Node-negative Invasive Breast Research Articles

Can mammographic and sonographic imaging features predict the Oncotype DX™ recurrence score in T1 and T2, hormone receptor positive, HER2 negative and axillary lymph node negative breast cancers?

To determine whether mammographic or sonographic features can predict the Oncotype DX™ recurrence scores (RS) in patients with TI-II, hormone receptor (HR) positive, HER2/neu negative and node negative breast cancers. Institutional board review was obtained and informed consent was waived for this retrospective study. Seventy-eight patients with stage I-II invasive breast cancer that was HR positive, HER2 negative, and lymph node negative for whom mammographic and or sonographic imaging and Oncotype DX™ assay scores were available were included in the study Four breast dedicated radiologists blinded to the RS retrospectively described the lesions according to BI-RADS lexicon descriptors. Multivariable logistic regression was used to test for significant independent predictors of low (<18) versus intermediate to high range (≥18). Two imaging features reached statistical significance in predicting low from intermediate or high risk RS: pleomorphic microcalcifications within a mass (P=0.017); OR 8.37, 95% CI (1.47-47.79) on mammography and posterior acoustic enhancement in a mass on ultrasound (P=0.048); OR 4.35, 95% CI (1.01-18.73) on multivariable logistic regression. A mass with pleomorphic microcalcifications on mammography or the presence of posterior acoustic enhancement on ultrasound may predict an intermediate to high RS as determined by the Oncotype DX(TM) assay in patients with stage I-II HR positive, HER2 negative, and lymph node negative invasive breast cancer.

Towards optimal management of the axilla in the context of a positive sentinel node biopsy in early breast cancer.

The sentinel lymph node biopsy (SLNB) was initially pioneered for staging melanoma in 1994 and it has been subsequently validated by several trials, and has become the new standard of care for patients with clinically node negative invasive breast cancer. The focussed examination of fewer lymph nodes in addition to improvements in histopathological and molecular analysis has increased the rate at which micrometastases and isolated tumour cells are identified. In this article we review the literature regarding the optimal management of the axilla when the SLNB is positive for metastatic disease based on level 1 evidence derived from randomised clinical trials.

Abstract A124: Characterization of claudin-low breast cancers

Abstract Molecular profiling of human breast cancers has defined 5 molecular subtypes: luminal A, luminal B, HER2 over-expressing, basal-like and claudin-low. The claudin-low subtype was identified in 2007 and is characterized by low expression of claudin proteins, E-cadherin and markers of luminal differentiation, and is reported to be associated with expression of mesenchymal and cancer stem cell (CSC) markers. The prevalence of this subtype is reported to be between 7-14% and there are an excess of tumors with metaplastic and medullary-like features, an association with poor prognosis and evidence that they may be resistant to conventional chemotherapies. Using information from publicly available gene expression microarray data, we sought to identify immunohistochemical markers of the claudin-low subtype and to further describe the morphological features of claudin-low breast tumors and the overall survival characteristics of patients with these tumors, along with any associations between these tumors and CSC markers. Using the gene expression microarray datasets, we performed hierarchical clustering to assign a molecular subtype to the tumors. Differential gene expression analysis was used to identify genes that were significantly upregulated and downregulated between claudin-low tumors and other tumor subtypes as candidates for immunohistochemical markers for our formalin fixed paraffin embedded (FFPE) tumor cohort. We utilized 943 stage I or stage II, lymph node negative primary invasive breast cancers treated with breast conserving surgery and adjuvant radiation, which had FFPE tumor blocks available for tissue microarray construction. On the basis of IHC expression of ER, PR, HER2, Ki67, EGFR, CK5/6, Claudin proteins and E-cadherin, tumors were classified as luminal A, luminal B, HER2 over-expressing, basal-like or claudin-low. Kaplan-Meier methods were used to estimate overall survival, and Fisher's exact tests were used to compare tumor characteristics and expression of CSC markers (ALDH1, CD44hi/CD24low) between claudin-low and luminal A tumors. Claudin-low tumors comprised 8% of our cohort with an overall survival of 73.6% at a median follow up of 12 years, similar to that of basal-like and HER2 over-expressing subtypes. Compared to luminal A type tumors, the claudin-low tumors were statistically more likely to have circumscribed tumor margins. However, there was no statistically significant association between claudin-low subtype and the expression of CSC markers. The claudin-low subtype represents a minority of invasive breast cancers, however this group is characterized by poor prognosis, and as such the identification of these tumors is useful to determine treatment options in the clinical setting. Citation Format: Kay Dias, Anna Dvorkin-Gheva, Greg Pond, Mark Levine, Timothy Whelan, Anita Bane. Characterization of claudin-low breast cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A124.

NSABP B-47: A randomized phase III trial of adjuvant therapy comparing chemotherapy alone to chemotherapy plus trastuzumab in women with node-positive or high-risk node-negative HER2-low invasive breast cancer.

TPS1139 Background: Adjuvant trastuzumab trials in HER2+ breast cancer (BC) demonstrated a large reduction in recurrence and death. Central testing showed HER2 non-amplified participants derived similar benefit. Among HER2-amplified patients (pts), multiple studies showed no effect on benefit by degree of amplification. Blinded internal and external review confirmed the non-amplified nature of the HER2 normal group. Based on these findings, NSABP B-47, sponsored by the NCI, was activated January 2011 and is actively accruing. The study is NCI central IRB approved, open via the CTSU, and endorsed by SWOG, ECOG, and RTOG. Methods: Study: Chemotherapy treatment is by physician choice: The non-anthracycline regimen is TC (docetaxel 75 mg/m2, cyclophosphamide (C) 600 mg/m2) IV q 3 wks for 6 cycles; the anthracycline regimen is AC → WP (doxorubicin 60 mg/m2 and C 600 mg/m2 IV either q 3 wks or q 2 wks [investigator discretion] for 4 cycles → paclitaxel 80 mg/m2 IV wkly for 12 doses). Pts are randomly assigned to chemotherapy with or without trastuzumab for 1 year. Pts receive adjuvant radiation therapy and endocrine therapy, as clinically indicated. Detailed menstrual history, concurrent medications, weight changes, and biomarkers (estrogen, stress, inflammation), are being collected. Eligibility: Eligibility includes: node positive or high risk node negative BC pts; HER2 IHC 1+ or 2+ scores, but non amplified by FISH; normal cardiac, renal, and liver function. Detailed eligibility will be provided. Statistical Design: The primary aim is to determine whether the addition of trastuzumab to chemotherapy improves invasive disease-free survival (IDFS). 3,260 pts will be enrolled to provide statistical power of 0.9 to detect a 33% reduction in the hazard rate of IDFS using a one-sided alpha level of 0.025. Progress: Protocol was activated in January 2011. First pt was entered in February 2011. As of January 23, 2013, 1,416 of 3,260 (43.4 %) pts have been enrolled. Updated information on enrollment and study background will be provided. Support: NCI U10-12027, -37377, 69651, 69974, and Genentech, Inc. Clinical trial information: NCT01275677.

A prospective clinical utility and pharmacoeconomic study of the impact of the 21-gene Recurrence Score® assay in oestrogen receptor positive node negative breast cancer

PurposeThe primary purpose of this study was to measure the impact of the 21-gene Recurrence Score® result on systemic treatment recommendations and to perform a prospective health economic analysis in stage I–II, node-negative, oestrogen receptor positive (ER+) breast cancer. MethodsConsenting patients with ER+ node negative invasive breast cancer and their treating medial oncologists were asked to complete questionnaires about treatment preferences, level of confidence in those preferences and a decisional conflict scale (patients only) after a discussion of their diagnosis and risk without knowledge of the Recurrence Score. At a subsequent visit, the assay result and final treatment recommendations were discussed prior to both parties completing a second set of questionnaires. A Markov health state transition model was constructed, simulating the costs and outcomes experienced by a hypothetical ‘assay naïve’ population and an ‘assay informed’ population. ResultsOne hundred and fifty-six patients across two cancer centres were enrolled. Of the 150 for whom successful assay results were obtained, physicians changed their chemotherapy recommendations in 45 cases (30%; 95% confidence interval (CI) 22.8–38.0%); either to add (10%; 95% CI 5.7–16.0%) or omit (20%; 95% CI 13.9–27.3%) adjuvant chemotherapy. There was an overall significant improvement in physician confidence post-assay (p<0.001). Patient decisional conflict also significantly decreased following the assay (p<0.001). The simulation model found an incremental cost-effectiveness ratio of Canadian Dollars (CAD) $6630/quality-adjusted life years (QALY). ConclusionWithin the context of a publicly funded health care system, the Recurrence Score assay significantly affects adjuvant treatment recommendations and is cost effective in ER+ node negative breast cancer.

Discrepancy between routine and expert pathologists' assessment of non-palpable breast cancer and its impact on locoregional and systemic treatment

Histopathological parameters are essential for deciding on adjuvant treatment following breast cancer surgery. We assessed the impact of inter-observer variability on treatment strategy in patients operated for clinically node negative, non-palpable breast carcinomas. In the context of a multicenter randomised controlled trial, clinical and histological data of 310 patients with clinically node negative non-palpable invasive breast cancer were prospectively collected. Histological assessment of the primary tumour and sentinel nodes was first performed in a routine setting, subsequently central review took place. In case of discordance between local en central assessments, we determined the impact on locoregional and systemic treatment strategy. Discordance between local and central review was observed in 13% of the patients for type (kappa 0.60, 95% CI 0.50–0.71), in 12% for grade (k=0.796, 95% CI 0.73–0.86), in 1% for ER status (k=0.898, 95% CI 0.80–1.0), in 2% for PR status (k=0.940 95% CI 0.89–0.99). Discrepancy in the assessment of the sentinel node(s) was seen in 2% of the patients (k=0.954, 95% CI 0.92–0.98). Applying current Dutch Guidelines, central review would have affected locoregional treatment in 2% (7/310), systemic treatment in 5% (16/310) and both in 1% (2/310) of the patients. For the 9 (3%) patients in whom central review would have led to additional systemic treatment, Adjuvant! predicted 10 years mortality and recurrence rate would have decreased with a median of 4.6% and 15%, respectively. Discordance between routine histological assessment and central review of non-palpable breast carcinoma specimens and sentinel nodes was observed in 24% of patients. This inter-observer variation would have impacted locoregional and/or systemic treatment strategies in 8% of the patients.

Abstract P5-01-14: Discrepancy between routine and expert pathologists in histological assessment of early stage non palpable breast cancer and its impact on loco regional and systemic treatment

Abstract Background: Histopathological parameters are essential for deciding on further adjuvant treatment in patients with breast cancer after surgery. Here, we assessed the impact of inter observer variability on further treatment strategy in patients with clinically node negative, non palpable breast carcinomas. Methods: Clinical and histopathological data of 314 patients with clinically node negative non palpable invasive breast cancer were analysed. Histological assessment of the primary tumour and sentinel nodes was first performed in a routine setting, subsequently central review took place. In cases of discordance between local en central assessment, we determined the impact on locoregional and systemic treatment strategy. Results: Discordance between local and central review was seen in 13% of the patients (kappa 0.60 95% CI 0.50–0.71) for type, in 12% (k = 0.796 95% CI 0.73–0.86) for grade, in 1% for ER status (k = 0.898 95% CI 0.80–1.0), in 2% for PR status(0.940 95% CI 0.89–0.99). Discrepancy in the assessment of the sentinel node(s) was seen in 2% of the patients. (k = 0.954 95% CI 0.92–0.98). Practising current Dutch Guidelines, loco regional treatment would be affected in 2% (7/310), systemic treatment in 5% (16/310) and both in 1% (2/310) of the patients. For patients in whom central review would have led to additional systemic treatment (3%), the 10 years mortality and recurrence rate would decrease with a median of 4.6% and 15% respectively. Conclusion: Inter observer variation in the histopathological assessment of non-palpable breast carcinoma specimens and sentinel nodes results in a different loco regional or systemic treatment advice in 8% of the patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-01-14.

Abstract S2-3: Disparities in the utilization of axillary sentinel lymph node biopsy among black and white patients with node-negative breast cancer from 2002–2007.

Abstract Background: Disparities exist in many aspects of standard breast cancer treatment in certain patient populations. In the mid-1990s, axillary sentinel lymph node biopsy (SLNB) was introduced as an alternative to axillary lymph node dissection (ALND) for staging clinically node-negative breast cancer. During the early 2000s, the validity of SLNB was being determined and its technique was being disseminated throughout the surgical community. By the mid to late-2000s, SLNB had been shown to provide accurate axillary staging with lower complications and no difference in survival compared to ALND in node-negative patients. SLNB has now replaced ALND as the accepted method for staging early breast cancer. The purpose of this study is to examine differences in the utilization of SLNB in pathologic node-negative invasive black breast cancer patients compared to white patients as SLNB became standard axillary staging and whether this difference impacted patient complications. Methods: Using the population-based Surveillance, Epidemiology, and End Results (SEER)-Medicare data, cases of incident, non-metastatic, pathologic node-negative breast cancer in women age≥66 were identified. Patients were considered to have undergone SLNB if specified by SEER records or if a billing claim for axillary lymphatic mapping was identified. Unadjusted associations of SLNB with race were evaluated using the chi-square test. The Cochran-Armitage test evaluated trends over time. Multivariate logistic regression tested whether race was associated with the use of SLNB after adjustment for clinicopathologic factors. Five-year cumulative incidence of lymphedema assessed via ICD-9 diagnosis codes was measured using the Kaplan-Meier method. Adjusted proportional hazards regression evaluated assiciations of race and ALND with lymphedema risk. Results: Of 31,274 women identified, 1,767 (5.7%) were Black, 27,856 (89%) were White and 1,651 (5.3%) were of other/unknown race. SLNB was performed in 74% of white patients compared to 62% of black patients (P&amp;lt;0.001). Although use of SLNB increased by year for both black and white patients (P&amp;lt;0.001), a fixed disparity in the use of SLNB persisted through 2007. In adjusted analysis, black patients were 33% less likely than white patients to undergo SLNB (relative risk = 0.74, 95% CI 0.67-0.81; P&amp;lt;0.001). Five-year cumulative incidence of lymphedema was 11.4% in patients undergoing ALND vs. 6.3% in patients undergoing SLNB (adjusted HR 1.92, 95% CI 1.75-2.10; P&amp;lt;0.001). Overall, black race was also associated with a higher risk of lymphedema (adjusted HR 1.40; 95% CI 1.20-1.63; P&amp;lt;0.001). However, among patients undergoing SLNB, whites and blacks had similar risks of lymphedema (6.2% and 7.7%, respectively; P=0.08). Conclusion: Even with the increased use of SLNB and its acceptance as standard axillary staging for node-negative breast cancer patients, disparities persist in its underutilization in appropriate black patients compared to white patients by as much as 26%. This racial disparity in SLNB use translated to a higher risk of lymphedema for black patients. Improving surgeon practices, the multidisciplinary team approach, and patient education are important in optimizing the beneficial impact of SLNB and reducing complications from unnecessary ALNDs in all patients with early stage breast cancer. Future research is needed to delineate mechanisms underlying this persistent disparity and to identify strategies to mitigate it. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr S2-3.

Abstract P2-10-23: Lymphovascular Invasion (LVI) and Overall Survival in Node-negative and Node-positive Breast Cancer Patients: A Meta-analysis.

Abstract Introduction: LVI is an important prognostic factor in patients with lymph node-negative invasive breast cancer. The prognostic value of LVI in lymph node-positive disease or in other subgroups remains unclear. Here we present a meta-analysis of studies assessing the impact of LVI on overall survival (OS) in different subgroups of breast cancer patients. Methods: A published data meta-analysis was conducted. Studies reporting hazard ratios (HR) for the association of LVI with OS in a multivariable model were included. Those not reporting HR or utilizing only univariable analyses were excluded. HR and 95% confidence intervals (CI) were extracted or computed and pooled using the DerSimonian and Laird random effects model. Subgroup analyses were conducted for the differential effect of lymph node involvement, estrogen receptor (ER) status, extent of LVI and decade in which the study was conducted (pre 1990, 1990–1999 and 2000 onwards). Results: Twenty studies comprising 40,417 patients were included in the analysis. There was marked heterogeneity in the effect size between studies (Cochran Q p &amp;lt; 0.001, I2=62%). When all studies were pooled, LVI was significantly associated with worse OS (HR:1.70 95% CI 1.49–1.95, p &amp;lt; 0.001). Among studies selecting patients by lymph node status, the association between LVI and OS was similar for lymph node positive and negative patients (HR:2.03 and 2.41 respectively, subgroup difference p = 0.56). There were no significant differences based on ER-status (subgroup difference p = 0.75) or decade of study (subgroup difference p = 0.21). Pooled assessment of the effect of extent of LVI was not possible as this parameter was not consistently reported. Conclusion: LVI is associated with similarly detrimental OS regardless of lymph-node or ER status. Improvements in adjuvant therapies over the past 20 years do not appear to have diluted the impact of LVI on OS. The marked heterogeneity in the effect size may result from differences in the extent of LVI. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-23.

Abstract P1-01-08: A Nomogram for predicting two or less axillary lymph node involvement for breast cancer

Abstract BACKGROUND: Completion axillary lymph node dissection (ALND) is currently the standard of care in the event of a positive sentinel lymph node biopsy (SLNB). However, result from Z0011 indicate that women with a one or two involved axillary nodes and clinical T1-T2 tumors undergoing lumpectomy with radiation therapy followed by systemic therapy do not benefit from completion of ALND in terms of survival. As a result, the use of intraoperative frozen section during SLNB will be declined. OBJECTIVE: To define possible predictors of having two or less involved axillary node using preoperative image study to provide information for surgeons making decision about sparing intraoperative frozen section analysis of sentinel lymph node. METHODS: We reviewed the records of 1214 patients with clinical T1-T2 and clinical node negative invasive breast cancer. None of these patients were in situ cancer on initial gun biopsy nor received neoadjuvant chemotherapy. Factors associated with having three or more involved axillary nodes were evaluated by univariate and multivariate logistic regression analysis. RESULTS: Among 1214 patients, 203 patients (16.7%) had three or more positive nodes. On a multivariate analysis, having three or more positive nodes was associated with primary tumor size by breast US, axillary LN grade according to cortical thickness by US and presence of axillary LN enlargement on chest CT. The area under ROC curve corresponding to the multivariate logistic regression model applied to dataset of 1214 patients was 0.827 (95% CI: 0.793–0.860). When applied to a validation dataset of 378 patients, it was 0.899 (95% CI: 0.841–0.957). A nomogram to predict the probability of having two or less axillary nodes involvement based on patients' age and preoperative image findings was developed from the multivariate logistic regression model. CONCLUSION: Patients with a low probability of having three or more positive nodes can be identified from preoperative image finding. The nomogram developed will be helpful to surgeons making decision about sparing intraoperative frozen section analysis of sentinel lymph. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-01-08.

Vascular Invasion as an Independent Prognostic Factor in Lymph Node Negative Invasive Breast Cancer

Identification of simple and measurable prognostic factors is an important issue in treatment evaluation of breast cancer. The present study was conducted to evaluate the prognostic role of vascular invasion in lymph node negative breast cancer patients. in a retrospective design, we analyzed the recorded profiles of the 1,640 patients treated in the breast cancer department of Motahari clinic affiliated to Shiraz University of Medical Sciences, Shiraz, Iran, from January 1999 to December 2012. Overall and adjusted survivals were evaluated by the Cox proportional hazard model. All the hypotheses were considered two-sided and a p-value of 0.05 or less was considered as statistically significant. Mean age in lymph node negative and positive patients was 50.0 and 49.8 respectively. In lymph node negative patients, the number of nodes, tumor size, lymphatic invasion, vascular invasion, progesterone receptor, and nuclear grade were significant predictors. In lymph node and lymphatic negative patients, vascular invasion also played a significant prognostic role in the survival which was not evident in lymph node negative patients with lymphatic invasion. The results of our large cohort study, with long term follow up and using multivariate Cox proportional model and comparative design showed a significant prognostic role of vascular invasion in early breast cancer patients. Vascular invasion as an independent prognostic factor in lymph node negative invasive breast cancer.

Small node-negative (T1b-cN0) invasive hormone receptor-positive breast cancers: Is there a subpopulation that might have benefit from adjuvant chemotherapy?

The aims of the study were to identify a subpopulation more likely to be at greater risk of recurrence in small T1b-c node-negative hormone receptor (HR)-positive breast cancer, and which would benefit from adjuvant chemotherapy. Clinico-pathologic characteristics and clinical outcomes of 538 postoperative HR-positive T1b-cN0 breast cancer patients were retrospectively analyzed. High Ki67 index and a young age (< 35 years) were identified as independent risk factors for relapse (p < 0.0001 and 0.015, respectively). A nomogram based on Cox-regression model showed an area under the curve (AUC) of 0.73 in the training set. The validation set showed a good discrimination with an AUC of 0.65. In patients with high nomogram scores (≥ 100, n = 24, 4.5%) who had high Ki67 index with more than 75%, or young age (< 35 years) and a Ki67 index > 50%, the relapse-free survival curve of patients who had received anthracycline-containing adjuvant chemotherapy showed a better outcome than those who had not (p = 0.029). Ki67 index and age are valuable surrogate markers to predict recurrence and as indicators of tumors that could benefit from adjuvant chemotherapy in small T1b-c node-negative HR-positive breast cancer.

Prognosis in women with small (T1mic,T1a,T1b) node-negative operable breast cancer by immunohistochemically selected subtypes.

546 Background: Knowledge is limited about prognostic significance of breast cancer subtypes among women with small invasive node-negative breast tumors. Methods: We explored patterns of recurrence...

Abstract 318: Hierarchical clustering analysis of array CGH data identifies clinicopathologically distinct groups of lymph-node-negative invasive breast cancer

Abstract If high-risk group of lymph node-negative (pN0) invasive breast cancer is accurately identified by a molecular diagnosis of biopsy or surgically resected tumor specimens, it would be beneficial for choosing the most appropriate adjuvant or neoadjuvant therapies. In this study, we examined genomic copy-number alterations of the 800 cancer-related genes in 51 pN0 primary invasive breast cancers (20 relapsed and 31 non-relapsed cases) surgically resected from 51 patients at the NCCH, Tokyo, between 1990 and 1994. DNA isolated from acetone-fixed paraffin-embedded tissues using microdissection were labeled directly with fluorescence dyes and subjected to array-based comparative genomic hybridizaton (array-CGH). Although copy number differences between relapsed and non-relapsed cases were unclear, unsupervised hierarchical clustering analysis was by using these 800 cancer-related genes resulted in identification of three cluster groups with significant differences in clinical outcome. Group 1 was mostly (9 of 13) composed ER(−)/HER2(−) cases, group 2 comprised 9 ER(+)/HER2(−), 8 ER(+)/HER2(+), and 6 ER(−)/HER2(+) cases, and group 3 was mostly (12 of 15) composed of ER(+)/HER2(−) cases. By the log-rank test for overall survival (OS), group 1 showed significantly worse outcome than group 2 (p = 0.0138), but no significant difference was observed in relapse-free survival (RFS) between these groups (p = 0.17). Among 20 patients who suffered relapsed later, both OS and RFS rates of group 1 were significantly higher than those of group 2 (p = 0.0083 and 0.0018). Representative genes that amplified in group 1 were CCND2(12p13.32), CDKN1B(12p13.1), whereas those kind genes in group 2 were ERBB2(17q12), NGFR(17q21.33). This work demonstrates that hierarchical clustering of array-CGH data by using 800 cancer related genes can classify pN0 invasive breast cancers into clinically relevant groups and might pave the way to develop a new tool to estimate the risk of relapse and death in patients with pN0 breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 318. doi:10.1158/1538-7445.AM2011-318

MicroRNA-301 Mediates Proliferation and Invasion in Human Breast Cancer

Several microRNAs have been implicated in human breast cancer but none to date have been validated or utilized consistently in clinical management. MicroRNA-301 (miR-301) overexpression has been implicated as a negative prognostic indicator in lymph node negative (LNN) invasive ductal breast cancer, but its potential functional impact has not been determined. Here we report that in breast cancer cells, miR-301 attenuation decreased cell proliferation, clonogenicity, migration, invasion, tamoxifen resistance, tumor growth, and microvessel density, establishing an important oncogenic role for this gene. Algorithm-based and experimental strategies identified FOXF2, BBC3, PTEN, and COL2A1 as candidate miR-301 targets, all of which were verified as direct targets through luciferase reporter assays. We noted that miR-301 is located in an intron of the SKA2 gene which is responsible for kinetochore assembly, and both genes were found to be coexpressed in primary breast cancer samples. In summary, our findings define miR-301 as a crucial oncogene in human breast cancer that acts through multiple pathways and mechanisms to promote nodal or distant relapses.

D2-40/p63 defined lymph vessel invasion has additional prognostic value in highly proliferating operable node negative breast cancer patients

Phosphohistone H3 assessed proliferation has strong prognostic value. Lymph vessel invasion by D2-40 is also prognostic, but D2-40+ myoepithelial expression in small ducts completely filled by solid-pattern ductal carcinoma in situ can mimic lymphovascular invasion. As myoepithelial cells are also p63 positive, we have investigated whether lymph vessel invasion identified by combined D2-40/p63 is stronger prognostically than by D2-40 alone, and whether it has independent prognostic value to phosphohistone H3. In 240 operable T1–2N0M0 node negative invasive breast cancer patients <71 years, phosphohistone H3 was determined by quantitative immunohistochemistry and lymph vessel invasion by D2-40/p63 double immunostaining. Correlation analysis between the clinico-pathologic factors and lymph vessel invasion, and univariate and multivariate prognostic survival analysis were performed. With median 117 (range: 12–192) months follow-up, 36 patients (15%) developed and 28 (12%) died of distant metastases. Ten of the 61 patients (16%) with cancer cells surrounded by D2-40 were p63 positive and none of these ‘false lymph vessel invasion’ recurred. D2-40+/p63− lymph vessel invasion occurred in 51/239 (21%) cases and correlated with grade, mitotic activity index, phosphohistone H3, ER, cytokeratin14, and HER2. D2-40+/p63− lymph vessel invasion was strongly prognostic, but far more in women ≥55 than those <55 years (P<0.0001 and 0.04). With multivariate analysis, phosphohistone H3 proliferation was the strongest single prognosticator. Lymph vessel invasion had additional prognostic value to phosphohistone H3 only in women ≥55. This group of patients, without/with lymph vessel invasion, had 10-year survival rates of 83 and 50%, respectively (hazard ratio-lymph vessel invasion=3.0, P=0.04; hazard ratio-phosphohistone H3=6.9, P=0.002). Where age was <55 years, only phosphohistone H3 had independent prognostic value. Combinations of other features had no additional value. In conclusion, T1–2N0M0 invasive breast cancer patients ≥55 years with phosphohistone H3≥13, D2-40+/p63− defined lymph vessel invasion identifies a subgroup with a high risk of distant metastases.

Prognosis in women with small (T1mic,T1a,T1b) node-negative operable breast cancer by immunohistochemically selected subtypes

Knowledge is limited about prognostic significance of breast cancer subtypes among women with small invasive node-negative breast tumours. We explored patterns of recurrence in 1691 women with pT1mic/T1a/T1b, pN0 and M0 breast cancer according to four immunohistochemically defined tumour subtypes: (i) Luminal A (ER-positive, PgR-positive, HER2-negative and Ki-67 < 14%); (ii) Luminal B (ER-positive and/or PgR-positive, HER2-positive and/or Ki-67 ≥ 14%); (iii) HER2-positive, both endocrine receptors absent; and (iv) Triple Negative. At multivariate analysis, women with the Triple Negative breast cancer subtype had an increased risk of loco-regional relapse (LRR) (Hazards Ratio (HR) 3.58; 95%CI: 1.40-9.13) and breast cancer related events (HR 2.18; 95%CI: 1.04-4.57). Overall, Luminal B subtype was not associated with a statistically significant increased risk of recurrence compared with Luminal A, while patients with Luminal B subtype tumours overexpressing HER2 had a 2 fold risk of reduced breast cancer related survival (BCS), but not an increased risk of LRR and distant metastases. Women with HER2 breast cancer subtype had a statistically significant increased risk of LRR (HR 4.53; 95%CI: 1.56-13.1), distant metastases and reduced BCS (HR 3.22; 95%CI: 1.44-7.18) and overall survival (HR 2.87; 95%CI: 1.05-7.89) when compared with the Luminal A subtype, at multivariate analysis. In conclusion, women with small size, node-negative, breast cancer are at higher risk of relapse if with HER2-positive endocrine receptor absent or Triple Negative disease.

Can Features Evaluated in the Routine Pathologic Assessment of Lymph Node–Negative Estrogen Receptor–Positive Stage I or II Invasive Breast Cancer Be Used to Predict the Oncotype DX Recurrence Score?

Oncotype DX is a multigene reverse transcription-polymerase chain reaction assay used to quantify recurrence risk in patients with stage I or II estrogen receptor-positive, lymph node-negative invasive breast cancer. The results are reported as a Recurrence Score (RS). The 16 cancer genes evaluated include a proliferation set, hormone receptor set, and HER2 set. The activity of these genes is addressed by pathologic assessment of breast cancers. To determine if factors evaluated in pathologic evaluation of breast cancer could be used to predict Oncotype DX results. We studied 138 cases of invasive breast cancer for which Oncotype DX results and pathology data were available. Grading was performed by using Nottingham grading system. For hormone receptor immunostaining, 10% nuclear staining was considered a positive result. Oncotype DX RS was low in 81 cases, intermediate in 44 cases, and high in 13 cases. All 6 cases with both a negative progesterone receptor (PR) and a mitotic count score of 3 had a high RS. All 12 cases with both a negative PR and a mitotic count score greater than 1 had either an intermediate or high RS. Although Nottingham grade, PR status, mitotic count score, tumor size, and nuclear grade were each significantly associated with RS, in bivariate analyses the only variables that remained independently predictive of an intermediate or high RS score in a multivariate logistic regression model were negative PR and mitotic count score greater than 1. Our study suggests that a mitotic count score greater than 1 combined with a negative PR result, as determined by pathologic assessment, could serve as a marker for an intermediate or high Oncotype DX RS.

Effect of the novel oncomir MiR-301 on tumor proliferation and invasion in human breast cancer.

1076 Background: It is increasingly apparent that micro-RNAs contribute to biological progression in almost all human malignancies. Although several studies have reported the association of dysregulated miRs in human breast cancer, the specific role of miRs in mediating tumor invasion remain to be clarified. We have previously conducted global miR profiling on 74 lymph node negative invasive ductal breast cancer samples, and observed that miR-301 over-expression was strongly associated with either nodal or distant relapse. Hence, the objective of this study is to better understand the function of miR-301 in breast cancer biology, along with identification of its mRNA targets. Methods: MiR-301 expression was evaluated in four breast cancer cell lines. The biological effects of miR-301 knock down were examined in vitro and in vivo using cell viability, clonogenic, cell migration and tumor formation assays. Downstream target genes were also evaluated. Results: MiR-301 was over-expressed in all breast cancer cell lines. AntagomiR caused significant reduction in miR-301 expression, associated with reduced viability, decreased clonogenicity, cell migration and invasion, as well as suppressed tumor growth, demonstrating a strong oncogenic role for miR-301. A tri-pronged approach combining prediction algorithms, experimentally mRNA profiling, along with primary cancer Affymetrix mRNA data identified FOXF2, BBC3, and PTEN as potential targets for miR-301, which could explain the observed phenotypes of reduction. Luciferase reporter assays confirmed that these genes are direct targets of miR-301. In addition, miR- 301 is located within the intron of SKA2, a spindle-kinetochore complex, and these two genes were corroborated to be co-expressed in cancer cell lines as well as primary cancer tissues. Conclusions: We have identified miR-301 as a novel oncomir in human breast cancer, which promotes growth, proliferation, invasion, and metastases, mediated at least by FOXF2, BBC3, and PTEN. Furthermore, the co-expression of miR-301 with SKA2 likely further co-operatively increases genetic instability with failed spindle check-points, thereby accounting for the more aggressive breast cancer clinical behavior. No significant financial relationships to disclose.

Progesteron receptor status as a predictor of progression-free survival and overall survival in patients with early breast cancer.

677 Background: The frequency of small (pT1a,b,c) node negative invasive breast cancers (BC) is increasing because of wider implementation of BC screening. When cancer is detected early, patients (pts) show an overall better prognosis than pts with more advanced disease, however, even in this group a subset of pts will experience local or distant recurrences. The aim of our study was to evaluate in this subgroup of BC cases the association between disease-free survival (DFS), overall survival (OS) and pts clinicopathological characteristics. Methods: Six hundred twenty eight pts with early BC (pT1a,b,cN0M0) were observed between March 1996 and December 2007 (median follow-up 80 months - IQR 45-118) and were retrospectively investigated in our Institution. Tumours were classified histologically according to the WHO histological classification on BC. Tumour grading was assessed according to Elston and Ellis. Estrogen receptor (ER) and progesteron peceptor (PgR) status and HER2neu overexpression were evaluat...