Shorter Breast Cancer-specific Survival Research Articles

Neighborhood Disadvantage and Breast Cancer–Specific Survival in the US

Despite improvements in breast cancer screening, treatment, and survival, disparate breast cancer-specific survival outcomes persist, particularly in disadvantaged neighborhoods. Most of these disparities are attributed to disparities in individual, tumor, and treatment characteristics. However, a critical knowledge gap exists as to whether disparities in breast cancer-specific survival remain after accounting for individual, tumor, and treatment characteristics. To evaluate if neighborhood disadvantage is associated with shorter breast cancer-specific survival after controlling for individual, tumor, and treatment characteristics in a national population. This national retrospective cohort study included patients with breast cancer diagnosed from 2013 to 2018 from the Surveillance, Epidemiology, and End Results 17 Census tract-level socioeconomic status and rurality database of the National Cancer Institute. Data analysis was performed from September 2022 to December 2023. Neighborhood disadvantage measured by Yost index quintiles. Breast cancer-specific survival was evaluated using a competing risks cause-specific hazard model controlling for age, race, ethnicity, rurality, stage, subtype, insurance, and receipt of treatment. A total of 350 824 patients with breast cancer were included; 41 519 (11.8%) were Hispanic, 39 631 (11.3%) were non-Hispanic Black, and 234 698 (66.9%) were non-Hispanic White. A total of 87 635 patients (25.0%) lived in the most advantaged neighborhoods (group 5) and 52 439 (14.9%) lived in the most disadvantaged neighborhoods (group 1). A larger number of non-Hispanic White patients (66 529 patients [76.2%]) lived in advantaged neighborhoods, while disadvantaged neighborhoods had the highest proportion of non-Hispanic Black (16 141 patients [30.9%]) and Hispanic patients (10 168 patients [19.5%]). Breast cancer-specific survival analysis found the most disadvantaged neighborhoods (group 1) had the highest risk of mortality (hazard ratio, 1.43; 95% CI, 1.36-1.50; P < .001) compared with the most advantaged neighborhoods. In this national cohort study of patients with breast cancer, neighborhood disadvantage was independently associated with shorter breast cancer-specific survival even after controlling for individual-level factors, tumor characteristics, and treatment. This suggests potential unaccounted-for mechanisms, including both nonbiologic factors and biologic factors.

Predictors of successful neoadjuvant treatment in HER2‑positive breast cancer.

The prognosis of local or locally advanced human epidermal growth factor receptor 2 (HER2)-positive breast cancer after a complete response from neoadjuvant systemic treatment (NAT) is excellent. However, some of the patients succumb to their disease, so novel predictive factors to identify these patients at risk are needed. Retrospective data from 119 patients treated at the Helsinki University Hospital Comprehensive Cancer Centre (Helsinki, Finland) were collected. All patients had in situ hybridization-confirmed HER2-positive breast cancer and underwent NAT with a curative intention. The primary tumours were relatively large, most patients had cytologically confirmed lymph node metastases and the treatments used were current regimens. A total of 63 (52.1%) patients had a pathological complete response (pCR) to neoadjuvant therapy. Achieving pCR predicted longer disease-free survival (DFS; P=0.0083) but not overall survival (P=0.061). The patients with a pCR had an estimated DFS rate of 96.8% at 5 years, compared with only 59.7% of the patients with non-pCR. Radiological complete response (CR) was associated with pCR (P=0.00033), although imaging yielded 30.4% false-negative and 36.9% false-positive results. The association between the radiological CR and pCR was more obvious in oestrogen receptor-negative tumours. Moderate (compared with strong) immunohistochemical HER2 expression predicted a lower chance of pCR (P=0.0078) and worse breast cancer-specific survival (P=0.0015). In conclusion, pCR after NAT served as an important prognostic factor in women with high-risk HER2-positive breast cancer. The patients with only moderate immunohistochemical HER2 expression had a lower chance of reaching a pCR, as well as a shorter breast cancer-specific survival.

Neighborhood Disadvantage and Breast Cancer–Specific Survival

Neighborhood-level disadvantage is an important factor in the creation and persistence of underresourced neighborhoods with an undue burden of disparate breast cancer-specific survival outcomes. Although studies have evaluated neighborhood-level disadvantage and breast cancer-specific survival after accounting for individual-level socioeconomic status (SES) in large national cancer databases, these studies are limited by age, socioeconomic, and racial and ethnic diversity. To investigate neighborhood SES (using a validated comprehensive composite measure) and breast cancer-specific survival in a majority-minority population. This retrospective multi-institutional cohort study included patients with stage I to IV breast cancer treated at a National Cancer Institute-designated cancer center and sister safety-net hospital from January 10, 2007, to September 9, 2016. Mean (SD) follow-up time was 60.3 (41.4) months. Data analysis was performed from March 2022 to March 2023. Neighborhood SES was measured using the Area Deprivation Index (tertiles), a validated comprehensive composite measure of neighborhood SES. The primary outcome was breast cancer-specific survival. Random effects frailty models for breast cancer-specific survival were performed controlling for individual-level sociodemographic, comorbidity, breast cancer risk factor, access to care, tumor, and National Comprehensive Cancer Network guideline-concordant treatment characteristics. The Area Deprivation Index was calculated for each patient at the census block group level and categorized into tertiles (T1-T3). A total of 5027 women with breast cancer were included: 55.8% were Hispanic, 17.5% were non-Hispanic Black, and 27.0% were non-Hispanic White. Mean (SD) age was 55.5 (11.7) years. Women living in the most disadvantaged neighborhoods (T3) had shorter breast cancer-specific survival compared with those living in the most advantaged neighborhoods (T1) after controlling for individual-level sociodemographic, comorbidity, breast cancer risk factor, access to care, tumor, and National Comprehensive Cancer Network guideline-concordant treatment characteristics (T3 vs T1: hazard ratio, 1.29; 95% CI, 1.01-1.65; P < .04). In this cohort study, a shorter breast cancer-specific survival in women from disadvantaged neighborhoods compared with advantaged neighborhoods was identified, even after controlling for individual-level sociodemographic, comorbidity, breast cancer risk factor, access to care, tumor, and National Comprehensive Cancer Network guideline-concordant treatment characteristics. The findings suggest potential unaccounted mechanisms, including unmeasured social determinants of health and access to care measures. This study also lays the foundation for future research to evaluate whether social adversity from living in a disadvantaged neighborhood is associated with more aggressive tumor biologic factors, and ultimately shorter breast cancer-specific survival, through social genomic and/or epigenomic alterations.

Abstract 5405: Expression of cathepsin D in early-stage breast cancer and its prognostic and predictive value

Abstract Purpose: Cathepsin D is a proteolytic enzyme that is normally localized in the lysosomes and is involved in the malignant progression of breast cancer. The expression of Cathepsin D is regulated by steroid hormones including oestrogen and growth factors. There are conflicting results between different studies regarding Cathepsin D significance as prognostic and predictor marker in breast cancer. Furthermore, Cathepsin D has not been recommended for clinical use by the American Society of Clinical Oncology because of insufficient evidence. This study was aimed to fill the gap in the lack of recent and large studies on prognostic significance of Cathepsin D expression by studying a large well-characterized series of primary breast cancer for cathepsin D expression using immunohistochemistry. Methods: Expression of Cathepsin D was assessed by immunohistochemical staining of tissue microarrays, in a large well-characterized series of early-stage operable breast cancer (n=954) from Nottingham Primary Breast Carcinoma Series between the period of 1988 and 1998 who underwent primary surgery. Correlation of cathepsin D expression with clinicopathological parameters and prognosis was evaluated. Results: Cathepsin D expression was positive in 28.8% (275/954) of breast cancer tumors. Positive expression of cathepsin D was significantly associated with high histological grade (p=0.007), pleomorphism (p=0.002), poor Nottingham Prognostic Index score (NPI) (p&amp;lt;0.002), recurrence (p=0.005) and distant metastasis (p&amp;lt;0.0001). Kaplan-Meier analysis showed that Cathepsin D expression was significantly associated with shorter breast cancer specific survival (BCSS) (p=0.001), higher risk of recurrence (p=0.001) and distant metastasis (p&amp;lt;0.0001). Cathepsin D positive tumors with ER positive staining who were treated with tamoxifen were associated with lower BCSS and higher risk of developing distant metastasis (p=0.001). Conclusion: This study shows Cathepsin D expression as a promising prognostic biomarker for early operable invasive breast cancer. Our results indicate that Cathepsin D expressing tumors are associated with shorter breast cancer specific survival and higher risk of recurrence and metastasis. Cathepsin D could be clinically useful to predict response to tamoxifen treatment. Targeting Cathepsin D either through inhibition of action or interference with the proteolytic pathway could be used to treat metastatic breast cancers expressing Cathepsin D. Citation Format: Inas M. Alhudiri, Christopher C. Nolan, Ian O. Ellis, Emad A. Rakha, Adam Elzagheid, Andrew R. Green, Caroline J. Chapman. Expression of cathepsin D in early-stage breast cancer and its prognostic and predictive value [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5405.

EP.TH.275The role of amino acid transporters SLC1A5 and SLC3A2 in primary breast cancer in older women

Abstract Aims Breast cancer in older women has more favourable biology, compared to younger women. Increased glutamine metabolism is a hallmark of cancer. The prognostic role of amino acid transporters involved with glutamine flux, SLC1A5 and SLC3A2, has been shown in breast cancer in younger women. This study aimed to investigate the role of SLC1A5 and SLC3A2 in breast cancer in older women as possible prognostic markers. Methods Surgical specimens were obtained from an existing series of 1,758 older women (≥70 years) with primary breast cancer, treated in a single institution with long-term (37+ years) follow-up. Of this cohort, 813 had primary surgical treatment. As part of previous work, it was possible to construct good quality tissue microarrays (TMAs) in 575 cases. Immunohistochemical staining for SLC1A5 and SLC3A2 was performed. H-score was considered as a continuous variable as well as using positivity cut-offs of ≥ 45 for SLC1A5 and ≥15 for SLC3A2, using X-tile software. Association between H-score and tumour size, grade, ER status, local-recurrence-free-survival (LRFS), overall survival (OS) and breast-cancer-specific-survival (BCSS) was investigated. Results No correlation was seen between neither marker and LRFS, OS, or BCSS in older women with breast cancer. Both markers were associated with high tumour grade and negative ER status (both p &amp;lt; 0.001). Conclusions These findings are contrary to those found in younger women, where these amino acid transporters are associated with shorter BCSS. This may suggest that breast cancer in older women is less reliant on glutamine metabolism, which is consistent with an overall less aggressive phenotype.

Abstract 3709: miR-21-3p: A potential oncomir in breast cancer

Abstract MIR21 is a well known oncomir in many cancer types including breast cancer. Most studies that have described the oncogenic properties of MIR21 have focused on the leading strand miR-21-5p. Fewer studies have focused on the passenger strand miR-21-3p, but in a few types of cancer it has been shown to have oncogenic properties. A handful of studies suggest that miR-21-3p could be a biomarker in breast cancer. MIR21 is located at 17q23.1, a region which is often amplified in breast tumors. It overlaps the 3´ end of VMP1 but it has its own distinct promoter. The aim of this study was to analyze whether miR-21-3p associated with prognostic factors in breast cancer. The levels of miR-21-5p and miR-21-3p were measured by qPCR in 144 breast tumors from a cohort of breast cancer patients (cohort 1). The association of expression levels with clinical and pathological factors was analyzed by Student´s t-test and Anova, and with survival by Kaplan-Meier, the log-rank test, and the Cox proportional hazards test. To confirm the results, available data were used from breast cancer cohorts from The Cancer Genome Atlas (TCGA) (n=256) and METABRIC (n=1286). The mean expression levels of miR-21-5p were significantly higher than those of miR-21-3p in breast tumors from cohort 1 (p=2.2e−10). However, only miR-21-3p was more highly expressed in tumors than normal breast tissue (p=0.003), and in all three cohorts there was a strong positive correlation between MIR21 copy number and miR-21-3p expression levels. There was a significant higher expression of miR-21-3p in HER2 positive tumors compared to HER2 negative tumors in the two larger cohorts (p &amp;lt; 0.05), and high miR-21-3p levels associated with higher histograde in the largest cohort (9.2e−14). A shorter breast cancer specific survival was associated with higher miR-21-3p expression in the METABRIC cohort (HR 1.338, CI 1.104-1.622, p=0.003). HER2 was the largest confounding factor but even after adjustment for HER2 positivity the effect remained significant (HR 1.260, CI 1.036-1.532, p = 0.021). Our data show that miR-21-3p is expressed in breast tissue and tumors. Furthermore, our preliminary data suggest that miR-21-3p may be an indicator of worse outcome for breast cancer patients. However, additional work is needed to elucidate the association of miR-21-3p expression with distinct prognostic factors in breast cancer. Citation Format: Arsalan Amirfallah, Adalgeir Arason, Bjarni A. Agnarsson, Oskar Th Johannsson, Bylgja Hilmarsdottir, Rosa Bjork Barkardottir, Inga Reynisdottir. miR-21-3p: A potential oncomir in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3709.

Retrospective assessment of cyclin-dependent kinase 5 mRNA and protein expression and its association with patient survival in breast cancer.

Cyclin‐dependent kinase 5 (Cdk5) is an atypical member of the cyclin‐dependent kinase family and functions as a serine/threonine kinase that can be activated by non‐cyclin binding activators p35 or p39. Cdk5 expression and activity has been linked with the development and progression of cancer; however, its expression in breast cancer has not been fully described. Protein expression of Cdk5 was determined in a large cohort of early‐stage invasive breast cancer tumours (n = 1110) with long‐term follow‐up data using immunohistochemistry. Expression of CDK5 mRNA was assessed in the METABRIC cohort (n = 1980). Low nuclear and cytoplasmic expression of Cdk5 expression was significantly associated with shorter breast cancer‐specific survival (P = .004 and P = .001, respectively). Importantly, low nuclear and cytoplasmic expression of Cdk5 remained associated with survival in multivariate analysis, including potentially confounding factors (hazard ratio (HR) = 0.612, 95% confidence interval (CI) = 0.418‐0.896, P = .011 and HR = 0.507, 95% CI = 0.318‐0.809, P = .004, respectively). In addition, low nuclear and cytoplasmic expression of Cdk5 was significantly associated with clinicopathological criteria associated with adverse patient prognosis. Low CDK5 mRNA expression was associated with shorter patient survival (P = .005) in the METABRIC cohort; no associations between copy gain or loss and survival were observed. These data suggest that low Cdk5 expression is associated with poor clinical outcome of breast cancer patients and may be of clinical relevance.

Abstract P6-09-08: High circulating levels of Periostin are associated with a poor survival in primary, non-metastatic breast cancer patients

Abstract Background: Periostin, also known as osteoblast-specific factor OSF-2, functions as a ligand for integrins to support adhesion and migration of tumor cells which leads to increased cell survival, invasion, angiogenesis and metastasis in different cancer types including breast cancer (BC). Assuming that metastasis requires a dissemination of tumor cells, associated with worse outcome in BC, the aim of this study was to determine the expression of Periostin in blood samples of patients with primary, non-metastatic BC and to correlate the results with clinical parameters including the presence of disseminated tumor cells (DTCs) in the bone marrow (BM), survival and the risk of developing metastatic disease. Patients and Methods: BM and blood sampling were performed before surgery in an adjuvant setting in 509 patients with first diagnosis of BC between Aug 2006 and Dec 2009. Two BM aspirates were analyzed for DTCs using density centrifugation followed by immunocytochemistry applying the pan-cytokeratin antibody A45-B/B3. Blood was collected from each patient and Periostin serum levels were measured by ELISA (Biomedica, Vienna, Austria). Results: Periostin levels were detectable (504.8 ± 178.7 pmol/l) in all BC patients. There were no significant differences between serum Periostin levels when stratifying according to tumor stage, lymph node involvement or grading. Periostin levels were significantly increased in women above the age of 60 (468.6±166.6 pmol/l vs. 540.1±184.2 pmol/l; p&amp;lt;0.0001) and significantly enhanced in postmenopausal compared to peri- or premenopausal women (p&amp;lt;0.05 and p&amp;lt;0.001, respectively). No differences were observed between DTC-positive and DTC-negative patients. When separating patients according to high (top 50%) or low (low 50%) Periostin levels, patients with low Periostin levels had a significantly shorter BC specific survival (HR 0.61; 95%CI 0.39-0.96; p=0.03). Conclusion: In summary, while Periostin levels were unchanged in patients with and without DTCs, high levels of Periostin were associated with a poorer BC specific survival. These results warrant further studies on the role of Periostin in BC. Citation Format: Hoffmann O, Goebel A, Bittner A-K, Rauner M, Hofbauer LC, Kimmig R, Kasimir-Bauer S, Rachner TD. High circulating levels of Periostin are associated with a poor survival in primary, non-metastatic breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-09-08.

Expression of Lamin A/C in early-stage breast cancer and its prognostic value.

Lamins A/C, a major component of the nuclear lamina, play key roles in maintaining nuclear integrity, regulation of gene expression, cell proliferation and apoptosis. Reduced lamin A/C expression in cancer has been reported to be a sign of poor prognosis. However, its clinical significance in breast cancer remains to be defined. This study aimed to evaluate expression and prognostic significance of lamin A/C in early-stage breast cancer. Using immunohistochemical staining of tissue microarrays, expression of lamin A/C was evaluated in a large well-characterised series of early-stage operable breast cancer (n = 938) obtained from Nottingham Primary Breast Carcinoma Series. Association of lamin A/C expression with clinicopathological parameters and outcome was evaluated. Positive expression rate of lamin A/C in breast cancer was 42.2% (n = 398). Reduced/loss of expression of lamin A/C was significantly associated with high histological grade (p < 0.001), larger tumour size (p = 0.004), poor Nottingham Prognostic Index score (p < 0.001), lymphovascular invasion (p = 0.014) and development of distant metastasis (p = 0.027). Survival analysis showed that reduced/loss of expression of lamin A/C was significantly associated with shorter breast cancer-specific survival (p = 0.008). This study suggests lamin A/C plays a role in breast cancer and loss of its expression is associated with variables of poor prognosis and shorter outcome.

Clinicopathological and Functional Significance of RECQL1 Helicase in Sporadic Breast Cancers.

RECQL1, a key member of the RecQ family of DNA helicases, is required for DNA replication and DNA repair. Two recent studies have shown that germline RECQL1 mutations are associated with increased breast cancer susceptibility. Whether altered RECQL1 expression has clinicopathologic significance in sporadic breast cancers is unknown. We evaluated RECQL1 at the transcriptomic level (METABRIC cohort, n = 1,977) and at the protein level [cohort 1, n = 897; cohort 2, n = 252; cohort 3 (BRCA germline deficient), n = 74]. In RECQL1-depleted breast cancer cells, we investigated anthracycline sensitivity. High RECQL1 mRNA was associated with intClust.3 (P = 0.026), which is characterized by low genomic instability. On the other hand, low RECQL1 mRNA was linked to intClust.8 [luminal A estrogen receptor-positive (ER+) subgroup; P = 0.0455] and intClust.9 (luminal B ER+ subgroup; P = 0.0346) molecular phenotypes. Low RECQL1 expression was associated with shorter breast cancer-specific survival (P = 0.001). At the protein level, low nuclear RECQL1 level was associated with larger tumor size, lymph node positivity, high tumor grade, high mitotic index, pleomorphism, dedifferentiation, ER negativity, and HER-2 overexpression (P < 0.05). In ER+ tumors that received endocrine therapy, low RECQL1 was associated with poor survival (P = 0.008). However, in ER- tumors that received anthracycline-based chemotherapy, high RECQL1 was associated with poor survival (P = 0.048). In RECQL1-depleted breast cancer cell lines, we confirmed doxorubicin sensitivity, which was associated with DNA double-strand breaks accumulation, S-phase cell-cycle arrest, and apoptosis. We conclude that RECQL1 has prognostic and predictive significance in breast cancers. Mol Cancer Ther; 16(1); 239-50. ©2016 AACR.

Clinical and biological significance of RAD51 expression in breast cancer: a key DNA damage response protein

Impaired DNA damage response (DDR) may play a fundamental role in the pathogenesis of breast cancer (BC). RAD51 is a key player in DNA double-strand break repair. In this study, we aimed to assess the biological and clinical significance of RAD51 expression with relevance to different molecular classes of BC and patients' outcome. The expression of RAD51 was assessed immunohistochemically in a well-characterised annotated series (n=1184) of early-stage invasive BC with long-term follow-up. A subset of cases of BC from patients with known BRCA1 germline mutations was included as a control group. The results were correlated with clinicopathological and molecular parameters and patients' outcome. RAD51 protein expression level was also assayed in a panel of cell lines using reverse phase protein array (RPPA). RAD51 was expressed in the nuclei (N) and cytoplasm (C) of malignant cells. Subcellular co-localisation phenotypes of RAD51 were significantly associated with clinicopathological features and patient outcome. Cytoplasmic expression (RAD51C(+)) and lack of nuclear expression (RAD51N(-)) were associated with features of aggressive behaviour, including larger tumour size, high grade, lymph nodal metastasis, basal-like, and triple-negative phenotypes, together with aberrant expression of key DDR biomarkers including BRCA1. All BRCA1-mutated tumours had RAD51C(+)/N(-) phenotype. RPPA confirmed IHC results and showed differential expression of RAD51 in cell lines based on ER expression and BRCA1 status. RAD51N(+) and RAD51C(+) tumours were associated with longer and shorter breast cancer-specific survival (BCSS), respectively. The RAD51N(+) was an independent predictor of longer BCSS (P<0.0001). Lack of RAD51 nuclear expression is associated with poor prognostic parameters and shorter survival in invasive BC patients. The significant associations between RAD51 subcellular localisation and clinicopathological features, molecular subtype and patients' outcome suggest that the trafficking of DDR proteins between the nucleus and cytoplasm might play a role in the development and progression of BC.

TP53 Mutational Analysis Enhances the Prognostic Accuracy of IHC4 and PAM50 Assays.

IHC4 and PAM50 assays have been shown to provide additional prognostic information for patients with early breast cancer. We evaluated whether incorporating TP53 mutation analysis can further enhance their prognostic accuracy. We examined TP53 mutation and the IHC4 score in tumors of 605 patients diagnosed with stage I–III breast cancer at National Taiwan University Hospital (the NTUH cohort). We obtained information regarding TP53 mutation and PAM50 subtypes in 699 tumors from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort. We found that TP53 mutation was significantly associated with high-risk IHC4 group and with luminal B, HER2-enriched, and basal-like subtypes. Despite the strong associations, TP53 mutation independently predicted shorter relapse-free survival (hazard ratio [HR] = 1.63, P = 0.007) in the NTUH cohort and shorter breast cancer-specific survival (HR = 2.35, P = <0.001) in the METABRIC cohort. TP53 mutational analysis added significant prognostic information in addition to the IHC4 score (∆ LR-χ2 = 8.61, P = 0.002) in the NTUH cohort and the PAM50 subtypes (∆ LR-χ2 = 18.9, P = <0.001) in the METABRIC cohort. We conclude that incorporating TP53 mutation analysis can enhance the prognostic accuracy of the IHC4 and PAM50 assays.

Abstract 3264: IGF-axis protein expression and survival in Native Hawaiian breast cancer patients

Abstract Obesity-induced and related aberrations in insulin signaling may contribute to differences in breast cancer risk and outcome across racial/ethnic populations. The mechanisms underlying breast cancer disparities among Asian and Pacific Islander populations in the U.S. (specifically the high risk and poor survival of Native Hawaiians) are unknown. We examined the expression of IGF-axis proteins (IGF1, IGF1R, IGFBP2, and IGFBP3) in archived tumor tissue from 358 invasive breast cancer patients diagnosed in Hawaii. Overall and breast cancer-specific survival were evaluated via Kaplan-Meier curves and Cox's proportional hazards regression. Covariates included demographic and clinical factors, ER/PR/HER2 status, and for a subset of patients participating in the Multiethnic Cohort (MEC) (n=104), history of obesity, diabetes, and estrogen use at baseline. Across all cases, expression of the IGF-axis proteins in tumors did not differ by race/ethnicity. In the MEC subset, IGF1 expression was observed in 22% of estrogen users compared to 46% of non-users (p=0.04) and expression of IGFBP2 was observed in 71% of estrogen users compared to 93% of non-users (p=0.02). Protein expression did not vary by body mass index (BMI) or diabetes history. Obesity was most prevalent in Native Hawaiians (72%) and lowest in Japanese Americans (2%) (p&amp;lt;0.0001). History of estrogen use was highest in whites (73%) and lowest in Native Hawaiians (28%) (p=0.008). Across all MEC patients, IGFBP2 expression was associated with shorter breast cancer-specific survival after adjustment for age, stage, first-course of treatment, ER, PR, HER2, and estrogen use (HR=2.23, 95% CI 1.03-4.85). When examined by individual race/ethnic groups across all patients, overall and breast cancer-specific survival did not vary by IGF-axis protein expression for whites, Japanese, Filipinos, or Chinese. Among Native Hawaiians, IGFBP2 and IGFBP3 expression was associated with shorter overall survival when adjusted for age, stage, first-course of treatment, ER, PR, and HER2 (HR=10.04, 95% CI 2.13-47.50 and HR=3.40, 95% CI 1.05-10.99, respectively). In the MEC subset, the associations remained after additional adjustment for BMI (HR=16.30, 95% CI 2.84-93.40 and HR=4.64, 95% CI 1.30-16.59, respectively). IGFBP3 was also associated with a poorer breast cancer-specific survival in Native Hawaiian women when adjusted for age, stage, first-course of treatment, ER, PR, and HER2 (HR=2.79, 95% CI 1.12-6.93), and additional adjustment for BMI (HR=3.03, 95% CI 1.19-7.34). IGF1R expression was significantly inversely associated with overall survival among Native Hawaiians after adjustment for age, stage, first-course of treatment, ER, PR, HER2, and BMI (HR=14.46, 95% CI 2.26-92.57). In the analyses of individual race/ethnic groups, null cell sizes precluded the evaluation of estrogen use as a covariate. Aberrant insulin-signaling may influence outcomes in Native Hawaiian breast cancer patients. Citation Format: Brenda Y. Hernandez, Lynne R. Wilkens, Loic Le Marchand, David Horio, Clayton D. Chong, Lenora W. M. Loo. IGF-axis protein expression and survival in Native Hawaiian breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3264. doi:10.1158/1538-7445.AM2014-3264

Abstract P5-07-05: Oncogene PELP1 regulates alternative splicing in breast cancer through PRMT6

Abstract Background: Alternative splicing (AS) is essential in regulating gene expression and protein diversity; however, dysregulation leads to cancer development with tumor-specific splice variants that act by disrupting a tumor suppressor function. Protein arginine methylation is a modification involved in signal transduction, pre-RNA metabolism and transcriptional activation and deregulation of arginine methylase expression or functions can contribute to cancer progression via alternate splicing. PELP1 is a proto-oncogene, whose expression is associated with higher histological grade and shorter breast cancer specific survival, and PELP1 localization is a determinant of hormone sensitivity. This study focuses on examining the role of PELP1 in regulating gene expression and its possible role in alternative splicing. Methods: ZR75 and MCF7 cells stably expressing PELP1-shRNA or overexpressing PELP1 were used in RNASeq and ChipSeq analysis. Gene differential expression lists were generated using DEseq and PELP1 regulated pathways were analyzed using Ingenuity Pathway Analysis (IPA). Cell proliferation, migration and ERE reporter gene assays were used to test the role of PRMT6 in PELP1's oncogenic functions. Immunoprecipitation, confocal microscopy, ChIP, and GST pull down assays were used to demonstrate PELP1-PRMT6 interactions. Exon specific RTqPCR assays and CD44 minigene reporter assays were used to demonstrate PELP1's role in splicing. Results: RNA- and ChIP-sequencing results showed that the ERa coregulator PELP1 is a novel regulator of alternative splicing in breast cancer through the modulation of several genes involved in splicing including protein arginine methyltransferase 6 (PRMT6). PRMT6 plays a key role in coupling of transcription and alternative splicing by functioning as a transcriptional coactivator that can also regulate alternative splicing in a hormone independent manner. We discovered the novel complex of PELP1 and PRMT6 by co-immunoprecipitation. Biochemical assays revealed that PELP1 binds RNA, colocalizes with the splicing factor SC35 and promotes alternative splicing in reporter gene assays. PELP1 regulates the exon inclusion:skipping ratio of alternatively spliced exons in endogenous vascular endothelial growth factor (VEGF). PELP1's regulation of VEGF splicing is affected by the status of PRMT6. Interestingly, PRMT6 is needed for optimal oncogenic functions of PELP1. Further PELP1 has the potential to regulate the histone methyltransferase activity of PRMT6 and chromatin immunoprecipitation showed that PELP1 affects the enzyme recruitment and enrichment of the histone modification H3R2me2 at estrogen responsive genes. Conclusions: Our findings show a role of PELP1-PRMT6 axis in ERα-mediated RNA splicing and their deregulation has implications for targeted therapeutics in ERα-driven breast cancer. This work was supported by the NIH grant CA095681 and NIH F31 Fellowship 1F31CA173909-01A1. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-07-05.

Survival of patients with operable breast cancer (Stages I-III) at a Brazilian public hospital - a closer look into cause-specific mortality

BackgroundBreast cancer incidence is increasing. The survival rate varies and is longer in high-income countries. In Brazil, lower-income populations rely on the Unified Public Health System (Sistema Único de Saude, SUS) for breast cancer care. The goal of our study is to evaluate the survival of patients with operable breast cancer stages I-III at a Brazilian public hospital that treats mostly patients from the SUS.MethodsA cohort study of patients who underwent surgery for breast cancer treatment at the Clinical Hospital of the Federal University of Minas Gerais from 2001 to 2008 was performed, with a population of 897 cases. Information on tumor pathology and staging, as well as patients’ age and type of health coverage (SUS or private system) was collected. A probabilistic record linkage was performed with the database of the Mortality Information System to identify patients who died by December 31th, 2011. The basic cause of death was retrieved, and breast cancer-specific survival rates were estimated with the Kaplan-Meier method. The Cox proportional hazards model was used for univariate and multivariate analysis of factors related to survival.ResultsA total of 282 deaths occurred during the study’s period, 228 of them due to breast cancer. Five-year breast cancer-specific survival rates were 95.5% for stage I, 85.1% for stage II and 62.1% for stage III disease. Patients from the SUS had higher stages at diagnosis (42% was in stage III, and from the private system only 17.6% was in this stage), and in the univariate but not multivariate analysis, being treated by the SUS was associated with shorter survival (hazard ratio, HR = 2.22, 95% CI 1.24-3.98). In the multivariate analysis, larger tumor size, higher histologic grade, higher number of positive nodes and age older than 70 years were associated with a shorter breast cancer-specific survival.ConclusionsFive-year breast cancer survival was comparable to other Brazilian cohorts. Patients treated by the SUS, rather than by the private system, had shorter survival times, mostly due to higher initial stage of the disease.

Abstract P1-05-13: CLIC3 is associated with invasive behaviour and poorer prognosis in estrogen receptor-negative breast cancer.

Abstract Background: We previously described a Chloride Intracellular Channel-3 (CLIC3)-dependent recycling pathway which trafficked active integrins from late endosomes to the cell surface and which was required for the migratory and invasive behaviour of A2780 ovarian cancer cells in vitro. We also demonstrated that elevated expression of CLIC3 was associated with poor prognosis in pancreatic cancer. We therefore set out to investigate the role of CLIC3 in breast cancer. Materials and Methods: CLIC3 expression was assessed by immunohistochemistry and the weighted histoscore method in a tissue microarray (TMA) consisting of triplicate cores from 141 patients diagnosed with invasive estrogen receptor (ER)-negative early breast carcinoma between 1995 and 1998. Full clinicopathological and follow-up data were available. Further data were obtained from publicly available gene expression datasets (Desmedt, GSE7390) and Oncomine™. Knockdown of CLIC3 in the ER-ve MDA-MB231 breast cancer cell line was achieved by nucleofection of 2 different siRNA sequences (Dharmacon). Inverse invasion assays measured invasion into a plug of matrigel supplemented with fibronectin over 72 hours whilst organotypic invasion assays measured invasion into a fibroblast-containing collagen matrix over 6 days. Results: CLIC3 mRNA expression was significantly elevated in breast cancer in comparison to normal breast tissue in two independent data sets. High CLIC3 protein levels were associated with significantly shorter breast cancer specific survival (p = 0.026) in a TMA of 141 ER-ve patients. This finding was corroborated by the observation that high CLIC3 mRNA levels were associated with shorter overall survival in 198 patients in the Desmedt dataset (p = 0.038). Transient silencing of CLIC3 expression using two independent siRNA sequences had no effect on proliferation of MDA-MB231 cells but significantly reduced their invasiveness by 46% and 93% respectively in an inverse invasion assay and by 36% and 42% respectively in an organotypic invasion assay. CLIC3 was found to co-localise with Rab7 and LAMP1 in late endosomes/lysosomes in MDA-MB231 cells. Conclusions: Our clinical and in vitro data indicate an important role for CLIC3 in the invasive and metastatic behaviour of some breast cancers. Further work to elucidate molecular mechanisms is underway and will be presented. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-13.

The microRNA maturation regulator Drosha is an independent predictor of outcome in breast cancer patients

Drosha is a protein that plays a key role in the biogenesis of microRNAs which are well known to be deranged in human breast cancer (BC). The purpose of the current study was to assess the biological and prognostic value of Drosha protein expression in BC. Drosha protein expression was assessed immunohistochemically in two sets of BC: (1) full-face sections of selected BC series with distinct stages of tumour progression (Normal parenchymal cells, ductal carcinoma in situ (DCIS), primary invasive BC and nodal metastases) to evaluate its differential expression, (2) tissue microarray comprising a large and well-characterised series of unselected clinically annotated invasive BC to investigate its correlation with clinicopathological features and patient outcome. A gradual loss of Drosha cytoplasmic expression was observed along tumour progression from DCIS, to invasive and to metastatic cancer cells. In invasive BC, loss of Drosha cytoplasmic expression was associated with BRCA1 and ER expression and with shorter BC specific survival (BCSS), disease free interval (DFI) and distant metastasis free interval (DMFI). This correlation was maintained in ER negative, HER2 negative, triple negative and LN negative cases. Moreover, loss of cytoplasmic Drosha was predictive of better response to chemotherapy and endocrine therapy. This study provides evidence that Drosha protein potentially plays an important role in BC progression and assessment of its expression provides an independent predictor of patient outcome. These observations provide further evidence that alterations in miRNA regulation influence tumour behaviour.

Lack of expression of the proteins GMPR2 and PPARα are associated with the basal phenotype and patient outcome in breast cancer

Basal-like tumours (BP) are a poor prognostic class of breast cancer but remain a biologically and clinically heterogeneous group. We have previously identified two novel genes PPARα (positive) and GMPR2 (negative) whose expression was significantly associated with BP at the transcriptome level. In this study, using a large and well-characterised series of operable invasive breast carcinomas (1,043 cases) prepared as TMAs, we assessed these targets at the protein level using immunohistochemistry and investigated associations with clinicopathological variables and patient outcome. Lack of PPARα and GMPR2 protein expression was associated with BP, as defined by the expression of cytokeratin (CK) 5/6 and/or CK14, (p = 0.023, p = 0.001, respectively) or as triple-negative (ER-, PR-, HER2-) phenotype (p < 0.001 for both proteins). Positive expression of both markers was associated ER and PR positive status (p < 0.05) and with the good Nottingham Prognostic Index group (p = 0.012, p < 0.001, respectively). Univariate survival analysis showed an association between lack of expression of PPARα and GMPR2 and poor outcome in terms of shorter disease-free survival and shorter breast cancer-specific survival, respectively. However, multivariate analysis showed that these associations were not independent of other prognostic variables, namely tumour size, grade, and nodal stage. In conclusion, this study demonstrates that loss of expression of GMPR2 and PPARα is associated with BP at the protein level; indicating that they may play a role in carcinogenesis of this molecularly complex and clinically important subtype. Further studies into their relevance in further classification of BP are warranted.

Histone demethylase GASC1 - a potential prognostic and predictive marker in invasive breast cancer

BackgroundThe histone demethylase GASC1 (JMJD2C) is an epigenetic factor suspected of involvement in development of different cancers, including breast cancer. It is thought to be overexpressed in the more aggressive breast cancer types based on mRNA expression studies on cell lines and meta analysis of human breast cancer sets. This study aimed to evaluate the prognostic and predictive value of GASC1 for women with invasive breast cancer.MethodsAll the 355 cases were selected from a cohort enrolled in the Kuopio Breast Cancer Project between April 1990 and December 1995. The expression of GASC1 was studied by immunohistochemistry (IHC) on tissue microarrays. Additionally relative GASC1 mRNA expression was measured from available 57 cases.ResultsIn our material, 56% of the cases were GASC1 negative and 44% positive in IHC staining. Women with GASC1 negative tumors had two years shorter breast cancer specific survival and time to relapse than the women with GASC1 positive tumors (p=0.017 and p=0.034 respectively). The majority of GASC1 negative tumors were ductal cases (72%) of higher histological grade (84% of grade II and III altogether). When we evaluated estrogen receptor negative and progesterone receptor negative cases separately, there was 2 times more GASC1 negative than GASC1 positive tumors in each group (chi2, p= 0.033 and 0.001 respectively). In the HER2 positive cases, there was 3 times more GASC1 negative cases than GASC1 positives (chi2, p= 0.029). Patients treated with radiotherapy (n=206) and hormonal treatment (n=62) had better breast cancer specific survival, when they were GASC1 positive (Cox regression: HR=0.49, p=0.007 and HR=0.33, p=0.015, respectively). The expression of GASC1 mRNA was in agreement with the protein analysis.ConclusionsThis study indicates that the GASC1 is both a prognostic and a predictive factor for women with invasive breast cancer. GASC1 negativity is associated with tumors of more aggressive histopathological types (ductal type, grade II and III, ER negative, PR negative). Patients with GASC1 positive tumors have better breast cancer specific survival and respond better to radiotherapy and hormonal treatment.

Abstract 958: PELP1 regulation of mTOR Axis: Role in breast cancer progression and resistance

Abstract Hormonal therapy resistance is a major clinical problem. Emerging evidence suggests that estrogen (ER) participates in extra-nuclear signaling events in the cytoplasm / membrane and such actions may play a role in therapy resistance. Recent studies suggested that mTOR pathway play a critical role in ER-mediated cell proliferation and growth factor signaling crosstalk. Proline, Glutamic-acid and Leucine-rich Protein 1 (PELP1) participates in ER extra-nuclear actions. PELP1 is a prognostic indicator of shorter breast cancer specific survival and PELP1 expression is predominantly in the cytoplasm in a subset of breast tumors. The objective of this application is to test whether cross talk occurs between mTOR-PELP1 signaling axis and whether mTOR targeting drugs can be used to target PELP1 oncogenic functions leading to therapy resistance. We have tested this hypothesis using ER-positive breast cancer cells that over express PELP1 (MCF7-PELP1, ZR75-PELP1) and models cells lacking PELP1 (MCF7-PELP1 shRNA, ZR-75-PELP1 shRNA). Vector transfected ZR75 and MCF7 cells were used as controls. Rapamycin and AZD8055 were used as pharmacological inhibitors to block mTOR pathway. Over expression of PELP1 enhanced the estrogen and Heregulin mediated cell proliferation in breast cancer cells. Rapamycin (10−7M) or AZD8055 (10−8M) treatment significantly reduced PELP1 driven growth in both MCF7 and ZR75 cells that are treated with estrogen. Similarly, Rapamycin and AZD8055 treatment of MCF7 and ZR75 cells significantly reduced PELP1 mediated increase in cell growth of Heregulin stimulated cells. Mechanistic studies using yeast two hybrid screen and reciprocal immunoprecipitations demonstrated that PELP1 directly interacts with mTOR and PP2A. PELP1cyto model cells that uniquely express PELP1 in the cytoplasm, exhibit excessive activation of mTOR signaling pathway upon estrogen and growth factor stimulation. Over expression of PELP1 in the breast cancer model cells increased phosphorylation of mTOR axis components (phos-mTOR, -S6K, -4EBP1,-AKT). Further, Knock down of PELP1 with siRNA significantly reduced the activation of mTOR signaling components upon estrogen and Heregulin stimulation. Immunohistochenistry studies using xenograft tumor tissues with PELP1 overexpression and PELP1cyto driven tumors with PELP1 deregulation showed correlation of PELP1 expression with the activation of mTOR signaling components and that PELP1cyto driven tumors have excessive activation of mTOR signaling components. Combination therapy of Tamoxifen and Rapamycin or AZD8055, sensitized PELP1 deregulated cells to hormonal therapy. Our results suggest that PELP1 driven oncogenic functions involve PELP1 modulation of mTOR signaling and blockade of mTOR signaling render PELP1 driven tumors highly sensitive to therapeutic inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 958. doi:1538-7445.AM2012-958