ER-positive Tumors Research Articles

The Landmark Series: Neoadjuvant Endocrine Therapy for Breast Cancer.

Patients with estrogen receptor (ER)-positive breast cancer generally do not have significant response to neoadjuvant chemotherapy. In these patients, neoadjuvant endocrine therapy (NET) is an alternative for those who may benefit from tumor downsizing prior to surgery. This article reviews clinical trials that have defined the role of NET. Cumulatively, these trials demonstrate that NET is effective in downsizing ER-positive breast tumors. Aromatase inhibitors are preferred in postmenopausal patients. An aromatase inhibitor with ovarian suppression is effective in premenopausal patients. While trials to date have shown the effectiveness of NET to facilitate breast conservation, they have provided little data regarding optimal axillary management.

Distinct temporal trends in breast cancer incidence from 1997 to 2016 by molecular subtypes: a population-based study of Scottish cancer registry data

BackgroundWe describe temporal trends in breast cancer incidence by molecular subtypes in Scotland because public health prevention programmes, diagnostic and therapeutic services are shaped by differences in tumour biology.MethodsPopulation-based cancer registry data on 72,217 women diagnosed with incident primary breast cancer from 1997 to 2016 were analysed. Age-standardised rates (ASR) and age-specific incidence were estimated by tumour subtype after imputing the 8% of missing oestrogen receptor (ER) status. Joinpoint regression and age–period–cohort models were used to assess whether significant differences were observed in incidence trends by ER status.ResultsOverall, ER-positive tumour incidence increased by 0.4%/year (95% confidence interval (CI): −0.1, 1.0). Among routinely screened women aged 50–69 years, we observed an increase in ASR from 1997 to 2011 (1.6%/year, 95% CI: 1.2–2.1). ER-negative tumour incidence decreased among all ages by 2.5%/year (95% CI: −3.9 to −1.1%) over the study period. Compared with the 1941–1959 birth cohort, women born in 1912–1940 had lower incidence rate ratios (IRR) for ER+ tumours and women born in 1960–1986 had lower IRR for ER− tumours.ConclusionsFuture incidence and survival reporting should be monitored by molecular subtypes to inform clinical planning and cancer control programmes.

Evaluating the Relationship Between Dynamic Contrast-Enhanced MRI (DCE-MRI) Parameters and Pathological Characteristics in Breast Cancer.

Dynamic contrast-enhanced MRI (DCE-MRI) is used to evaluate tumor microvasculature. However, studies demonstrating an association between perfusion parameters derived from DCE-MRI and histopathologic characteristics are limited to a small set of histopathologic factors, and the results are inconsistent. To evaluate the relationship between DCE-MRI perfusion parameters and common histopathologic tumor characteristics used to predict angiogenesis and determine prognosis in breast cancer. Retrospective. In all, 105 breast cancer patients with invasive ductal carcinoma (122 lesions). 3.0T, turbo spin-echo (TSE) T1 -weighted, fat-suppressed T2 -weighted, TSE T2 -weighted, and dynamic unenhanced and contrast-enhanced 3D T1 high-resolution isotropic volume examination. One reviewer obtained perfusion parameters (Ktrans , kep , ve , and vp ) of each breast cancer from DCE MRI using the extended Tofts model with a fixed baseline T1 value and a population-based arterial input function. The relationship between DCE-MRI perfusion parameters and histopathologic tumor characteristics used to predict angiogenesis and determine prognosis was evaluated. Student's t-test, Mann-Whitney U-test, analysis of variance (ANOVA), and Kruskal-Wallis test were used. Triple-negative breast cancers exhibited higher Ktrans and kep than luminal cancers (P < 0.05). Estrogen receptor (ER)-negative tumors showed higher Ktrans than ER-positive tumors (P < 0.05). Progesterone receptor (PR)-negative tumors presented higher ve than PR-positive tumors (P < 0.05). Tumors with higher Ki-67 showed higher kep than tumors with lower Ki-67 (P < 0.05). P53-positive tumors exhibited higher Ktrans and kep than p53-negative tumors (P < 0.05). Higher histologic grade tumors (grade II/III) presented higher Ktrans , kep , vp (P < 0.05) than grade I tumors. Tumors with LVSI presented higher Ktrans and kep than tumors without LVSI (P < 0.05). Breast cancer presenting higher Ktrans and kep on DCE-MRI was associated with poor prognostic histopathologic factors. Therefore, pretreatment DCE-MRI perfusion parameters may be useful imaging biomarkers for the evaluation of tumor prognosis and angiogenesis. 3 TECHNICAL EFFICACY STAGE: 2.

Abstract C078: Racial/ethnic disparities in breast cancer survival by subtypes: The role of obesity

Abstract Background: Previous studies have established a strong association of higher body mass index (BMI) with increased risk of postmenopausal breast cancer (BC) but with decreased risk for premenopausal BC. Additionally, a handful of studies have found higher BMI at diagnosis to be associated with increased mortality for both ER positive and negative BC subtypes, but associations have been inconsistent. We re-examined associations of BMI with tumor subtypes and BC-specific death, and whether the disproportionate prevalence of obesity in non-Hispanic (nH) black women mediated racial disparities in survival. Methods: We included 6884 breast cancer cases from the MCBCR from 2001-2014. Continuous BMI was categorized as under/normal weight (10&amp;lt;bmi&amp;lt;bmi&amp;lt;bmi35). We ran age and race adjusted logistic regression models for associations of BMI as a nominal categorical variable with ER subtype overall and by menopausal status. We then modeled the hazard of BC death in Cox proportional hazard regression against race/ethnicity (nH Black versus nH White) while adjusting for age at diagnosis, before and after including BMI in our models. Survival analysis models were stratified on menopausal status and ER subtype. All analyses were conducted using STATA, v.15 (Stata Corp LLC, College Station, Texas). Results: nH Black women were less likely to be normal weight (8% vs. 15%) and more likely to be overweight (17% vs. 11%) compared to nH Whites, but there was no disparity in morbid obesity in this sample (50% vs. 50%). Higher BMI was not associated with prevalence of ER subtypes. Morbid obesity was associated with BC-specific death for both pre-menopausal (HR=3.55, 95% CI: 1.62, 7.79) and postmenopausal women (HR=2.02, 95% CI: 1.30, 3.17) and for both ER positive (HR=2.58, 95%CI: 1.55, 4.31) and negative tumor subtypes (HR=0.73, 95%CI: 0.93, 3.23). There was no evidence that BMI mediated BC survival disparities in any of the strata defined by menopausal status or ER subtype. However, the association of morbid obesity with BC death was much stronger for nH White (HR=3.81, 95%CI: 2.67, 5.44) than nH Black patients (HR=1.51, 95%CI: 0.98, 2.33). Among ER positive BC patients, the association of morbid obesity with BC death was even stronger for nH White patients (HR=4.54, 95%CI: 2.81, 7.32) but disappeared among nH Black patients (HR=1.10, 95%CI: 0.63, 1.93). Conclusions: In this clinical population, morbid obesity (defined here as a BMI&amp;gt;35) is very prevalent (50%) and it has negative implications for survival from a BC diagnosis regardless of menopausal status or subtype. Contrary to our expectation, the association of morbid obesity with BC death was considerably larger for nH White than for nH Black patients. Patients who are morbidly obese should be targeted for more detailed follow-up to improve our understanding of the mechanisms involved in the role of morbid obesity in BC death and how these mechanisms may differ for by race. &amp;lt;/bmi Citation Format: Carola T Sánchez Díaz, Garth H Rauscher. Racial/ethnic disparities in breast cancer survival by subtypes: The role of obesity [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr C078.

Prediagnostic 25-Hydroxyvitamin D Concentrations in Relation to Tumor Molecular Alterations and Risk of Breast Cancer Recurrence.

Although vitamin D inhibits breast tumor growth in experimental settings, the findings from population-based studies remain inconclusive. Our goals were to investigate the association between prediagnostic plasma 25-hydroxyvitamin D [25(OH)D] concentration and breast cancer recurrence in prospective epidemiologic studies and to explore the molecular underpinnings linking 25(OH)D to slower progression of breast cancer in the Nurses' Health Studies (NHS, N = 659). Plasma 25(OH)D was measured with a high-affinity protein-binding assay and a radioimmunoassay. We profiled transcriptome-wide gene expression in breast tumors using microarrays. Hazard ratios (HR) of breast cancer recurrence were estimated from covariate-adjusted Cox regressions. We examined differential gene expression in association with 25(OH)D and employed pathway analysis. We derived a gene expression score for 25(OH)D, and assessed associations between the score and cancer recurrence. Although 25(OH)D was not associated with breast cancer recurrence overall [HR = 0.97; 95% confidence interval (CI), 0.88-1.08], the association varied by estrogen-receptor (ER) status (P interaction = 0.005). Importantly, among ER-positive stage I to III cancers, every 5 ng/mL increase in 25(OH)D was associated with a 13% lower risk of recurrence (HR = 0.87; 95% CI, 0.76-0.99). A null association was observed for ER-negative cancers (HR = 1.07; 95% CI, 0.91-1.27). Pathway analysis identified multiple gene sets that were significantly (FDR < 5%) downregulated in ER-positive tumors of women with high 25(OH)D (≥30 ng/mL), compared with those with low levels (<30 ng/mL). These gene sets are primarily involved in tumor proliferation, migration, and inflammation. 25(OH)D score derived from these gene sets was marginally associated with reduced risk of recurrence in ER-positive diseases (HR = 0.77; 95% CI, 0.59-1.01) in the NHS studies; however no association was noted in METABRIC, suggesting that further refinement is need to improve the generalizability of the score. Our findings support an intriguing line of research for studies to better understand the mechanisms underlying the role of vitamin D in breast tumor progression, particularly for the ER-positive subtype. Vitamin D may present a personal-level secondary-prevention strategy for ER-positive breast cancer.

Potential roles of lymphovascular space invasion based on tumor characteristics provide important prognostic information in T1 tumors with ER and HER2 positive breast cancer

Patients presenting with lymphovascular space invasion (LVSI) had an absolute decrease in survival. In our present study, the potential roles of LVSI on tumor characteristics was explored to predict the difference in the prognosis of ER and HER2 positive T1 tumors. A total of 142 breast cancer patients diagnosed with ER+ and HER2+ tumors whose tumor size was ≤ 2cm were included in this analysis. One hundred forty-two patients were divided into four groups, group 1 (lymph nodes+ and LVSI+), group 2 (lymph nodes+ and LVSI-), group 3 (lymph nodes- and LVSI+), group 4 (lymph nodes- and LVSI-). Univariate and multivariate Cox proportional hazard models were used to identify independent prognostic factors and calculate the HR and 95% CI. Kaplan-Meier and Cox regression models were used to test the prognostic significance. LVSI positivity was significantly associated with patient age, menopausal status, tumor size, lymph node status, Ki67, PR, and tumor grade. In the univariate and multivariate model, LVSI, PR, and Ki67 were significantly associated with DFS, and LVSI, lymph node status, PR, and Ki67 were significantly associated with OS. LVSI was significantly related to increased risk of DFS and OS only in the PR-negative and low-positive subgroups. It was a prognostic factor for DFS but not for OS in women with low Ki67 and was associated with DFS and OS in high-Ki67 tumors. Furthermore, patients who presented with only LVSI had a significantly worse survival rate than those with lymph node metastasis without LVSI in small tumors. The presence of LVSI was highlighted as a variable significant to survival. In further clinical practice, patients with LVSI may need more intensive treatment in certain populations.

Targeting the IRE1-XBP1 axis to overcome endocrine resistance in breast cancer: Opportunities and challenges.

Estrogen receptor 1 (ESR1, which encodes estrogen receptor-alpha) is a key driver gene for the initiation and progression of hormone receptor-positive breast cancer. Estrogen receptor-alpha (ER) is expressed in up to 70% of cases, and patients are routinely treated with endocrine therapies. However, the development of resistance over time is common and occurs in one-third of ER-positive breast tumors, leading to disease progression and death. X-box binding protein 1 (XBP1), a key component of the unfolded protein response (UPR) and ER signaling pathway, generates a positive feedback regulatory loop that leads to increased expression of XBP1 and ER in luminal breast cancer. In this review, we highlight new insights into the mechanisms of crosstalk between XBP1 and ER signaling and its clinical implications. Next, we describe the key signaling nodes that play an important role in XBP1-mediated endocrine resistance in breast cancer. Further, we discuss XBP1 gene mutations in breast cancer and the role of these mutations in the emergence of endocrine resistance and response to treatment. Finally, we discuss the current state and future directions for targeting XBP1 in combination with standard endocrine therapy to improve clinical outcomes in endocrine-resistant breast cancer patients.

Phenotype variants of invasive breast cancer differ in associations between the primary tumor size and its Ki-67 value.

e12588 Background: Epidemiological studies report that Ki-67 values are higher in larger invasive breast cancers (IBC). This relation was investigated in 1040 consecutive IBC patients with various immunohistological phenotypes (ER+/-; HER2 0, 1+ to 3+), all treated at a single Croatian hospital. Methods: A scatter plot of 1040 Ki-67 values with their cancer diameter (in cms) have produced an LOWESS nonparametric curve that almost overlapped with the logarithmic function of the same data set (Ki-67% = 18.39+20.63*log(D), where D is a tumor diameter in cm). The next step in using this logarithmic function as a model, was to convert D values into log(D) for linear correlations, amenable to tests of significance. Results: Among all 1040 pts, only ER positive phenotype variants Ki-67% correlated with log(D): Among ER positive tumors, the HER2 expression has decreased the correlation significance and the slope of regression lines, suggesting that the HER2 presence increases Ki67% in small tumors, while larger tumors have Ki67% less augmented than among HER2 absent tumors. Thus the highest slope of the line was found among HER2 absent ER+ patients. Conclusions: Since only ER positive tumors have shown correlation of Ki67% with the log of their tumor size, tumor biology (estimated by Ki67%) of the ER negative IBCs seems unrelated with their size. This possibly suggests that the clinical course of ER negative IBC patients is less predictable due to some etiological distinction the ER positive and ER negative breast cancers. [Table: see text]

Statistical machine learning model to predict Oncotype DX risk category in women over age 50.

524 Background: The 21-gene Oncotype DX Breast Recurrence Score multigene assay (RS) identifies women with ER positive, HER negative, axillary node-negative breast cancer (BC) for whom chemotherapy provides no invasive disease-free survival benefit compared to endocrine therapy alone. International adoption of RS testing is limited by cost and resource availability. We created a supervised statistical machine learning model using standard clinicopathologic data to predict RS risk category in women &gt; 50 years old. Methods: From 2012 to 2018, women with ER positive, HER2 negative, pathologically node-negative BC of all ages were retrospectively identified from a prospective institutional database. Standard clinicopathologic data and RS were collected. Per institutional protocol, RS are ordered for all early-stage, ER positive tumors &gt; 5 mm. Data were randomly split into training (n=3755) and validation sets (n=1609). A random forest model with 500 trees was developed on the training set, then evaluated on the validation set. Model predictors included age, tumor size, histologic subtype, hormone receptor status, lymphovascular invasion, and overall grade. The model was used to predict RS category (low risk: RS ≤ 25, high risk: RS &gt; 25) in women &gt; 50 years old. Results: 5364 unique tumors in 5189 women were identified. 3731 (70%) of tumors were identified in women &gt; 50 years; median age was 63 years (IQR 57-69). In women &gt; 50, median tumor size was 12 mm (IQR 9-17). Most tumors were invasive ductal (79%), low or intermediate grade (79%), and LVI was absent in 82% of tumors. Median ER staining by IHC was 95%; 28% of tumors had negative or weakly positive PR staining (1-20%). The model correctly classified 96.8% of patients as low risk (95% CI: 95.7-97.7). Negative predictive value for identifying low risk category was also high (92.3%, 90.7-93.6). Sensitivity for identifying high-risk women was 44.7% (37.4-52.1) and positive predictive value was 67.2% (58.2-75.3). A classification table on the validation set includes tumors with complete data available, including predictors and RS. Conclusions: Our model was highly specific (96.8%) for identifying women &gt; 50 with RS ≤ 25 who do not benefit from adjuvant chemotherapy. This model may be utilized in lieu of RS testing if cost and availability are prohibitive. True RS &gt; 25 was not as well predicted. The model will be refined following pathologic review of discordant cases to reduce false negatives. [Table: see text]

The impact of estrogen receptor status on EGFR/TP53/CTNNB1 axis in the evolution of non-small cell lung cancer.

e21035 Background: The role of estrogen receptor status in the carcinogenesis of lung cancer remains elusive. A census of clonal and sub-clonal mutations has been defined through the analyses of evolutionary histories of cancers. We previously reported a prospective multicenter molecular epidemiology study (JME study; Kawaguchi T, J Clin Oncol 2016), which included the expression levels of estrogen receptors β (ER) using immunohistochemistry(IHC) and the mutational profile using next generation sequencing as well as solid smoking information and reproductive/ hormonal risk factors from the detailed questionnaire. Methods: Utilizing the data of the JME study, the impact of ER in lung cancer development was explored. All the patients underwent surgery. In 441 ever- and 435 never-smokers, ER were observed in 46.4% and 53.5%, respectively. The cancer-associated 72 gene mutations and 5 gene amplifications examined in this study included EGFR, SMAD4, APC, FBXW7, BRAF, STK11, PIK3CA, TP53, PTEN, KRAS, CTNNB1, NFE2L2, NF1, and MET. ALK fusion was detected by IHC. Patients were enrolled between July 2012 and December 2013, with follow up until November 30th, 2017.Cox proportional hazards models were used to assess the ER expression on relapse free survival (RFS) and overall survival (OS). A logistic regression model was applied to assess the impact of ER (positive vs. negative) on gene alterations, using sex, smoking history, age and stage as independent variables. Results: ER expression was significantly higher in never smokers (vs. ever smokers; p = 0.022) and earlier stage (stage I vs. II-IV; p = 0.002). Patients with ER positive tumors had a longer RFS than those with negative tumors (RFS rate at 4 years: 33.7 vs. 26.5%; p = 0.021), however, there was no significant difference in OS between the two groups (p = 0.108). In the impact of ER status on the gene alterations, mutations in EGFR (p = 0.003), TP53 (p = 0.007) and CTNNB1 (p = 0.027) were significantly associated with ER expression. Multivariate analysis showed that EGFR mutations (OR = 1.394, 95%CI: 1.029-1.890, p = 0.032) and CTNNB1 mutations (OR = 0.272, 95%CI: 0.087-0.853, p = 0.026) were significantly associated with ER expression, while there was a trend for significance with TP53 mutations (OR = 0.737, 95%CI: 0.537-1.011, p = 0.059). Conclusions: ER positive status triggered the clonal EGFR mutations and suppressed the sub-clonal mutations of TP53 and CTNNB1. It is suggested that ER plays a critical role in the alterations of EGFR/TP53/ CTNNB1 axis in lung cancer evolution.

Metastasis by semi-quantitative oestrogen receptor expression in stage I-III early diagnosed triple positive breast cancer patients.

e13031 Background: Some breast cancer subtype-specific demographics and histopathological characteristics can be associated with metastatic outcome. Semi-quantitative oestrogen receptor (sqER) is prognostic in ER-positive HER2-negative tumours. Here, we studied sqER for outcome in early diagnosed triple positive breast cancers (TPBC;ER+/PR+/HER2+). Methods: Retrospective monocentric study of all consecutive early diagnosed TPBC (July 2002 - Dec 2017). Immunohistochemistry (IHC) was used for ER, PR and HER2; IHC ≥1% ER/PR was pos; HER2 was FISH-confirmed. Low sqER was defined as Allred score &lt; 7/8. Local therapy was surgery +/- radiotherapy; systemic treatment was (neo)-adjuvant chemotherapy and trastuzumab and endocrine treatment. The clinicopathological variables we studied apart from sqER were age, menopausal status, BMI, parity, tumor grade and clinical stage at diagnosis. Variables were analysed by a Fishers Exact or Mann-Whitney U test. All reported p-values are two-sided. Cox regression was used to investigate howqER is associated with distant metastasis. Results: We included 415 female patients. 18% had low sqER; these were younger and more likely premenopausal at diagnosis; p &lt; 0.001. Other studied variables didn’t differ by sqER-expression. 7% developed metastasis after a mean follow up of 86 months. High sqER-TPBCs as compared to low sqER-TPBCs were less likely to metastasize (HR 0.42; 95 CI 0.19-0.91). Conclusions: Low sqER in TPBC, present in about a fifth of these tumours, is typically associated with younger age and premenopausal status at diagnosis. Low sqER TPBC are more likely to metastasize when compared to TPBC with high sqER levels. [Table: see text]

Survival after treatment for American Joint Committee on Cancer Stage III breast cancer in Ahmadu Bello University Teaching Hospital Zaria

Background: Breast cancer is the most common malignancy in women worldwide. Patients with American Joint Committee on Cancer Stage III breast cancer have been noted to have good response to neoadjuvant chemotherapy. The aim of this study is to evaluate survival of such patients after treatment at our hospital. Methods: A 5-year prospective study was conducted between January 2009 and December 2013. Patients had four courses of neoadjuvant chemotherapy (cyclophosphamide, adriamycin, paclitaxel), Auchincloss' modified radical mastectomy, two courses of adjuvant chemotherapy and adjuvant tamoxifen (premenopausal) or letrozole (post-menopausal) for ER-positive tumours. All patients had radiotherapy. Follow-up was on outpatient basis and through phone calls. Statistical analysis was done using SPSS version 21.0, survival analyses with Kaplan–Meier method, multivariate analyses done with P ≤ 0.05 regarded as statistically significant. Results: Data from 303 patients (291 females and 12 males) were analysed. One-year, 2-year, 3-year, 4-year and 5-year disease-free survival (DFS) was 79.80%, 56.57%, 51.51%, 47.47% and 47.47%, respectively. One-year, 2-year, 3-year, 4-year and 5-year overall survival (OS) was 80.80%, 61.61%, 55.56%, 51.51% and 51.50%, respectively. Initial tumour size, response to neoadjuvant chemotherapy, number of involved nodes, tumour grade and receptor status had statistically significant relationship with DFS and OS. The relationship between the patients' age, gender and both DFS and OS was not statistically significant. Conclusion: Five-year DFS and OS were 47.47% and 51.50%, respectively. Initial lesion size, response to therapy, involved nodes, tumour grade and receptor status had statistically significant relationship with DFS and OS.

Causal associations of thyroid function and dysfunction with overall, breast and thyroid cancer: A two-sample Mendelian randomization study.

Whether thyroid dysfunction plays a causal role in the development of cancer remains inconclusive. We conducted a two-sample Mendelian randomization study to investigate the associations between genetic predisposition to thyroid dysfunction and 22 site-specific cancers. Single-nucleotide polymorphisms associated with four traits of thyroid function were selected from a genome-wide association meta-analysis with up to 72,167 European-descent individuals. Summary-level data for breast cancer and 21 other cancers were extracted from the Breast Cancer Association Consortium (122,977 breast cancer cases and 105,974 controls) and UK Biobank (367,643 individuals). For breast cancer, a meta-analysis was performed using data from both sources. Genetically predicted thyroid dysfunction was associated with breast cancer, with similar patterns of associations in the Breast Cancer Association Consortium and UK Biobank. The combined odds ratios of breast cancer were 0.94 (0.91-0.98; p = 0.007) per genetically predicted one standard deviation increase in TSH levels, 0.96 (0.91-1.00; p = 0.053) for genetic predisposition to hypothyroidism, 1.04 (1.01-1.07; p = 0.005) for genetic predisposition to hyperthyroidism and 1.07 (1.02-1.12; p = 0.003) per genetically predicted one standard deviation increase in free thyroxine levels. Genetically predicted TSH levels and hypothyroidism were inversely with thyroid cancer; the odds ratios were 0.47 (0.30-0.73; p = 0.001) and 0.70 (0.51-0.98; p = 0.038), respectively. Our study provides evidence of a causal association between thyroid dysfunction and breast cancer (mainly ER-positive tumors) risk. The role of TSH and hypothyroidism for thyroid cancer and the associations between thyroid dysfunction and other cancers need further exploration.

OP1 “First do no harm”: Significance of delays to surgery in patients with non-metastatic breast cancer

Background The majority of patients in Australia with non-metastatic breast cancer will undergo primary surgery with curative intent. This involves many complex decisions that inevitably increase time from diagnosis to surgery. Current guidelines suggest surgery should occur within 30 days of a decision to treat1. However, there may be appropriate reasons to justify a delay to surgery. This study aims to analyse factors that contribute to an increased time to surgery (TTS) and establish whether the associated wait time is justifiable in the context of improved individualised breast cancer management. Methods This is a retrospective analysis of all patients at Austin Health surgically managed for non-metastatic invasive breast carcinoma between 20013 and 2019. TTS was defined as time between informed diagnosis and cancer surgery. Patients were categorised into TTS groups of ≤30 and >30 days. Kaplan-Meier survival analysis was used to evaluate the impact of TTS on survival outcomes. Results A total of 842 patients were included. Median number of days to surgery was 34 days. 43.9% of the total cohort received surgery within the recommended 30 days. Factors identified to be associated with an increased TTS were screening, transfer of care, ER positive tumour, mastectomy, immediate reconstruction and use of pre-operative imaging including MRI and staging scans. Median follow up for the cohort was 30 months. Between wait groups of ≤30 and >30 days, there were no significant association found between TTS and survival outcomes for DFS (HR 1.20 95% CI 0.56 to 2.60) and OS (HR 1.58 95% CI 0.82 to 3.03). Conclusions Breast cancer management involves many complex factors that significantly increases time from diagnosis to surgery. Surgery within 30 days of diagnosis is not associated with improved DFS and OS. Time delays associated with integral element of care should be used to guide a revision of current TTS recommendations. 1. Royal Australasian College of Surgeons. Elective Surgery Urgency Categories. 2019.

Abstract A85: Prediagnostic 25-hydroxyvitamin D concentrations in relation to tumor molecular alterations and risk of breast cancer recurrence

Abstract Although vitamin D inhibits breast tumor growth and development in experimental settings, the findings from population-based studies remain inconclusive. Our goals were to investigate the association between prediagnostic plasma 25-hydroxyvitamin D [25(OH)D] concentration and breast cancer recurrence in prospective epidemiologic studies and to explore the molecular underpinnings linking 25(OH)D to slower progression of breast cancer. Our study included 659 women with invasive breast cancer from the Nurses’ Health Studies (NHS) diagnosed from 1989 to 2010. Plasma 25(OH)D was measured with a high-affinity protein binding assay or a radioimmunoassay. We profiled transcriptome-wide gene expression in breast tumors using microarrays. Hazard ratios (HRs) of breast cancer recurrence were estimated from covariate-adjusted Cox proportional hazards models. We examined differential gene expression regulation in association with 25(OH)D with covariate-adjusted linear regression and employed pathway analysis. We derived a gene expression score for 25(OH)D, and further assessed associations between the standardized score and breast cancer recurrence. Although 25(OH)D was not associated with breast cancer recurrence overall (hazard ratio (HR)=0.96; 95% confidence interval (CI): 0.86, 1.08) (median follow-up=12.8 years), the association varied by estrogen receptor (ER) status (p for interaction=0.005). Importantly, among ER-positive stage I to III cancers, every 5 ng/ml increase in prediagnostic plasma 25(OH)D was associated with a 13% lowered risk of recurrence (HR=0.87; 95% CI: 0.76; 0.99). No association was observed in women with stage I to III ER-negative cancers (HR=1.07; 95% CI: 0.91; 1.27). Pathway analysis identified multiple gene sets that were significantly (FDR&amp;lt;5%) downregulated in ER-positive breast tumors of women with high prediagnostic 25(OH)D (≥30 ng/ml), compared to those with low levels (&amp;lt;30 ng/ml). These gene sets are primarily involved in tumor proliferation, apoptosis, migration and invasion, inflammation, and cancer cell metabolism. The standardized score of 25(OH)D signatures derived from these gene sets was marginally significantly associated with reduced risk of recurrence in ER-positive diseases (HR=0.77; 95% CI: 0.59, 1.01). Using TIMER, a mathematical algorithm to deconvolute tumor cellular heterogeneity, we found more infiltrated CD8+ T cells in ER-negative tumors as compared to ER-positive tumors, and this higher immune infiltration in ER-negative tumors may contribute to the observed heterogeneous response. Our findings support an intriguing line of research for future studies to better understand the biologic mechanisms underlying the role of vitamin D in breast tumor progression, particularly for the ER-positive subtype. Citation Format: Yujing Heng, Donghao Lu, Natalie DuPre, Kimberly Glass, Peter Kraft, David Feldman, Susan Hankinson, Kathryn Rexrode, Heather Eliassen, Rulla Tamimi, Cheng Peng. Prediagnostic 25-hydroxyvitamin D concentrations in relation to tumor molecular alterations and risk of breast cancer recurrence [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A85.

Abstract P5-04-18: Unfavourable tumour stromal immune cell infiltrates but not circulating immune cell profiles in Aboriginal women with breast cancer

Abstract Breast cancer (BrCa) in Aboriginal Australian women carries more than double the risk of death relative to non-Aboriginal Patients. This higher death is partially explained by factors such as higher stage at diagnosis, younger age, increased co-morbidities, remoteness, socioeconomic disadvantage, and access to culturally sensitive health services. Aboriginal women in a Western Australian cohort also more frequently had tumours with adverse biological features including high grade and luminal B or HER2-enriched phenotype. However, an unexplained detriment remained relative to non-Aboriginal women after accounting for these factors. The immune environment of a tumour as well as of the patient in general can impact on native tumour behaviour as well as response to treatment. Studies in many tumour types including meta-analyses in breast cancer correlate higher circulating neutrophil: lymphocyte ratios (NLR) and neutrophil:platelet ratios (NPR) with poorer outcome. Further to this, tumour infiltrating lymphocyte (TIL) levels also have prognostic significance, associating with improved outcome in triple negative and HER2 positive cancers receiving chemotherapy but with adverse outcomes in estrogen receptor positive (ER+) cancers, potentially through to links to anti-estrogen resistance. To further explore drivers of poor outcome in Aboriginal women we studied circulating NLRs and NPRs in a cohort of Indigenous women matched by remoteness of residence and age to non-Indigenous women in Western Australia. Considering the impact of incidental inflammatory conditions over time only levels from counts taken within 90 days either side of diagnosis were considered. Tissue sections where available were assessed for TILs by standard published methods. NLR was available on 214 patients. The median ratio for Indigenous patients (2.46) was not different to non-Indigenous patients (2.13). Considering patients with NLR &amp;gt; 3.0, a cut-off noted to have poorer prognosis in meta-analysis (ref), there was a stronger trend to higher relapse in non-Indigenous (HR 2.14, p=0.07) than Indigenous patients (HR =1.38, p=0.43) for higher NLR. High NLR was not more common in Indigenous relative to non-Indigenous patients (32% v 25%, p=0.37). Considering very high NLR, significantly more Indigenous women had NLRs &amp;gt;5.0 (4.8 v 6.3%, p=0.04). PLR was available on 225 patients. The median for Indigenous patients (126) was not different to non-Indigenous patients (130). Considering patients with PLR &amp;gt; 185, a cut-off noted to have poorer prognosis in meta-analysis (ref), there were non-significant trends to higher relapse in both Indigenous (HR 1.94, p=0.1) and non-Indigenous patients (HR =1.67, p=0.26) for higher PLR. However, high PLR was not more common in Indigenous relative to non-Indigenous patients (15.4 v 21.6%, p=0.24). Stromal TILs were available on 86 patients. Mean % TIL count was significantly higher in Indigenous women (10.7 v 6.6%, p=0.003). This difference was significant for ER+ (9.6 v 3.5%, p=0.004) but not ER- tumours (20.0 v 21.6%, p=0.79). Significantly more Aboriginal patients with ER positive tumours had &amp;gt;=10% TIL infiltrates (41.7 v 9.8%, p=0.0025) but not those with ER- cancers (70 v 40%, p=0.18). In summary, measured circulating immune or inflammatory profiles did not substantially differ between Aboriginal and non-Aboriginal breast cancer patients. In contrast, although numbers are small, ER+ tumours in Aboriginal women had a higher incidence of significant TIL infiltrates which may both contribute to poorer prognosis and predict for higher utility from immunotherapy. An expansion of the cohort is underway along with immune cell sub-typing and immune checkpoint molecule assessments. Citation Format: Andrew David Redfern, Jacinta J Fong, Lisa J Spalding, Sherene Loi, Benjamin Dessauvagie, Jennet Harvey, Katie Meehan. Unfavourable tumour stromal immune cell infiltrates but not circulating immune cell profiles in Aboriginal women with breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-18.

Abstract P1-10-08: Assessing immune biomarkers of response to anthracyclines in breast cancer

Abstract Background: Pathologists have long recognized that the interaction between immune and tumour cells is critical in the development and progression of breast cancer. Studies have demonstrated the presence of tumour-infiltrating lymphocytes (TILs) correlates with improved clinical outcome in breast cancer especially in the triple negative and HER-2 positive subtypes. TILs predict for improved response to certain therapies including chemotherapy and trastuzumab. The predictive value of TILs in ER positive tumours is less clear. It has been demonstrated the higher presence of the immune microenvironment is associated with a better prognosis and as a result a higher likelihood of benefit from chemotherapy and possibly from immunotherapy, whereas cold immune microenvironment carries greater risk of relapse and lower benefit from chemotherapy and possibly immuno-therapies. In this study, we evaluated whether TILs could be used to predict chemotherapy response and characterize the pre-existing tumour microenvironment (TME) using NanoString’s GeoMx Digital Spatial Profiling (DSP) platform. Methods: We assessed haematoxylin and eosin stained slides from the phase III BR9601 adjuvant breast cancer trial using software used in the international ring study 2 for standardized evaluation of TILs integrated in VMscope slide explorer. Evaluation of stromal TILs was based on international guidelines. NanoString’s DSP platform was used to analysis 256 patient samples from the BR9601 clinical trial. For analysis, region of interest were selected and compared for the TME (CD45+ve) and tumour rich (pan cytokeratin) in tissue microarrays. A panel of 56-antibodies were analysed in each ROI. Results: The mean TIL score in this cohort of patients was 15.58% (ranging from 0 to 66.67%). The presence of higher levels of TILs was significantly associated with ER negativity (p&amp;lt;0.001), high grade (p=0.01) and increased lymph nodal involvement (p=0.002). In multivariate analysis, patients whose tumours had medium/high levels of TILS expression had better DRFS (HR: 0.49, 95%CI 0.24-1.02, p=0.057) when treated with E-CMF than those treated with CMF alone. Highest levels of TILs were found in Basal and HER2-like tumours. A T-cell score was generated using the average expression of CD3, CD4 and CD8. The T-cell score was examined in both the tumour and TME. Using the T cell score it was apparent that the cohort had a range of immune “hot” and immune “cold” tumours. It was demonstrated that immune “hot” TME doesn’t not always correlate with immune “hot” tumour expression. Proteins that were most associated with T-cell exclusion (p&amp;lt;0.01) in the TME were Fibronectin, B7-H3, PTEN, ER-α, TGFB1, FAPα and CD34. This would indicate that these proteins are causal inhibitors of T-cell invasion. Conclusion: In conclusion, this study highlights the significance of assessing the entire tumour since TILs, tumour and stromal cells collectively engage in a complex interplay that contributes to disease development and progression. NanoString’s GeoMx DSP is a promising technology for multiplexed analysis. TILs, whether measured using automated software, or estimation by protein profiling, are predictive of chemotherapy benefit. Citation Format: Melanie Spears, Carsten Denkert, Sonia L Villagas, Nicola Lyttle, Linda Liao, Mary Anne Quintayo, Christopher J Twelves. Assessing immune biomarkers of response to anthracyclines in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-10-08.

Abstract P4-10-16: Reactive stroma and trastuzumab resistance in HER2-positive early breast cancer

Abstract Purpose We investigated the value of reactive stroma as a predictor for trastuzumab resistance in patients with early HER2-positive breast cancer receiving adjuvant therapy. Experimental Design The pathological reactive stroma and the mRNA gene signatures that reflect reactive stroma in 209 HER2-positive breast cancer samples from the FinHer adjuvant trial were evaluated. Levels of stromal gene signatures were determined as a continuous parameter, and pathological reactive stromal findings were defined as stromal predominant breast cancer (SPBC; ≥50% stromal) and correlated with distant disease-free survival (DDFS). Results Gene signatures associated with reactive stroma in HER2-positive early breast cancer (N=209), were significantly associated with trastuzumab resistance in estrogen receptor (ER)-negative tumors (HR=1.27 p-interaction=0.014 [DCN], HR=1.58, p-interaction=0.027 [PLAU], HR=1.71, p-interaction=0.019 [HER2STROMA, novel HER2 stromal signature]), but not in ER-positive tumors (HR=0.73 p-interaction=0.47 [DCN], HR=0.71, p-interaction=0.73 [PLAU], HR=0.84; p-interaction=0.36 [HER2STROMA]). Pathological evaluation of HER2-positive/ER-negative tumors suggested an association between SPBC and trastuzumab resistance. Reactive stroma did not correlate with tumor-infiltrating lymphocytes (TILs), and the expected benefit from trastuzumab in patients with high levels of TILs was pronounced only in tumors with low stromal reactivity (SPBC &amp;lt;50%). Conclusions Reactive stroma in HER2-positive/ER-negative early breast cancer tumors may predict resistance to adjuvant trastuzumab therapy. Citation Format: Amir Sonnenblick, Mali Salmon-Divon, Roberto Salgado, Noam Pondé, Sibylle Loibl, Carsten Denkert, Heikki Joensuu, Pirkko-Liisa Kellokumpu-Lehtinen, Amos Azaria, Sherene Loi, Stefan Michiels, Christos Sotiriou. Reactive stroma and trastuzumab resistance in HER2-positive early breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-16.

Abstract P4-10-29: Identifying novel molecular markers of response to trastuzumab in metastatic breast cancer

Abstract Background: The survival of women with HER2+ metastatic breast cancer (MBC) has improved with the introduction of the anti-HER2 antibody trastuzumab, but responses vary greatly among individuals. A predictive signature of response (and lack thereof) to trastuzumab may help personalize therapy for women with HER2+ MBC and enable the discovery of new “druggable” targets. Methods: In this retrospective cohort study, 256 patients with HER2+ MBC at the Sunnybrook Odette Cancer Centre were identified sequentially from January 1999 to December 2013 using the Cancer Care Ontario Registry. In total, 53 patients (21%) had archived metastatic tissue available for analysis, 29 (55%) of whom had samples obtained &amp;lt;3 years prior to the initiation of trastuzumab. Nanostring molecular testing was used to measure the expression of commonly known oncogenes and gene signatures using the Nanostring technologies nCounter Analysis System TM (Seattle, Washington, USA). Descriptive statistics were used to summarize patient results and prognostic scores. Patients were categorized into short-term survivors (died within 12 months) and long-term survivors (survived &amp;gt;30 months after starting trastuzumab). Results were compared between these two groups of patients using a Fisher’s exact test (dichotomous groups) and Wilcoxon rank sum tests (continuous scores). Univariable Cox regression models were evaluated using all patients, to evaluate if risk scores were prognostic of overall survival (OS). Statistical significance was set at the p=0.05 level and all tests were two-sided. Results: The median patient age was 60 years (interquartile range 53-65 years), 24% (n=7) had de-novo MBC and the majority of patients (52%) had ER or PR positive tumors. Among 29 included patients, the median OS was 23.4 months (95%CI 10.3-34.3 months); 10 patients died within 12 months and 10 patients survived &amp;gt;30 months after starting trastuzumab-based therapy. A median of 155 days (range 4-812) elapsed between metastatic biopsy and initiation of trastuzumab. The distribution of the Prosigna ROR Score, Basal Cor, HER2 Cor, Luminal A Cor, Luminal B Cor, Mammaprint Subtype/Risk Group, Oncotype Risk Score/Risk Group, IHC4 mRNA Risk Group and GGI Risk Score/Risk Group did not differ between the short- and long-term survivors. However, the Mammaprint Predictor Score was significantly higher [mean 0.34 (std 5.50) versus -4.95 (std 5.24), p=0.033] and the IHC4 Risk Score was significantly lower among long-term as compared to short-term survivors [mean 99.3 (std 67.1) versus 160.3 (std 40.9), p=0.044]. The association between molecular gene signatures and OS are described in Table 1. Conclusion: Several gene signatures were associated with OS in this exploratory analysis of women with HER2+ MBC who received trastuzumab-based therapy. The Mammaprint Predictor and the IHC4 Risk Scores differed among short- versus long-term survivors, warranting validation in an independent dataset. Table 1. Univariable Survival Results (n=29)ComparatorHR (95% CI)p-valueProsigna ROR Score/ 10 units1.23 (0.92, 1.65)0.17ProsignaHER2 vs Other1.25 (0.50, 3.13)0.64Basal Cor/ unit3.61 (1.10, 11.78)0.034Her2 Cor/ unit0.99 (0.15, 6.40)0.99Luminal A Cor/ unit0.22 (0.05, 0.95)0.042Luminal B Cor/ unit0.26 (0.06, 1.06)0.060Mammaprint Predictor Score/ unit1.10 (1.02, 1.19)0.015Mammaprint SubtypeHER2 vs Other0.85 (0.37, 2.00)0.72Mammaprint RiskHigh vs Low4.20 (1.37, 12.86)0.012Oncotype Risk Score/ 10 units1.27 (0.95, 1.70)0.11Oncotype Risk GroupHigh vs Intermediate4.83 (0.60, 39.07)0.14IHC4 Risk Score/ 10 units1.09 (1.00, 1.18)0.046IHC4 mRNA Risk GroupHigh vs Intermediate4.83 (0.60, 39.07)0.14GGI Risk Score/ 10 units1.63 (1.02, 2.61)0.043GGI Risk GroupHigh vs Low5.14 (1.13, 23.51)0.035 Citation Format: Katarzyna Joanna Jerzak, Danielle N Desautels, Phillip S Blanchette, Jane Bayani, Sharon Nofech-Mozes, Martin J Yaffe, Gregory Pond, Kathleen I Pritchard, John MS Bartlett. Identifying novel molecular markers of response to trastuzumab in metastatic breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-29.

Abstract P2-11-20: Endocrine resistance in breast cancer: A dynamic gene expression analysis approach reveals potential new responsible genes

Abstract While ER-targeted endocrine therapies are the most successful therapies for ER-positive breast cancer, tumor recurrence and endocrine-resistance happens in many of the cases. Despite efforts, the underlying genes and mechanisms of this acquired resistance remain largely unknown. Here, we utilized a recently developed pipeline for dynamic gene regulatory network analysis1to re-analyze the publically available datasets related to endocrine resistance in breast cancer in order to find new target genes related to endocrine resistance. The pipeline exploits the time-resolved expression pattern of genes to identify dynamic responsive genes (DRG) and group them into multiple modules based on the similarity in their dynamic activities, which leads us to identify more significant genes compared with previous gene expression analysis models that solely focus on expression value of the genes. Here, we analyzed a dataset of time-resolved gene expression during MCF-7 cell transition to endocrine-resistant cells by long-term deprivation of estrogen, which is an accepted model of endocrine resistance2. The dataset is publically available as GES20361 in the Gene Expression Omnibus (GEO) database. Based on their dynamic expression pattern over time, our method of analysis grouped genes into three main modules. Interestingly, the majority of well-studied genes in the literature related to endocrine resistance are part of one of these modules, while there are many genes such as GABPA, TFDP1, MCM7, MDC1, and FOXC1 that are potential novel targets for treatments against endocrine resistance. Module One included genes related to endocrine resistance, mainly those related to cell cycle, such as CCNA1, CCNA2, CCND1, and CDK family, and DNA replication and repair, such as MDC1 and PARP1, as well as EGFR. Consistent with the previous literature on endocrine resistant breast cancer cells, these genes all showed similar up-regulated expression patterns over time. Furthermore, our model confirmed the identified genes shown in the previous paper2 (POLD1, TERF1, RAF1, and E2F1) as part of Module One and as important genes in endocrine resistance regulation. Module Two was enriched for genes related to PI3K/AKT/mTOR signaling pathway, while Module Three mostly contained tyrosine kinase related genes, which not only confirms the previous findings related to the importance of these genes in acquiring endocrine resistance, but also indicates the difference between the expression patterns of these genes compare to cell cycle genes and those in Module One. Our analysis revealed that the genes in Module One continued their up-regulating trend beyond day 90, when the marked transition toward resistance happens and resistance phenotype starts to arise, while genes in Module Two got up-regulated before the 90 days and down-regulated afterward, suggesting different roles from the genes in Module One. Lastly, we studied the gene regulatory networks, transcription factors, and miRNAs upstream of the identified genes, and discovered several interesting targets, such as MiR-193b and MiR-16, which have integral parts in the regulation of our identified genes and thus can be of great importance toward endocrine resistance.