Breast Tumor Characteristics Research Articles

Abstract P5-06-03: Effect of obesity on molecular characteristics of invasive breast tumors: Gene expression analysis of 405 tumors by BMI

Abstract Background: Obesity is a risk factor for breast cancer in postmenopausal women and weight gain after diagnosis is associated with decreased survival and less favorable clinical characteristics such as greater tumor burden, higher grade, and poor prognosis, regardless of menopausal status. Despite the negative impact of obesity on clinical outcome, the molecular mechanisms by which excess adiposity influences breast cancer are not well-understood. Methods: Affymetrix U133 2.0 gene expression data was available for 405 primary breast tumors and 20 tumor-adjacent adipose tissues; RNA was isolated from tumor epithelia or adipose using laser microdissection. Patients were classified as lean (BMI<25), overweight (BMI 25-29.9) or obese (BMI>30). Statistical analysis was performed by ANOVA using Partek Genomics Suite version 6.6. Results: Obese patients were significantly more likely to be diagnosed >50 years and to be of African American ancestry compared to lean or overweight women, while pathological characteristics including tumor stage, size or grade, lymph node status and intrinsic subtype did not differ significantly between groups. Principal component analysis could not effectively cluster the tumors by weight and no genes were differentially expressed using a false discovery rate <0.05. Using a less stringent unadjusted P-value of <0.01, 329 genes were differentially expressed, however, this gene expression profile could not discriminate patient tumors by BMI. In contrast, BMI was effective in clustering tumor-adjacent adipose samples by BMI based on differential expression of 156 genes. Conclusions: Although tumor epithelial cells from obese women do not differ significantly from those of lean and/or overweight women at the gene expression level, tumor-adjacent adipose did differ by weight. These data demonstrate that less favorable outcomes in obese patients are not be attributable to the tumor itself but to influences from the microenvironment and suggest that decreasing breast adiposity may be an effective strategy to reducing risk and improving outcomes in obese women. Citation Format: Ellsworth RE, Toro AL, Costantino NS, Shriver CD, Ellsworth DL. Effect of obesity on molecular characteristics of invasive breast tumors: Gene expression analysis of 405 tumors by BMI. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-06-03.

Abstract P1-07-06: Weight gain during pre- and postmenopausal years results in earlier onset of breast cancer. The Tromsø cohort study

Abstract Background: Obesity is both an independent risk factor, and a prognostic factor of postmenopausal breast cancer. In contrast, the association between premenopausal obesity/leanness, and subsequent weight gain and breast cancer outcomes, is still unclear. Furthermore, the association between adult weight gain, weight and age at diagnosis, and tumor characteristics is less studied. Methods: During 1979-2007, a total of 18 990 women, aged 18-87 years, answered questionnaires and underwent clinical examination at a total of five repeated health surveys (attendance rate 68-82%). Height and weight were measured at each survey, and before surgery, among those women diagnosed with breast cancer during follow-up. Careful review of the respective medical records, including histopathological workup, was performed. Multivariate Cox Proportional Hazard models were used to study the importance of Body Mass Index (BMI kg/m2) and weight change on breast cancer risk, and to evaluate variation in breast tumor characteristics. Results: During a median follow-up of 23.3 years, 579 women with invasive breast cancer were identified, and the cases were histologically verified. These breast cancer cases had a mean age at diagnosis of 56.3 years, and mean BMI at diagnosis of 25.3 kg/m2. Most (67 %) of the breast cancer patients had estrogen receptor (ER) positive tumors, 48 % had progesterone receptor (PgR) positive tumors, and 41 % had lymph node positive disease. We divided all participating women in three groups of weight change (< 5kg, 5-15 kg, >15 kg). When we compared women with less than 5 kg weight gain, to women with weight gain 5-15 kg, and to women with weight gain above >15kg, we observed a RR of 1.43 (95% CI 1.07-1.90) and a RR of 1.86 (95% CI 1.30-2.68), respectively, for postmenopausal breast cancer. We divided women by quartiles of BMI (kg/m2) at entry, and observed that women in the lowest quartile of BMI (≤ 21.45 kg/m2), who had a subsequent weight gain >15 kg, had a RR of 2.40 (95% CI 1.07-5.38) for postmenopausal breast cancer compared to women with the same BMI at entry, but who remained stabile in weight. We observed a 6 year difference in age at diagnosis for women diagnosed with breast cancer, who at study entry were in the same BMI group (< 25kg/m2), but subsequently either experienced a large weight gain (>15 kg), or remained stabile in weight (59.5 years vs. 64.4 years, p=0.007). Furthermore, we observed a 15 year difference in age at diagnosis for women diagnosed with breast cancer, who at study entry were in the same BMI group (≥ 25kg m2), but subsequently either experienced a large weight gain (> 15 kg), or remained stabile in weight (60.3 years vs. 74.9 years, p=0.007). Conclusion: Avoiding large weight gain during pre- and postmenopausal years may both protect against, and delay onset of postmenopausal breast cancer. Our findings support the importance of weight gain as a modifiable lifestyle factor for early onset of breast cancer. Citation Format: Lofterød T, Frydenberg H, Flote VG, Risberg T, Eggen AE, McTiernan A, Mortensen E, Wist EA, Akslen LA, Reitan JB, Wilsgaard T, Thune I. Weight gain during pre- and postmenopausal years results in earlier onset of breast cancer. The Tromsø cohort study. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-07-06.

Abstract P6-10-12: Volumetric breast density better predicts tumour characteristics associated with poor prognosis compared to visual BI-RADS

Abstract Purpose: Early detection of breast cancer through mammography screening reduces breast cancer mortality. To improve outcomes from screening, more than half of invasive cancers and a third of high grade cancers should be small at detection (<15mm), and more than 70% of all cancers should be node negative at diagnosis. Although breast density is associated with reduced mammographic sensitivity, it is unclear whether certain tumor characteristics associated with poorer prognosis are more prevalent in women with denser breasts. The study investigated associations between visually- or volumetrically-assessed breast density and tumor characteristics related to poor prognosis. Methods: Our IRB-approved study included 755 DCIS, invasive ductal or invasive lobular breast cancers diagnosed in women (aged over 40) between January 2009 and December 2012. Information on the patients' tumor characteristics including stage, size, receptor, grade and lymph node status was collected retrospectively. Women were excluded if they had a previous history of breast cancer or breast surgery, or if they were missing tumour size data or raw digital mammograms taken within 24 months of the cancer diagnosis. For women with multiple cancers, only the first diagnosed cancer was included. Breast density was assessed using visual BI-RADS density categories and Volpara Density Grades (VDG; an automated equivalent to 4th Edition BI-RADS, assigned using preset cut-offs of volumetric breast density: 4.5, 7.5, 15.5 and >15.5%). VDG was calculated from both breasts if the prior negative mammogram was available, and the contralateral breast for positive mammograms. Results: Overall, 55% of invasive tumors and 33% of grade 3 tumors were smaller than 15 mm and 83% were node negative. Mean tumor size increased significantly with increasing VDG (VDG 1 = 12.8, 2 = 14.7, 3 = 16.1, 4 = 20.4 mm, p<0.001) and increasing BI-RADS (1 = 12.2, 2 = 13.9, 3 = 16.8, 4 = 18.2 mm, p=0.01). The proportion of node positive tumors also increased significantly with increasing VDG (5.1%, 12.8%, 19.3%, 26.1%, p<0.001) and BI-RADS (0%, 10.6%, 19.5%, 26%, p=0.01). There was a significant increase in grade 3 tumors (11%, 16.1%, 21.7%, 21.7%, p =0.02) and HER-2 positive tumors (5.6%, 10.5%, 13.3%, 14.3%, p=0.02) with increasing VDG that was not seen with visual BI-RADS assessment (p=0.4). Increasing VDG was also significantly associated with increased proportions of larger (i.e. >15mm) node positive cancers (1.7%, 6.3%, 10.5%, 16%, p=0.004) that were not seen with BI-RADS (p=0.2). Conclusion: We found that tumour size and node status differed significantly with breast density as measured by VDG and visual BI-RADS. However, grade 3, HER-2 positive and large/node-positive cancers were all significantly associated with increasing VDG, but not BI-RADS density grade. Further research is needed to investigate whether automated volumetric breast density can be used to predict which women are more likely to be diagnosed with tumours that have poorer prognostic features. Citation Format: Johnston L, Chan A, Arieno A, Morgan R, Highnam R, Destounis S. Volumetric breast density better predicts tumour characteristics associated with poor prognosis compared to visual BI-RADS. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-10-12.

Abstract P2-12-12: MarginProbe device use and re-excision rates for breast conservation surgeries

Abstract Background: Current methods of intraoperative assessment of lumpectomy margins are limited. Previous studies have found a lower rate of re-excisions with the adjunctive use of the MarginProbe device (Dune Medical Devices Ltd, Israel). The purpose of this study was to compare the tumor characteristics and re-excision rates before and after the use of MarginProbe for patients who had breast conservation surgery (BCS) at our institution. Methods: The Breast Cancer Database of our medical center was queried for patients who underwent BCS from 1/2010-3/2015 by three breast surgeons. 2 surgeons used the MarginProbe to direct excision of additional margins at the time of primary lumpectomy surgery and 1 surgeon performed routine 6-surface cavity shavings. We compared our historical data (1/2010-12/2014) to MarginProbe data (1/2015-4/2015). The following variables were included: age, mammographic breast density, tumor characteristics, and re-excision rates. Statistical analyses were performed using Pearson's Chi-Square and Fisher's Exact Tests. Results: We had a total of 1201 women who had BCS among the 3 breast surgeons. The median age was 61 years. The median invasive size was 1.2 cm. Majority of cancers were early stage (stage 0, I), invasive ductal carcinoma (61%), ER-positive (86%), PR-positive (74%), and Her2Neu-negative (88%). These tumor characteristics were not statistically different in the pre- and post-MarginProbe groups. The majority of patients had dense breasts (51%) and density did not differ among the pre- and post-MarginProbe groups (p=0.86). For the surgeons who used the MarginProbe for margin assessment at the time of surgery, the re-excision rate fell from 17% to 0% and 35% to 20% during the 4-month period. In contrast, the surgeon who routinely performed 6-surface shavings had a re-excision rate that fell from 13% to 12% in the same time period. 88% of MarginProbe readings were false positive. There was one false negative reading. Table 1. Tumor CharacteristicsVARIABLESTotal N=1201%No MarginProbe (N=1144, 95%)%MarginProbe (N=57, 5%)%P-valueTUMOR STAGE0292242782414240.95I64454614543053 IIA, IIB22319211191221 IIIA, IIIB, IIIC39338312 IV30.330.300 HISTOLOGYDuctal carcinoma in situ305252892516280.75Invasive ductal carcinoma73061695613561 Invasive lobular carcinoma1129107959 Invasive other54553512 ESTROGEN RECEPTOR STATUSNegative16314157146110.46Positive101486963865189 PROGESTERONE RECEPTOR STATUSNegative307262942613230.56Positive86774823744477 HER2-NEU STATUSNegative788887538835810.22Positive92108610614 Equivocal19217225 Conclusions: Routine use of the MarginProbe device was associated with lower re-excision rates compared to historical data and concurrent 6-cavity shaving approach. Better intraoperative margin assessment and lower re-excision rates will decrease the burden of breast cancer on patients and the health care system and support the practice of breast conserving surgery. Citation Format: Schnabel F, Guth A, Axelrod D, Chun J, Schwartz S, Shapiro R. MarginProbe device use and re-excision rates for breast conservation surgeries. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-12-12.

DEVELOPMENT OF A MULTIPLEX ALLELE-SPECIFIC REAL-TIME PCR METHOD FOR DETECTION OF PIK3CA GENE SOMATIC MUTATIONS AND ITS VALIDATION IN THE TUMORS OF BREAST CANCER PATIENTS

Aim: To develop a highly sensitive real-time polymerase chain reaction (PCR) system for detection of somatic mutations in 542 and 545 codons of exon 9 and 1047 codon of exon 20 of PIK3CA gene comprising more than 80% of all somatic mutations in this gene for application on histological material without macroand microdissection, and to analyze associations between these mutations and clinical and pathological characteristics of breast tumors. Materials and methods: The Allele-specific real-time PCR method with signal detection by TaqMan probes was used. For determination of its analytical sensitivity, plasmids carrying the mutations studied were constructed by standard genetic engineering methods using mutagenesis. DNA samples carrying various mutated/wild type DNA ratios (5.0; 2.0; 1.0; 0.5, 0.25%) were prepared. Results: Multiplex allele-specific real-time PCR method for detection of most common mutations in PIK3CA gene: p.E542K c.1624G>A, p.E545K c.1633G>A, p.H1047R c.3140A>G, p.H1047L c.3140A>T – was developed and optimized. Analytical sensitivity of mutation detection comprised 0.5% for p.E542K and 0.25% for p.E545K, p.H1047R and H1047L enzyme. Conclusion: The method developed for detection of somatic mutations in PIK3CA gene is sufficiently sensitive, specific and efficient, that allows to propose it for a routine screening in clinical diagnostic laboratories for evaluation of disease prognosis and monitoring of response to therapy and its modification.

Serum irisin levels are lower in patients with breast cancer: association with disease diagnosis and tumor characteristics.

BackgroundIrisin is a recently discovered myokine, involved in the browning of white adipose tissue. To date, its function has been mainly associated with energy homeostasis and metabolism, and it has been proposed as a promising therapeutic target for obesity and metabolic diseases. This is the first study investigating the role of irisin in human breast cancer.MethodsParticipants included one hundred and one (101) female patients with invasive ductal breast cancer and fifty one (51) healthy women. Serum levels of irisin, leptin, adiponectin and resistin were quantified in duplicates by ELISA. Serum levels of CEA, CA 15–3 and Her-2/neu were measured on an immunology analyzer. The association between irisin and breast cancer was examined by logistic regression analysis. The feasibility of serum irisin in discriminating breast cancer patients was assessed by ROC curve analysis. Potential correlations with demographic, anthropometric and clinical parameters, with markers of adiposity and with breast tumor characteristics were also investigated.ResultsSerum levels of irisin were significantly lower in breast cancer patients compared to controls (2.47 ± 0.57 and 3.24 ± 0.66 μg/ml, respectively, p < 0.001). A significant independent association between irisin and breast cancer was observed by univariate and multivariate analysis (p < 0.001). It was estimated that a 1 unit increase in irisin levels leads to a reduction in the probability of breast cancer by almost 90 %. Irisin could effectively discriminate breast cancer patients at a cut-off point of 3.21 μg/ml, with 62.7 % sensitivity and 91.1 % specificity. A positive association with tumor stage and marginal associations with tumor size and lymph node metastasis were observed (p < 0.05, p < 0.01, p < 0.01, respectively).ConclusionsOur novel findings implicate irisin in breast cancer and suggest its potential application as a new diagnostic indicator of the presence of disease.

Mammographic and clinical characteristics of different phenotypes of screen-detected and interval breast cancers in a nationwide screening program.

In the context of a population-based screening program, we aimed to evaluate the major mammographic features and clinicopathological characteristics of breast tumors at diagnosis and the associations between them, focusing on tumors with the worst prognosis. We analyzed cancers diagnosed in a cohort of 645,764 women aged 45-69 years participating in seven population-based screening programs in Spain, between January 1, 2000 and December 31, 2006 and followed up until June 2009. We included all interval cancers and a sample of screen-detected cancers, whether invasive or in situ. We compared tumor-related information and breast density for different phenotypes (Triple-negative (TN), HER2+, Luminal B and Luminal A) in screen-detected and interval cancers. We used Chi-square or Fisher's exact test to compare major mammographic features of invasive versus in situ tumors, of screen-detected versus interval cancers, and of different types of interval cancers. We included 2582 tumors (1570 screen-detected and 1012 interval cancers). There were significant differences in the distribution of most clinicopathological variables between screen-detected and interval cancers. Invasive TN interval tumors were more common than other phenotypes in breasts with low mammographic density; three-quarters of these tumors presented as masses without associated calcifications. HER2+ tumors were more common in denser breasts and were associated with calcifications and multifocality. Architectural distortion was more common in Luminal A and Luminal B tumors. Certain radiologic findings are associated with pre-invasive lesions; these differ among invasive tumor phenotypes. We corroborate that TN and HER2+ cancers have distinctive appearances also in the context of population-based screening programs. This information can be useful for establishing protocols for diagnostic strategies in screening units.

Clinical Report on the First Prototype of a Photoacoustic Tomography System with Dual Illumination for Breast Cancer Imaging.

Photoacoustic tomography is a recently developed imaging modality that can provide high spatial-resolution images of hemoglobin distribution in tissues such as the breast. Because breast cancer is an angiogenesis-dependent type of malignancy, we evaluated the clinical acceptability of breast tissue images produced using our first prototype photoacoustic mammography (PAM) system in patients with known cancer. Post-excisionally, histological sections of the tumors were stained immunohistochemically (IHC) for CD31 (an endothelial marker) and carbonic anhydrase IX (CAIX) (a marker of hypoxia). Whole-slide scanning and image analyses were used to evaluate the tumor microvessel distribution pattern and to calculate the total vascular perimeter (TVP)/area for each lesion. In this clinical study, 42 lesions were primarily scanned using PAM preoperatively, three of which were reported to be benign and were excluded from statistical analysis. Images were produced for 29 out of 39 cancers (visibility rate = 74.4%) at the median depth of 26.5 (3.25–51.2) mm. Age, menopausal status, body mass index, history of neoadjuvant treatment, clinical stage and histological tumor angiogenesis markers did not seem to affect the visibility. The oxygen saturation level in all of the measured lesions was lower than in the subcutaneous counterpart vessels (Wilcoxon test, p value<0.001), as well as in the counterpart contralateral normal breast region of interest (ROI) (Wilcoxon test, p value = 0.001). Although the oxygen saturation level was not statistically significant between CAIX-positive vs. -negative cases, lesional TVP/area showed a positive correlation with the oxygen saturation level only in the group that had received therapy before PAM. In conclusion, the vascular and oxygenation data obtained by PAM have great potential for identifying functional features of breast tumors.

Diffusion-Weighted Imaging in 3.0 Tesla Breast MRI: Diagnostic Performance and Tumor Characterization Using Small Subregions vs. Whole Tumor Regions of Interest.

IntroductionApparent diffusion coefficient (ADC) values are increasingly reported in breast MRI. As there is no standardized method for ADC measurements, we evaluated the effect of the size of region of interest (ROI) to diagnostic utility and correlation to prognostic markers of breast cancer.MethodsThis prospective study was approved by the Institutional Ethics Board; the need for written informed consent for the retrospective analyses of the breast MRIs was waived by the Chair of the Hospital District. We compared diagnostic accuracy of ADC measurements from whole-lesion ROIs (WL-ROIs) to small subregions (S-ROIs) showing the most restricted diffusion and evaluated correlations with prognostic factors in 112 consecutive patients (mean age 56.2±11.6 years, 137 lesions) who underwent 3.0-T breast MRI.ResultsIntra- and interobserver reproducibility were substantial (κ = 0.616–0.784; Intra-Class Correlation 0.589–0.831). In receiver operating characteristics analysis, differentiation between malignant and benign lesions was excellent (area under curve 0.957–0.962, cut-off ADC values for WL-ROIs: 0.87×10−3 mm2s-1; S-ROIs: 0.69×10−3 mm2s-1, P<0.001). WL-ROIs/S-ROIs achieved sensitivities of 95.7%/91.3%, specificities of 89.5%/94.7%, and overall accuracies of 89.8%/94.2%. In S-ROIs, lower ADC values correlated with presence of axillary metastases (P = 0.03), high histological grade (P = 0.006), and worsened Nottingham Prognostic Index Score (P<0.05). In both ROIs, ADC values correlated with progesterone receptors and advanced stage (P<0.01), but not with HER2, estrogen receptors, or Ki-67.ConclusionsADC values assist in breast tumor characterization. Small ROIs were more accurate than whole-lesion ROIs and more frequently associated with prognostic factors. Cut-off values differed significantly depending on measurement procedure, which should be recognized when comparing results from the literature. Instead of using a whole lesion covering ROI, a small ROI could be advocated in diffusion-weighted imaging.

Adiposity is associated with p53 gene mutations in breast cancer.

Mutations in the p53 gene are among the most frequent genetic events in human cancer and may be triggered by environmental and occupational exposures. We examined the association of clinical and pathological characteristics of breast tumors and breast cancer risk factors according to the prevalence and type of p53 mutations. Using tumor blocks from incident cases from a case-control study in western New York, we screened for p53 mutations in exons 2-11 using the Affymetrix p53 Gene Chip array and analyzed case-case comparisons using logistic regression. The p53 mutation frequency among cases was 28.1 %; 95 % were point mutations (13 % of which were silent) and the remainder were single base pair deletions. Sixty seven percent of all point mutations were transitions; 24 % of them are G:C>A:T at CpG sites. Positive p53 mutation status was associated with poorer differentiation (OR, 95 % CI 2.29, 1.21-4.32), higher nuclear grade (OR, 95 % CI 1.99, 1.22-3.25), and increased Ki-67 status (OR, 95 % CI 1.81, 1.10-2.98). Cases with P53 mutations were more likely to have a combined ER-positive and PR-negative status (OR, 95 % CI 1.65, 1.01-2.71), and a combined ER-negative and PR-negative status (OR, 95 % CI 2.18, 1.47-3.23). Body mass index >30 kg/m(2), waist circumference >79 cm, and waist-to-hip ratio >0.86 were also associated with p53 status; obese breast cancer cases are more likely to have p53 mutations (OR, 95 % CI 1.78, 1.19-2.68). We confirmed that p53 mutations are associated with less favorable tumor characteristics and identified an association of p53 mutation status and adiposity.

Abstract 1514: Acquired differentiation and loss of malignancy of pulmonary metastases in breast cancer

Abstract Cancer stem cells (CSCs) or tumor initiating cells (TICs) were demonstrated in a leukemia transplant experiment in 1930s with a single mouse leukemic cell as well as in recent studies of human leukemia and solid tumors. Our work demonstrated that breast CSCs are able to mediate spontaneous lung metastasis in patient-derived breast tumor xenografts (PDXs) in vivo (PNAS, 2010). We further characterized the phenotypic and molecular characteristics of spontaneous metastases versus primary breast tumors. We collected the tumor cells from the lungs and conducted orthotopic implantation back into the mouse mammary fat pads in order to make a more metastatic tumor model. Surprisingly, relative to the parental primary breast tumors, the lung metastasis (met)-derived mammary tumors exhibited a slower growth rate and a reduced metastatic potential with a more differentiated epithelial status based on gene array analyses. The up-regulated genes in the met-derived tumors included promoters of cell adhesion and differentiation as well as tumor suppressors and cell cycle inhibitors, such as KRT19 (CK19), CEACAM1 (CD66a), and CDKN1A. Many of the down-regulated genes were promoters of self-renewal, cell motility, and cell cycle, such as EZH2, SMAD2, and CCNB1. Clinically, this signature correlated with breast tumor subtypes. Consistently, the differentiation scores of the lung met-derived tumors were significantly higher than the primary tumors (p = 0.007). We then examined the functions of microRNA candidates in regulating CSC-mediated metastasis and therapy resistance as well as their clinical relevance. By miRNA analyses, thirty-two miRNAs were differentially expressed with twenty-seven miRNAs being up-regulated in the met-derived tumors, including miR-200 family members which induce epithelial phenotype and inhibits self-renewal, miR-30 family members, miR-138, and others. Further clinical studies demonstrated that miR-30c is independently associated with favorable distant relapse free survival of breast tumor patients (GSE22216 n = 210). Subsequent mechanistic work characterized that miR-30c inhibits both CSC-mediated metastasis and chemotherapy resistance by a direct targeting of cytoskeleton genes twinfilin TWF1 and vimentin (VIM). We also identified miR-138 as a novel regulator of invasion and epithelial-mesenchymal transition in breast cancer cells, acting by directly targeting the polycomb epigenetic regulator EZH2. Notably, based on the UNC breast tumor database analyses (n = 337), the miR-138 activity signature served as a novel independent prognostic marker for patient survival beyond traditional pathologic variables, intrinsic subtypes or a proliferation gene signature. Both miR-30c and miR-138 can be regulated by the transcription factor GATA3. Our results highlight the loss of malignant character in some lung micro-metastatic lesions and the epigenetic regulation of this phenotype. Note: This abstract was not presented at the meeting. Citation Format: Huiping Liu. Acquired differentiation and loss of malignancy of pulmonary metastases in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1514. doi:10.1158/1538-7445.AM2015-1514

Abstract 2307: Targeted agents and chemotherapeutic treatments of genetically engineered Basal-like breast cancer mouse model

Abstract Background: Genomic characterization of human breast tumors has resulted in the identification of at least five distinct tumor subtypes (Luminal A and B, HER2-enriched, Claudin-low, and Basal-like). These subtypes each show their own unique biology, which correlates with distinct patient outcomes. Basal-like Breast Cancer (BLBC) is associated with high grade, BRCA1 mutation status, younger patient age, and increased frequency in African Americans. In order to identify novel therapeutic strategies for BLBC, we developed a genetically engineered mouse model based upon two common human BLBC genetic events, namely BRCA1 and TP53 loss. Results: We developed a genetically engineered mouse (GEM) model of breast cancer by inactivating TP53 and BRCA1 genes using Cre-LOX technology in the mammary gland (K14-cre / TP53 L/L / BRCA1 L/L). As part of the characterization of this GEM model, we performed gene expression profiling and DNA copy number analysis using aCGH arrays on the Basal-like tumors from this model (25/27 total tumors were Basal-like). We found a number of chromosomal regions that were similarly altered and found to be specific to mouse and human BLBC including gains of mouse chromosome 1q and 12q and losses in chromosomes 3F1 and 15q. Subsequently two tumors from the murine Basal-like group were taken and expanded into an orthotopic syngeneic model where they passaged in wild type FVB mice. These two tumors lines were then tested extensively with chemotherapeutics (Doxorubicin, Carboplatin, Cytoxan, Paclitaxel) and biologically targeted agents (MEK inhibitor/AZD6244, PI3K Inhibitor/BEZ235, PARP Inhibitor/ABT888 and mTOR Inhibitor/Everolimus). The DNA damaging agents exhibited exceptional efficacy (i.e. ABT888, Cytoxan, and Carboplatin), whereas the biologically targeted agents tended to at best, only slow tumor growth, even in combination. Conclusions: Genetic engineering of mice using TP53 and BRCA1 loss results in tumors of predominantly the Basal-like subtype phenotype. As expected, these tumors were exquisitely sensitive to DNA damaging chemotherapeutics including cytoxan and carboplatin, yet generally insensitive to the 3 tested kinase inhibitors. Novel combinations of targeted agents and chemotherapeutics are now being tested. Citation Format: Cristina M. Contreras, Grace O. Silva, Adam D. Pfefferle, Charles M. Perou. Targeted agents and chemotherapeutic treatments of genetically engineered Basal-like breast cancer mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2307. doi:10.1158/1538-7445.AM2015-2307

Abstract 3330: Genomic profiling of breast cancers in women with protracted exposure to low doses of radiation from the 1986 Chernobyl nuclear accident

Abstract Exposure to ionizing radiation is one of the only known environmental causes of breast cancer, but most evidence for this relationship is derived from studies of high-dose exposures delivered at high-dose rates. Radiation exposure at low doses protracted over time is a more common pattern, yet the association of such exposure with breast cancer risk is understudied. In a population-based case-control study of women living in Bryansk Oblast, Russia, exposed to relatively low-doses of radiation from the 1986 Chernobyl nuclear accident over about 20 - 30 years, we characterized 468 breast cancer patients (25 - 55 years old, mean = 46.7 years). Preliminary estimates of individual radiation doses to the breast from internal and external exposure to Chernobyl fallout ranged from 0.1 mGy to 279 mGy (mean = 6.8 mGy, median = 1.1 mGy). The majority of the breast cancer patients in this cohort had node-positive disease (60%), were ER/PR positive (77% and 67% respectively) and HER2 negative (74%). We performed global profiling of gene expression using the Illumina WG-DASL assay on 442 tumors and defined their intrinsic subtypes (Hu Z, et al., 2003). Among those tumors, 22% were Basal-like, 11% Her2-enriched, 30% Luminal A, 20% Luminal B, and 17% Normal-breast-like. We compared gene expression between the quartile of patients with the highest individual doses (N = 109, mean = 23.7 mGy, median = 16.5 mGy) and the lowest (N = 109, mean and median = 0.5 mGy) and identified genes differentially expressed between the groups. Although there is evidence in the literature from studies of women exposed to high doses of radiation at high-dose rates that suggests a positive association between basal-like cancer and radiation exposure, our preliminary analyses found a higher rate of basal-like cancers in the low exposure group (26.9%) compared with the high exposure group (19.2%). Triple negative cancers identified by IHC were also more common in the low than in the high exposure group (17.0% vs. 6.7%), though neither of these trends was significant. EGFR expression, a marker of basal-like cancers, was seen in 17.7% of tumors in patients with low exposure compared with 7.4% in the high exposure group (p = 0.07). Further, studies suggest radiation exposure prior to puberty is associated with specific tumor marker expression. In our study, 14.4% of the patients were prepubescent at the time of the Chernobyl accident (ATA). We observed a statistically significant association between Her2 and pubescence: patients who were prepubescent ATA were more likely to have Her2-positive tumors than those who were not prepubescent (40% vs 24%, p = 0.007). This unique study of a large cohort of women with protracted exposure to low doses of radiation provides one of the first opportunities to assess the effect of such exposure on the molecular characteristics of breast tumors. Citation Format: Jamie Guenthoer, Lynn Onstad, Xiaoyu Chai, Nikolai B. Rivkind, Irina V. Kurnasova, Vladislav P. Troshin, Margarita Makarova, Elena A. Korchagina, Valeriy F. Stepanenko, Irina Beluhka, Sergei M. Kulikov, Nikita E. Shklovsky-Kordi, Li Hsu, Paul Voillequé, Kenneth J. Kopecky, Scott Davis, Peggy L. Porter. Genomic profiling of breast cancers in women with protracted exposure to low doses of radiation from the 1986 Chernobyl nuclear accident. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3330. doi:10.1158/1538-7445.AM2015-3330

Abstract 3212: Metastatic breast cancer cell communication within a pro-dormancy bone marrow niche

Abstract Distant metastases are the major cause of death from breast cancer. In estrogen receptor positive (ER+) breast cancer (BC) in particular, metastatic relapse can occur years after initial treatment, suggesting that disseminated tumor cells have a prolonged dormant phase before becoming proliferative. A major site of early disease dissemination and metastatic relapse of ER+ BC is the bone, although the critical signals that allow circulating breast cancer cells (BCCs) to identify bone marrow (BM) vasculature, enter the tissue, and tether to the BM microenvironment (BMM) are little understood. Investigating these interactions of BCCs with the BMM, however, could enable us to target the key biologic determinants allowing safe harbor for metastases within bone. To address the features of breast tumors associated with late recurrence, but not confounded by variations in systemic treatment, we compiled breast tumor gene expression data from 4,767 patients and established a discovery cohort consisting of 743 lymph node-negative patients who did not receive systemic neoadjuvant or adjuvant therapy. Using this data set, we found that expression of E-selectin ligands and CXCR4 were significantly elevated in ER+ tumors that had late recurrences. Based on these observations, we used intravital fluorescence microscopy to study the precise functions of E-selectin ligands and CXCR4 (SDF-1 receptor) in tumor metastasis in in vivo xenograft models and found that these play distinct roles in ER+ BCC migration in the BMM. Using highly specific E-selectin (GMI-1271) and CXCR4 (AMD3100) inhibitors, we determined that E-selectin interactions were critical for BCC entry into bone, while CXCR4/SDF-1 interactions anchored BCCs to the BMM. CXCR4 inhibition led to significant mobilization of BC micrometastases from the BMM into the peripheral circulation. Moreover, we found that dormant and proliferating BCCs occupy distinct regions of the BMM, with dormant BCCs predominantly found in SDF-1 and E-selectin rich regions and thus highly susceptible to AMD3100 mobilization. These findings shed light on how dormant and proliferating BCCs interact with the BMM and suggest new interventions to break the foothold of dormant BC micrometastases in bone. Citation Format: Trevor T. Price, Clara H. Lee, Qing Cheng, H. Kim Lyerly, William E. Fogler, John L. Magnani, Dorothy A. Sipkins. Metastatic breast cancer cell communication within a pro-dormancy bone marrow niche. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3212. doi:10.1158/1538-7445.AM2015-3212

Accurate Prediction and Validation of Response to Endocrine Therapy in Breast Cancer.

Aromatase inhibitors (AIs) have an established role in the treatment of breast cancer. Response rates are only 50% to 70% in the neoadjuvant setting and lower in advanced disease. Accurate biomarkers are urgently needed to predict response in these settings and to determine which individuals will benefit from adjuvant AI therapy. Pretreatment and on-treatment (after 2 weeks and 3 months) biopsies were obtained from 89 postmenopausal women who had estrogen receptor-alpha positive breast cancer and were receiving neoadjuvant letrozole for transcript profiling. Dynamic clinical response was assessed with use of three-dimensional ultrasound measurements. The molecular response to letrozole was characterized and a four-gene classifier of clinical response was established (accuracy of 96%) on the basis of the level of two genes before treatment (one gene [IL6ST] was associated with immune signaling, and the other [NGFRAP1] was associated with apoptosis) and the level of two proliferation genes (ASPM, MCM4) after 2 weeks of therapy. The four-gene signature was found to be 91% accurate in a blinded, completely independent validation data set of patients treated with anastrozole. Matched 2-week on-treatment biopsies were associated with improved predictive power as compared with pretreatment biopsies alone. This signature also significantly predicted recurrence-free survival (P = .029) and breast cancer -specific survival (P = .009). We demonstrate that the test can also be performed with use of quantitative polymerase chain reaction or immunohistochemistry. A four-gene predictive model of clinical response to AIs by 2 weeks has been generated and validated. Deregulated immune and apoptotic responses before treatment and cell proliferation that is not reduced 2 weeks after initiation of treatment are functional characteristics of breast tumors that do not respond to AIs.

SU‐E‐J‐248: Contributions of Tumor and Stroma Phenotyping in Computer‐Aided Diagnosis

Purpose:To gain insight into the role of parenchyma stroma in the characterization of breast tumors by incorporating computerized mammographic parenchyma assessment into breast CADx in the task of distinguishing between malignant and benign lesions.Methods:This study was performed on 182 biopsy‐proven breast mass lesions, including 76 benign and 106 malignant lesions. For each full‐field digital mammogram (FFDM) case, our quantitative imaging analysis was performed on both the tumor and a region‐of‐interest (ROI) from the normal contralateral breast. The lesion characterization includes automatic lesion segmentation and feature extraction. Radiographic texture analysis (RTA) was applied on the normal ROIs to assess the mammographic parenchymal patterns of these contralateral normal breasts. Classification performance of both individual computer extracted features and the output from a Bayesian artificial neural network (BANN) were evaluated with a leave‐one‐lesion‐out method using receiver operating characteristic (ROC) analysis with area under the curve (AUC) as the figure of merit.Results:Lesion characterization included computer‐extracted phenotypes of spiculation, size, shape, and margin. For parenchymal pattern characterization, five texture features were selected, including power law beta, contrast, and edge gradient. Merging of these computer‐selected features using BANN classifiers yielded AUC values of 0.79 (SE=0.03) and 0.67 (SE=0.04) in the task of distinguishing between malignant and benign lesions using only tumor phenotypes and texture features from the contralateral breasts, respectively. Incorporation of tumor phenotypes with parenchyma texture features into the BANN yielded improved classification performance with an AUC value of 0.83 (SE=0.03) in the task of differentiating malignant from benign lesions.Conclusion:Combining computerized tumor and parenchyma phenotyping was found to significantly improve breast cancer diagnostic accuracy highlighting the need to consider both tumor and stroma in decision making.Funding: University of Chicago Dean Bridge Fund, NCI U24‐CA143848‐05, P50‐CA58223 Breast SPORE program, and Breast Cancer Research Foundation. COI: MLG is a stockholder in R2 technology/Hologic and receives royalties from Hologic, GE Medical Systems, MEDIAN Technologies, Riverain Medical, Mitsubishi, and Toshiba. MLG is a cofounder and stockholder in Quantitative Insights.

Role of diffusion weighted imaging and dynamic contrast enhanced magnetic resonance imaging in breast tumors

PurposeTo evaluate the role of diffusion weighted imaging and dynamic contrast enhanced magnetic resonance imaging in characterization of breast tumors and comparing the results with the histological finding. Patients and methodsFrom January 2011 to January 2015, 71 patients with 74 suspicious breast lesions had performed breast DCE-MRI combined with DWI and the results were compared with the histopathological examination which was used as the standard diagnostic method. ResultsThe study included 71 patients with 74 suspicious breast lesions, there were 38 benign lesions ((51.35%) and 36 malignant lesions (48.65%)).DCE-MRI proved to have a sensitivity of 91.7%, and a specificity of 84.2%. ADC cutoff value to differentiate between benign and malignant lesions was 1.32×103mm2/s (P<0.001). The diffusion weighted MRI proved to have a sensitivity of 94.4%, and a specificity of 92.1%.The combined MRI protocol of DCEMRI and DWI proved to increase the sensitivity and specificity of breast MRI. ConclusionDWI had a higher sensitivity and specificity than DCE-MRI. The combined MRI protocol of DCEMRI and DWI proved to increase sensitivity and specificity of MRI in diagnosis and differential diagnosis of breast lesions.

Abstract S2-04: Comprehensive molecular characterization of invasive lobular breast tumors

Abstract Invasive lobular breast cancer (ILC) is the second most common histological subtype of breast cancer accounting for 10-15% of invasive breast tumors. ILC is typically ER+ and beyond the known mutation and/or loss of E-cadherin function, which contributes to a highly discohesive morphology, little is known about the additional mechanisms driving ILC tumorigenesis, or alterations that differentiate ILC from invasive ductal carcinomas (IDC). Methods A dataset of 817 breast tumors from the TCGA Project, including 490 IDC, 127 ILC and 88 samples with a mixed IDC-ILC histology, were profiled on six genomic platforms to develop a comprehensive atlas of mutational, epigenetic, transcriptional and proteomic data. Integrative genomic analyses, both supervised and unsupervised, of ILC tumors and across histological subtypes were performed to identify genomic drivers of ILC oncogenesis. Results Comprehensive multi-platform analyses identified distinct molecular events associated with ILC tumors. As expected, lack of E-cadherin protein, as determined by Reverse Phase Protein Array (RPPA), and CDH1 mRNA expression was uniformly observed in ILC cases associated with distinct alterations targeting CDH1. In addition to previously reported CDH1 and PIK3CA mutations, we identified a number of novel ILC-enriched recurrent mutations targeting PTEN, RUNX1, TBX3, and FOXA1. An increased incidence of PTEN inactivating events, both mutations and copy number changes, were identified in ILC (13%) compared to IDC ER+ (7%), which corresponded with altered PTEN protein expression. These alterations were largely mutually exclusive with PIK3CA mutations and correlate with increased Akt activation as evident by increased Akt phosphorylation (pS473 and pT308), thus identifying a potential therapeutic opportunity for ILC patients. GATA3 signaling, which regulates epithelial cell differentiation, is frequently altered in luminal/ER+ breast cancers. Our analyses determined GATA3 mutations are more frequent in IDC luminal tumors as compared to ILC (19 % vs 5%). ILC luminal tumors show significantly lower GATA3 protein expression, but a higher frequency of mutations in FOXA1 (9% vs 2% in Luminal IDC), a transcription factor required to promote ER transcriptional programs. Within ILC tumors, FOXA1 mutations were found to cluster into a specific region of the Forkhead (FK) DNA binding domain. A broader analysis of FOXA1 mutations in breast and prostate cancer confirm two specific hotspots in the FK domain and the C-terminal transactivation domain. Interestingly, these mutational classes are associated with distinct transcriptional changes suggesting different functional effects. Finally, mRNA-seq analyses identified three robust molecular subclasses that are characterized by distinct genetic, genomic and proteomic patterns, including an increased immune-related group (Class 2), as well as differences in prognosis. Conclusions In this study, we developed a comprehensive atlas of genomic alterations that reveals key molecular differences differentiating ILC (FOXA1) from IDC (GATA3) tumorigenesis, a potential therapeutic target for ILC (Akt), and novel ILC subclasses based on underlying biological events. These findings provide further insight into the molecular heterogeneity of ER+ breast cancer. Citation Format: Giovanni Ciriello, Michael L Gatza, Katherine A Hoadley, Hailei Zhang, Suhn K Rhie, Reanne Bowlby, Matthew D Wilkerson, Cyriac Kandoth, Michael McLellan, Andrew Cherniack, Peter W Laird, Chris Sander, Tari A King, Charles M Perou. Comprehensive molecular characterization of invasive lobular breast tumors [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S2-04.

Variations in compliance to quality indicators by age for 41,871 breast cancer patients across Europe: A European Society of Breast Cancer Specialists database analysis

ObjectiveThe aim of this study is to assess age-specific compliance to quality indicators (QIs) regarding the treatment of breast cancer as defined by European Society of Breast Cancer Specialists (EUSOMA) for patients across Europe. MethodsAll patients entered into this study were affected by in situ or invasive breast cancer, diagnosed and treated between 2003 and 2012 at 27 Breast Units across Europe, who were entered into the EUSOMA database. Patients were categorised according to age; compliance to thirteen QIs was assessed for each age group and per time period (2003–2007 and 2008–2012). Compliance to QIs was tested by multivariable logistic regression models adjusted for breast unit, incidence year and tumour characteristics. ResultsOverall, 41,871 patients with a mean age of 59.6years were available for analysis. The highest compliance was reached for patients aged 55–64years and in the time period 2008–2012, whilst the lowest compliance was observed for women aged over 74 or under 40years and in the earlier time period. In multivariable logistic regression models, a significant difference between age categories was shown for 12 out of 13 QIs (P<0.001). Compliance to the QIs for patients aged ⩾75years was significantly lower when compared to patients aged 55–64years for ten QIs, whilst for patients in the youngest age group this was true for seven QIs. ConclusionIn conclusion, we found that among the 27 included breast units across Europe, compliance to QIs for breast cancer treatment is often lower in the youngest and oldest breast cancer patients, with a tendency to overtreatment in the youngest patients, and to under-treatment in the elderly.

Women with breast cancer in the Veterans Health Administration: demographics, breast cancer characteristics, and trends.

An increasing number of women are being cared for within the Veterans Health Administration (VA). However, the demographics and trends of women with breast cancer at the VA has not been documented. We describe the demographics and breast cancer characteristics of the 4445 women enrolled in the VA and reported to the Department of Veterans Affairs Central Cancer Registry diagnosed with breast cancer from 1995 to 2012. The cases of breast cancer per year increased over time to 365 in 2012. Black women represented only 16% of women diagnosed with breast cancer in the VA in 1995-1999 but increased to 25% by 2010-2012 (P<0.001). The median age at diagnosis in 1995-1999 was 58.4 and decreased to 56.8 by 2010-2012 (P<0.02). The fraction of breast cancers that were node negative was 45% in 1995-1999 and increased to 64% in 2010-2012; correspondingly, women presented at an earlier stage in more recent years (P<0.001). Urban women with breast cancer cared for within the VA are more likely to be younger (P=0.04) and nonwhite (P<0.001) compared with rural women, but the breast tumor characteristics appear similar. Oncology physicians at the VA must be prepared to care for breast cancer among women as the number of cases is growing. With only 365 women diagnosed with breast cancer at the VA as per year 2012 and nearly 150 treating VA facilities, the number of breast cancer patients seen by a particular physician could be quite low, and this fact suggests a need for an evaluation of the quality and outcomes of breast cancer care at the VA.