen J , Nielsen D , Ejlertsen B
Published: August 2014

PURPOSE: The DBCG 77C trial compared one year of tamoxifen in postmenopausal, steroid-receptor unknown, high-risk breast cancer patients to no adjuvant systemic therapy. After a potential follow-up of 30 years we report overall efficacy for the study and results according to subtypes subsequently assessed by immunohistochemistry and FISH.

METHODS: Between 1977 and 1982, 1716 postmenopausal patients with tumors larger than 5 cm or positive axillary nodes were randomly assigned to no systemic therapy or tamoxifen 30 mg daily for one year. The main study is reported as an ITT analysis with the predefined DFS and BCM as endpoint. For multivariate analysis the Cox proportional hazards regression model was applied to assess the adjusted hazard ratio of treatment regimen, and to explore interactions. Formalin-fixed, paraffin-embedded primary breast tumor tissue blocks were available from 1548 (90%) of the 1716 participants enrolled and 1428 were assessable for ER, PR, HER2 and Ki67. The hormone receptor positive (ER and/or PR) cancers were defined as luminal A if Ki67 low and HER2-negative; as luminal B if Ki67 high or HER2-positive; and otherwise as HER2 positive or triple negative.

RESULTS: In the intent-to-treat (ITT) population one year of tamoxifen improved the disease-free-survival (DFS) (Hazard ratio (HR) = 0.87; 95% CI 0.77-0.98), the recurrence-free-survival (RFS) (HR = 0.79; 0.69-0.90) and reduced the breast-cancer-specific-mortality (BCM) (HR = 0.83; 0.73-0.93). Recurrence-free survivals were improved significantly by tamoxifen in luminal A (HR = 0.66; 0.53-0.84) and luminal B/HER2- (HR = 0.54; 0.39-0.74) but not in the other subsets, and with similar results for BCM with 30 years follow-up.

CONCLUSION: One year of treatment with tamoxifen significantly improves RFS and BCM in postmenopausal patients with ER positive breast cancers. The benefit from tamoxifen was not significantly different in luminal A and B subtypes.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-13-03.
Abstract P1-14-04: Subsequent endocrine therapy after becoming to resistance of ‘estrogen therapy’ is beneficial as a salvage treatment for the late stage metastatic breast cancer
Authors: Iwase H , Yamamoto Y , Yamamoto-Ibusuki M , Murakami K-I , Inao T , Okumura Y , Omoto Y
Published: August 2014

BACKGROUND: Aromatase inhibitor (AI) is a most commonly used as the endocrine therapy in postmenopausal hormone-dependent breast cancer. Paradoxically, estrogen additive therapy, high-dose estrogen using ethinylestradiol, can be useful after long-term estrogen deprivation therapies with AI. Furthermore, there is a possibility of the beneficial effect of AI or fulvestrant as a subsequent endocrine therapy after EE2 failure.

METHODS: Ethinylestradiol (EE2; 3mg/day, TID) therapy was performed in 22 patients with metastatic breast cancer (median; 62 years-old, the mean observation time: 13.6 months.), who were heavily treated by sequential endocrine therapies (3rd or more line) including cytotoxic chemotherapies. We examined the efficacy of a subsequent endocrine therapy using aromatase inhibitor or fulvestrant after becoming resistance to the EE2 therapy in the patients who got the disease control by the prior EE2 therapy. The primary endpoint was clinical benefit rate (CBR) and the secondary endpoint was time to treatment failure (TTF). (Registration number; UMIN 000002831, additional analysis)

RESULTS: Median of TTF of EE2 treatment was 46 weeks (23-62+, 2 cases were ongoing). The response rate was 41% (9/22), the clinical benefit rate was 50% (11/22). The stable disease (< 6 months) was 18% (4/22) and another 3 cases were judged as progressive disease. Four cases withdrew due to nausea, fatigue and muscle-skeletal pain. In 13 cases progressed after disease control (SD or more) of EE2, a subsequent endocrine therapy, fulvestrant for 6 cases and AI for 7 cases, was performed. Fulvestrant group showed 50% (3 in 6) of CBR and 15 weeks (5-55+) of TTF, and AI-treated group showed 43% (3 in 7) of CBR and 19 weeks (5-33+) of TTF. Two in three cases, who became resistance to anti-estrogen treatment, got long SD by further EE2 re-therapy.

DISCUSSION: EE2 therapy could be beneficial for heavily treated metastatic breast cancer. Some cases showed clinical benefits of a subsequent therapy by AI or fulvestrant after becoming to EE2 failure. Taken together, sequential use of estrogen and anti-estrogen therapy (vice versa) could be one of the options as a salvage endocrine therapy for the end stage breast cancer.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-14-04.
Abstract P2-06-06: ALU and LINE-1 hypomethylation is associated with HER2+/ER- subtype of breast cancer
Authors: Jung HY , Park SY , Jung N , Cho N-Y , Kang GH
Published: August 2014

The changes in DNA methylation status in cancer cells are characterized by hypermethylation of promoter CpG islands and diffuse hypomethylation of non-coding genomic regions. ALU and long interspersed nucleotide element-1 (LINE-1) are non-coding genomic repetitive sequences and methylation of these elements can be used as a surrogate marker for genomewide methylation status. This study was designed to evaluate the changes of ALU and LINE-1 hypomethylation during breast cancer progression from normal to pre-invasive lesions and invasive breast cancer (IBC), and their relations with characteristics of IBC. We analyzed the methylation status of ALU and LINE-1 in 145 cases of breast samples including normal breast tissue (n = 30), atypical ductal hyperplasia/ flat epithelial atypia (ADH/FEA, n = 30), ductal carcinoma in situ (DCIS, n = 35) and IBC (n = 50), and another set of 129 cases of IBC by pyrosequencing. LINE-1 methylation was significantly decreased from normal to ADH/FEA, while ADH/FEA, DCIS and IBC were not different each other. There was no difference in ALU methylation levels during progression of breast cancer. In IBC, ALU hypomethylation was correlated with negative estrogen receptor (ER) status (p = 0.007) and LINE-1 hypomethylation was associated with negative ER status (p<0.001), positive HER2 status (p = 0.005) and p53 overexpression (p = 0.024). ALU-1 and LINE-1 methylation status was significantly different between breast cancer subtype and the HER2+/ER- subtype had significantly lower methylation levels and frequencies than the other subtypes. Our findings suggest that LINE-1 hypomethylation is an early event during breast cancer progression and prominent hypomethylation of ALU and LINE-1 in HER2+/ER- subtype may be related to chromosomal instability of this specific subtype.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-06-06.
Abstract P2-09-05: The potent anti-androgen enzalutamide demonstrates broad anti-tumour activity across all androgen receptor-positive triple-negative breast cancer subtypes
Authors: Denne L , Shia A , Komulainen E , Haley V , Lenihan C , Cavicchioli F , O'Leary K , Schmid P
Published: August 2014

Background: Up to 30% of triple-negative breast cancers (TNBC) show expression of the androgen receptor (AR) and may therefore be candidates for AR-targeted therapy. Molecular profiling of TNBC has identified the luminal AR (LAR) subset, which shares many aspects of ER-positive luminal cancers but is resistant to ER-targeted therapy. LAR cancers highly express the AR, are heavily enriched in hormone-regulated pathways and demonstrate tumour growth in response to androgen stimulation that can be inhibited by the mixed AR agonist/antagonist bicalutamide. The objective of this study was to investigate the role of AR signalling in TNBC in more detail and to evaluate the potential of the second generation anti-androgen Enzalutamide in this setting relative to bicalutamide.

Methods: A panel of 13 TNBC breast cancer cell lines was selected, representing different molecular subtypes of TNBC (including 4 LAR cell lines). Baseline AR expression was determined using qPCR (Taqman) and Western-Blot (Antibody, Santa Cruz Biotechnology). The effects of enzalutamide and bicalumatide on cell proliferation and survival were determined using MTT assays with and without DHT stimulation. Comprehensive molecular profiling (including Illumina Human HT-12 v4 whole genome mRNA and Illumina Human 450K methylation microarrays) was applied before and after enzalutamide treatment to define the molecular signature associated with sensitivity to enzalutamide therapy. All statistical calculations and IC50 doses were calculated using Prism v6.0 (GraphPad).

Results: A wide range of AR expression was confirmed across the cell line panel defining 3 groups with complete lack of AR expression, low/moderate AR expressions levels, or very high expression in the LAR subtype (with up to 100-fold greater AR expression compared to other TNBC subtypes). Enzalutamide demonstrated broad anti-tumour activity across all AR+ TNBC subtypes (but not in AR-negative subtypes), with IC50 values being similar in LAR and non-LAR cell lines. Treatment with enzalutamide resulted in more effective growth inhibition and a broader range of activity compared to bicalutamide. Data on molecular profiles of enzalutamide sensitivity will be presented at the meeting.

Conclusion: AR inhibition is a promising therapeutic strategy for both LAR and non-LAR AR-expressing TNBC. The potent anti-androgen enzalutamide shows broad activity across all AR+ TNBC subtypes and warrants further clinical evaluation. A phase 2 study of enzalutamide in AR+ TNBC has been initiated.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-05.
Abstract P2-11-11: Clinical utility of a radiosensitivity molecular signature: Identification of breast cancer patients that benefit from RT boost dose
Authors: Torres-Roca JF , Servant N , Bollet MA , Eschrich SA
Published: August 2014

Objective: In previous studies we developed a radiosensitivity molecular signature (RSI) that has been independently validated in multiple disease sites (n = 652) including breast cancer 1-3. Here, we test whether RSI can be utilized to identify sub-populations in breast cancer that benefit from RT dose intensification (boost).

Methods: RSI was tested in two published breast cancer datasets (n = 342) 4. Patients were selected if they had experienced a local recurrence within the first 10 years after primary treatment or were free of local recurrence for a minimum of 10 years after treatment. Primary treatment was initial breast-conserving surgery plus whole breast RT with/without a boost (no boost, n = 94, low boost < = 66 Gy, n = 148, high boost >66 Gy, n = 100). Patients were treated between January 1984 and November 2002. RSI was generated as previously described 1-3. Time to local recurrence was used as the endpoint for the study.

Results: Increased RT dose (>66 Gy vs. < = 66 Gy) resulted in a slight decrease in local recurrence risk that did not reach statistical significance (p = 0.11). As hypothesized, RSI-high patients (more radioresistant) were sensitive to increased RT dose (high RT-boost) which resulted in a decrease in local recurrence (HR = 0.3418, CI 0.1195-0.9777, p = 0.036). In contrast, RSI-low and intermediate patients had similar local recurrence risk independent of RT dose (RSI-low HR = 0.7687, CI 0.3404-1.736, p = 0.53, RSI-intermediate, HR = 0.9115, CI 0.5125-1.621, p = 0.75). Importantly, there were no statistical differences in the distribution of known prognostic variables between RSI-determined sub-populations (RSI-low, intermediate, high) including age, T-stage, margin status, nodal status, adjuvant chemotherapy, hormonal therapy, grade and molecular subtype.

Conclusions: RSI identifies a sub-population of breast cancer patients that benefit from increased RT-dose. RSI may be utilized to support RT-based decisions (high boost vs. low boost vs. no boost) in breast cancer.

1. Eschrich S, Fulp WJ, Pawitan Y, et al: Validation of a Radiosensitivity Molecular Signature in Breast Cancer. Clin Cancer Res, 2012

2. Eschrich S, Zhang H, Zhao H, et al: Systems biology modeling of the radiation sensitivity network: a biomarker discovery platform. Int J Radiat Oncol Biol Phys 75:497-505, 2009

3. Eschrich SA, Pramana J, Zhang H, et al: A gene expression model of intrinsic tumor radiosensitivity: prediction of response and prognosis after chemoradiation. Int J Radiat Oncol Biol Phys 75:489-96, 2009

4. Servant N, Bollet MA, Halfwerk H, et al: Search for a gene expression signature of breast cancer local recurrence in young women. Clin Cancer Res 18:1704-15, 2012.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-11-11.
Abstract P2-11-14: Does the Nottingham prognostic index further refine survival within the breast cancer subtypes?
Authors: Caggiano V , Parise C
Published: August 2014

Background

The Nottingham Prognostic Index (NPI), based on tumor size, lymph node status, and histologic grade, is a well established method of predicting survival of operable breast cancer. Breast cancer survival of the luminal, triple negative, and HER2 overexpressing subtypes has been previously described. The purpose of this study is to determine if the NPI can further refine survival of these subtypes within the AJCC stages of breast cancer.

Method

We identified 118,923 cases of stages 1-3 first primary female invasive breast cancer from the California Cancer Registry diagnosed between January 1, 2000 and December 31, 2010 with known ER/PR/HER2 status. The subtypes were classified as Luminal A (ER and/or PR-positive, HER2-negative), Luminal B (ER and/or PR-positive, HER2-positive), triple negative (ER-/PR-/HER2-), or HER2-overexpressing (ER-/PR-/HER2+). The NPI was computed and cases were stratified as having good (NPI <3.4), moderate (3.4-5.4) or poor (>5.4) prognosis. Kaplan Meier survival analysis (95% CIs) was used to compare survival among the NPI categories within each subtype and stage.

Results

The table shows that in stage 1, there was a statistically significant difference in survival between the good versus the moderate and poor NPI categories for luminal A, luminal B, and the triple negative subtypes. For the HER2-overexpressing subtype, the only difference was between the good and poor NPI category. For stage 2, the only difference in survival was for Luminal A where both the moderate (94.6%; 93.4%, 95.8%) and poor (84.3%; 83.6%, 85.0%) NPI categories had worse survival than the good category (99.9%; 97.1%, 100.0%). For stage 3, there were no differences among the NPI categories for any of the subtypes.

Eight year survival of stage 1 breast cancer subtypes within the NPI categories 8-Year Survival95% LCL*95% UCL*Luminal A   Good95.695.196.2Moderate94.493.994.9Poor90.389.191.5Luminal B   Good95.894.097.5Moderate94.393.195.5Poor88.686.291.0Triple Negative   Good93.690.197.0Moderate88.186.090.1Poor86.084.287.8HER2-overexpressing   Good92.387.697.1Moderate89.786.992.4Poor86.483.389.5*CL = Confidence Limit

For stage 2, the only difference in survival was for Luminal A where both the moderate (94.6%; 93.4%, 95.8%) and poor (84.3%; 83.6%,85.0%) NPI categories had worse survival than the good category (99.9%; 97.1%, 100.0%). For stage 3, there were no differences among the NPI categories for any of the subtypes.

Conclusion

The NPI is a valuable addition to AJCC stage 1 for all breast cancer subtypes. The NPI and AJCC staging systems both incorporate lymph node status and tumor size. However, histologic grade, found only in the NPI, may improve the accuracy of survival, especially in stage 1.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-11-14.