Node-positive Breast Carcinoma Research Articles

Spatial Transcriptomic Profiling Reveals Gene Expression Characteristics in Lymph Node-positive Breast Carcinoma.

Studies on the differences in gene expression characteristics according to lymph node metastasis in breast carcinoma are lacking. This study aimed to compare the spatially resolved transcriptomic profiles between node-positive and negative breast carcinomas. Eight patients with breast carcinoma were included, and digital spatial profiling and bioinformatic analysis were conducted to investigate the spatial transcriptomes. In the epithelial compartment, the top two most up-regulated genes were nuclear receptor subfamily 4 group A member 1 (NR4A1) and Jun proto-oncogene, activating protein-1 transcription factor subunit (JUN). The gene ontology (GO) enrichment analysis of the node-positive group revealed a significant up-regulation of genes associated with myeloid differentiation, mononuclear cell differentiation, and hematopoietic regulation. The gene set enrichment analysis (GSEA) revealed significant enrichment of gene sets associated with the regulation of inflammatory cytokines in both the epithelial and stromal compartments of the node-positive group. Our study highlights significant differences in gene expression profiles and spatially resolved transcriptional activities between node-positive and negative breast carcinomas. These findings underscore the importance of developing personalized treatment strategies for lymph node metastasis of breast carcinoma.

HER2-low metastases of HER2-negative primary tumors: a single institution analysis of intertumoral and internodal heterogeneity in node-positive breast cancer.

HER2 status in breast cancer is an essential parameter in individual therapeutic decision-making and is routinely assessed in primary tumors in accordance with international recommendations. Reports of HER2 heterogeneity raise the question of basing treatment decisions on HER2 status in metastases, if present. We investigated the degree and clinical implications of HER2 heterogeneity in lymph node-positive breast cancer. Because of recent recognition of therapeutic opportunities in this group of tumors, we especially focused on cases involving low-level HER2 expression. The HER2 status of primary tumors and of corresponding lymph node metastases was determined in archived material at the protein and gene levels using the gene- protein assay and interpreted in accordance with 2018 ASCO/CAP criteria. HER2-low status was defined as protein expression levels 1+ or 2+ with negative amplification status. We analyzed a series of 43 cases of primary infiltrating breast cancer, each with at least two axillary nodes harboring macrometastases (>2 mm), in total 206 such nodes. In 7% of cases, we detected intertumoral HER2 heterogeneity. Three of nine HER2-positive primary tumors were associated with HER2-negative metastases. No cases with HER2-negative primary tumors had HER2-positive metastases, but 55% (6/11) of HER2 0 primary tumors had HER2 1+ and/or 2+ metastases, and 19% (3/16) HER2 1+ cases had exclusively HER2 0 metastases. All metastases in HER2 2+ cases showed HER2-low protein expression levels. Internodal HER2 heterogeneity at low protein expression levels (presence of HER2 0, HER2 1+, and/or HER2 2+ metastatic deposits within the same axilla) was seen in 40% (17/43) of cases. We found no statistically significant association between HER2 heterogeneity and other tumor-related parameters. Survival data indicated worse outcomes in the HER2-low group compared with the rest of the cohort. Our results indicate a substantial instability of HER2 protein expression, leading to considerable intertumoral and internodal HER2 heterogeneity in lymph node-positive breast carcinomas. This heterogeneity is particularly relevant in HER2-low tumors in which the corrective effects of HER2 gene copy number analysis definitionally is absent. Our findings suggest that determining HER2 status in metastatic lymph nodes may generate relevant information for therapeutic decision-making.

Axillary Staging after Neoadjuvant Chemotherapy for Initially Node-Positive Breast Carcinoma in Germany: Initial Data from the AXSANA study.

Introduction To date, the optimal axillary staging procedure for initially node-positive breast carcinoma patients after neoadjuvant chemotherapy (NACT) has been unclear. The aim of the AXSANA study is to prospectively compare different surgical staging techniques with respect to the oncological outcome and quality of life for the patients. Little is known about current clinical practice in Germany. Material and Methods In this paper we analyzed data from patients enrolled in the AXSANA study at German study sites from June 2020 to March 2022. Results During the period under investigation, 1135 patients were recruited at 143 study sites. More than three suspicious lymph nodes were initially found in 22% of patients. The target lymph node (TLN) was marked in 64% of cases. This was done with clips/coils in 83% of patients, with magnetic seeds or carbon suspension in 8% each, and with a radar marker in 1% of patients. After NACT, targeted axillary dissection (TAD) or axillary lymphadenectomy (ALND) were each planned in 48% of patients, and sentinel lymph node biopsy alone (SLNB) in 2%. Clinically, the nodal status after NACT was found to be unremarkable in 65% of cases. Histological lymph node status was correctly assessed by palpation in 65% of patients and by sonography in 69% of patients. Conclusion At the German AXSANA study sites, TAD and ALND are currently used as the most common surgical staging procedures after NACT in initially node-positive breast cancer patients. The TLN is marked with various markers prior to NACT. Given the inadequate accuracy of clinical assessment of axillary lymph node status after NACT, it should be questioned whether axillary dissection after NACT should be performed based on clinical assessment of nodal status alone.

Abstract 92: tGLI1 and IL-6/IL-6R/GP130/STAT3 pathways cooperate to promote breast cancer stem cells and are co-activated in HER2-enriched and triple-negative breast cancers

Abstract Breast cancer is the most frequently diagnosed cancer and second leading cause of cancer-related deaths in women; 30% of primary breast cancer cases will develop distant metastases. The majority (90%) of breast cancer-related deaths are due to distant metastases. HER2-enriched breast cancer and triple-negative breast cancer (TNBC) have the highest propensity to metastasize and have limited to no treatment options. Metastatic HER2-enriched breast cancer and TNBC patients commonly develop therapeutic resistance and, therefore, lack efficacious therapies. Due to dismal prognoses in metastatic HER2-enriched breast cancer and TNBC patients, there remains an urgent need to identify novel molecular pathways for actionable therapeutic targets. Truncated glioma-associated oncogene homolog 1 (tGLI1), an alternatively spliced variant of GLI1, is a gain-of-function tumor-specific terminal effector in the Hedgehog pathway. tGLI1 promotes breast cancer growth and circulating tumor cells to the brain. More recently, our lab has reported tGLI1 physically interacts with Signal Transducer and Activator of Transcription 3 (STAT3); tGLI1 and STAT3 are co-overexpressed in node-positive HER2-enriched and triple-negative breast carcinomas. The Interleukin-6 (IL-6) signaling axis is a primary regulator of STAT3 and is frequently dysregulated and hyperactivated in multiple cancers, including breast cancer. IL-6 functions through a hexameric receptor complex comprised of IL-6 receptor α (IL-6R) and glycoprotein 130 (GP130) to recruit Janus tyrosine kinases (JAK). In turn, JAKs phosphorylate STAT3 resulting in homodimerization, nuclear translocation, and transcriptional activity. Independently, tGLI1 and IL-6/IL-6R/GP130/STAT3 pathways have been previously investigated as mediators for breast cancer metastases; however, whether they cooperate to promote breast cancer progression remains unknown. Herein, we report tGLI1 and IL-6/IL-6R/GP130/STAT3 pathways to be frequently co-expressed and co-activated in HER2-enriched breast cancer and TNBC tumors and that the co-activation is associated with a shortened metastasis-free survival (MFS). Additionally, when stratified by metastatic site, breast tumors with high tGLI1 and IL-6/IL-6R/GP130/STAT3 co-activation are associated with a worse bone, brain, and lung MFS compared to tumors with low co-activation. Ectopic co-overexpression of tGLI1 and GP130 upregulates mRNA expression of stemness genes, Nanog, Sox2, and Oct4, leading to the subsequent promotion of the breast cancer stem cell (CSC) subpopulation in vitro. Taken together, our findings revealed a novel crosstalk between tGLI1 and the IL-6/IL-6R/GP130/STAT3 signaling axis to enhance the CSC subpopulation, suggesting the importance of the crosstalk in mediating metastasis of HER2-enriched breast cancer and TNBC. Citation Format: Sara G. Manore, Daniel L. Doheny, Dongqin Zhu, Grace L. Wong, Angelina T. Regua, Austin Arrigo, Calvin J. Wagner, Mariana Najjar, Hui-Wen Lo. tGLI1 and IL-6/IL-6R/GP130/STAT3 pathways cooperate to promote breast cancer stem cells and are co-activated in HER2-enriched and triple-negative breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 92.

Axillary ultrasonography combined with pre-operative wire localisation of clipped node in nodal restaging after neoadjuvant chemotherapy in node positive breast cancer patients: a pilot study.

To evaluate the role of axillary ultrasonography (axUS) and ultrasound-guided pre-operative wire localisation of pre-treatment positive clipped node (CN) for prediction of nodal response to neoadjuvant chemotherapy (NACT) in node positive breast carcinoma patients. A prospective study was conducted between June 2018 and August 2020 after Ethics Committee approval. Breast carcinoma patients (cT1-cT4b) with palpable axillary nodes (cN1-cN3) and suitable for NACT were recruited after written informed consent. Single, most suspicious node was biopsied and clipped. Nodal response to NACT was assessed on axUS. Wire localisation of CN was performed prior to axillary lymph node dissection (ALND). Diagnostic performances of axUS and CN excision were assessed. Of the 69 patients evaluated, 32 patients (mean age, 43.5 ± 11.8 years; females, 31/32 [97%]; pre-menopausal, 18/32 [56.3%]) with metastatic nodes who received NACT were included. Nodal pathological complete response rate was 34.4% (11/32) overall and 70% (7/10) in patients with ≤2 suspicious nodes on pre-NACT axUS. False-negative rates (FNRs) of axUS and CN excision were 4.8% and 28.6% respectively. Combination of post-NACT axUS and CN excision had an FNR of 4.8% overall and 0% in patients with ≤2 suspicious nodes on pre-NACT axUS. Combination of AxUS and ultrasound-guided wire localisation of pre-treatment positive CN has high diagnostic accuracy for nodal restaging after NACT in node positive breast cancer patients. Addition of axUS assessment to wire localisation of CN reduces its FNR for detecting residual metastasis after NACT.

Epithelial-mesenchymal plasticity and lymph node metastases in invasive breast carcinoma of no special type

Background: Breast cancer is the commonest cancer among women with distant metastases being the leading cause of mortality. Epithelial-mesenchymal plasticity (EMP) is a proposed mechanism involved in establishment of metastasis. E-cadherin and vimentin can be used as epithelial and mesenchymal markers, respectively, to identify EMP. Aims: To compare E-cadherin and vimentin expressions between node-negative and node-positive invasive breast carcinoma–no special type (IBC-NST) and their lymph node metastases. Methods: Forty node negative tumours and 44 axillary node positive tumours of IBC-NST with their metastatic lymph nodes were retrieved from patients who had undergone mastectomy at the Teaching Hospital, Peradeniya. Samples were subjected to dual immunohistochemical staining with anti-E-cadherin and anti-vimentin primary antibodies. Any cytoplasmic staining for vimentin and 70% or more of membranous staining for E-cadherin were taken as positive and cells counted in 10 high power fields. Intensity of staining was graded as high=3, moderate=2, low=1 and absent=0 by comparing with internal controls. Percentage of positive cells was multiplied by the intensity to obtain the immuno-score (IS) for both protein expressions. IS was compared using independent and paired t-test. Results and conclusions: A lower E-cadherin (p=0.001) and higher vimentin (p=0.008) expression was seen in node positive group compared to node negative group. A higher E-cadherin (p<0.001) and lower vimentin (p<0.001) expression was seen in lymph nodes compared to the corresponding primary tumours. A significant loss of epithelial properties and acquisition of mesenchymal properties in the node positive group indicates epithelial to mesenchymal transition of tumour cells, contributing to metastasis. In the lymph nodes, higher E-cadherin and lower vimentin expression suggests a reverse mechanism, i.e., mesenchymal to epithelial transition. These Results are suggestive of epithelial-mesenchymal plasticity of tumour cells in establishment of metastasis in IBC-NST.

The association of axillary lymph node-positive breast cancer with metabolic parameters of 18F-fluorodeoxyglucose PET / CT

Objective: This study aims to examine the association between 18F-fluorodeoxyglucose PET/CT (18F-FDG PET/CT) metabolic parameters of lymph node-positive and lymph node-negative breast carcinomas.&#x0D; Material and method: We included breast carcinomas patients who underwent 18F-FDG PET/CT imaging at our department between May 2018 and December 2019. A total of 108 female breast cancer patients were included (aged 48.8 ± 13.6years; range, 28-84 years). PET scanning was performed in 3D mode from the skull ceiling to the half of the thigh. According to pathology reports, we divided the patients into two groups: a lymph node-positive group of patients and a lymph node-negative group of patients. We calculated the sensitivity and specificity for determining the PET/CT pathological lymph node. Metabolic parameters like TLG (Total lesion glycolysis), MTV (Metabolic tumor volume), SUVmean, and SUVmax values were calculated.&#x0D; Result: The lymph node-positive group’s body weight and body mass index(BMI) were statistically higher than the lymph node-negative group (p=0,027,p=0,022 respectively). SUV max and SUV mean of the lymph node-positive group were statistically higher than the lymph node-negative group (p=0.008, p=0,009, respectively). Both TLG and MTV of the lymph node-positive group were statistically higher than the lymph node-negative group (p=0.01, P= 0.01, respectively). Ki-67(%) of the lymph node-positive group was not statistically different from the lymph node-negative group. We calculated the PET/CT’s sensitivity and specificity as 78,57% and 59,09%, respectively. For the positive predictive value of PET/CT, we found 55%, and for the negative predictive value, it was 81.25%.&#x0D; Conclusions: PET/CT metabolic parameters of patients with lymph node-positive breast cancer were higher than patients with lymph node-negative. High body weight and BMI appears to increase the possibility of metastases of lymph node. The sensitivity of PET/CT can be considered to be useful in determining the pathological lymph node, but the specificity of PET/CT is not very good.

Abstract P2-07-07: Development of a machine learning-based classifier for Oncotype DX® category prediction in a population of lymph node positive breast carcinoma patients

Abstract INTRODUCTION: Oncotype DX® (ODX) Assay is a valuable prognostic and predictive tool in ER+, Her2- invasive breast cancer (IBC). Initially tested and validated in lymph node (LN) negative patients, the indications of this test have been expanded to include patients with limited LN-positive disease. Several prediction systems have been developed to predict the ODX Recurrence Score (RS) with substantial performance in predicting a high vs low RS score but with low performance when predicting the 3 classes that compose the standard ODX. Additionally, many of these prediction systems have not been developed and/or tested for a population of LN+ patients. OBJECTIVES: The primary objective of this study was to evaluate the performance of several previously published ODX RS predictive systems in a population of LN+ patients. Furthermore, we developed a machine-learning based classification system to accurately predict the ODX 3 category RS for this specific population. METHODS: We conducted a retrospective search of Stanford's pathology database for all patients with LN+ IBC diagnosed between January 2013 and December 2017 with an ODX RS available. A total of 119 patients were identified for inclusion in this cohort. Our multivariate pathologic feature-based discriminatory model aimed to classify each case as belonging to the low, intermediate or high ODX RS category. We performed model validation by the 10-fold cross validation (10F-CV) method. The model's performance was assessed by comparing simple accuracy, balanced accuracy, F1 score (harmonic average of the precision and recall) and several concordance classification metrics. RESULTS: Of the evaluated methods, Magee equations performed well in this population of LN+ patients with the modified Magee equation 2 displaying the best accuracy (70.9%) which was surprisingly better than originally reported (55.8%, in Klein et al. Mod Path. 2013). After an initial screen of methods and tuning of the best performing model, our model achieved an overall accuracy of 78.1% on 10F-CV with a 79.1 % balanced accuracy and no two-step discordances. This corresponded to an increase of weighted Cohen's kappa of 30% versus the best performing Magee equation in this cohort and an increase of 103% versus the modified Magee 1 equation (which uses the same features as our model except for tumor grade). DISCUSSION: Classifiers aimed at providing an alternative to Oncotype DX testing are available and perform consistently across datasets. We are currently validating our approach in a population of 1000 LN-negative patients from the MSKCC and the SEER database. Due to the substantial performance of our machine learning-based classifier based on standard reported pathologic features, our model may be considered an alternative to the ODX standard testing or a screening method for ODX testing, especially for cases with where the cost and availability of the ODX test are a concern. Citation Format: Beca F, Yang S-R, Gruber JG, Barry-Holson K, West R, Wen HY, Allison KH. Development of a machine learning-based classifier for Oncotype DX® category prediction in a population of lymph node positive breast carcinoma patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-07-07.

Epigenetic changes found in uterine decidual and placental tissues can also be found in the breast cancer microenvironment of the same unique patient: description and potential interpretations.

Microenvironmental properties are thought to be responsible for feto-maternal tolerance. Speculatively, ectopic expression of placental gene programs might also be related to cancer cells’ ability to escape from immune vigilance mechanisms during carcinogenesis and cancer progression. Recently, we published the first human genomic evidence of similar immune related gene expression profiles in both placenta (placenta and decidual tissue) and cancer (both primary and metastatic) in the same patient with lymph-node positive breast carcinoma during pregnancy. Here we report the first epigenomic analysis of these tissue samples and describe their main findings, with respect to immune related genes regulation (over or under expressed) in cancer cells with regards placental tissues. We confirm significant similarities, and hierarchical clustering (both unsupervised and supervised), in CpG island methylation patterns between decidual/placental and cancer microenvironments, which cannot be easily explained by simple models or unique pathways. Several different cell types are probably involved in these complex immune regulation mechanisms. Cancers may somehow “hijack” gene programs evolved over millions of years to allow for feto-maternal tolerance in placental mammals in order to escape from immune vigilance and spread locally or to distant sites.

Histone deacetylase inhibitor-induced cancer stem cells exhibit high pentose phosphate pathway metabolism

PurposeWe recently demonstrated that histone deacetylase (HDAC) inhibitors can “reprogram” differentiated triple-negative breast cancer cells to become quiescent stem-like cancer cells. We hypothesized that the metabolic state of such cells differs from that of their differentiated progeny.ResultsIn untreated cells, glucose uptake was higher in ALDH+ cells than in ALDH− cells (p = 0.01) but lactate production was not different; treating ALDH− or ALDH+ cells with VA or SAHA similarly increased glucose uptake without changing lactate production but upregulated G6PD, a rate-limiting enzyme in pentose phosphate pathway metabolism. NADPH production was higher in HDAC inhibitor-treated stem-like cells than in vehicle-treated cells (p < 0.05). Two G6PD inhibitors, 6-aminonicotinamide and dehydroepiandrosterone, decreased mammosphere formation efficiency and ALDH activity and 6-aminonicotinamide reduced the VA-induced increase in ALDH+ cells. Finally, patients expressing high G6PD mRNA had significantly worse overall survival (p < 0.001), and patients with high G6PD protein showed a similar trend towards worse disease-specific survival (p = 0.06).MethodsGlucose consumption, lactate and NADPH production, and reactive oxygen species generation were compared in aldehyde dehydrogenase (ALDH)-positive and –negative cells in the presence or absence of the HDAC inhibitors valproic acid (VA) or suberoylanilide hydroxamic acid (SAHA). Glucose-6-phosphate dehydrogenase (G6PD) expression was evaluated in a tissue microarray from 94 patients with node-positive invasive breast carcinoma and in two publically available databases and correlated with overall survival.ConclusionsEnergy metabolism in HDAC inhibitor-induced stem-like cancer cells differed sharply from that of differentiated cell types. HDAC inhibitor-induced dedifferentiation promoted metabolic reprogramming into the pentose phosphate pathway, which is targeted effectively by G6PD inhibition. These findings highlight a potential dual-therapy approach to targeting bulk differentiated cells with HDAC inhibitors and CSCs with G6PD inhibitors.

Prognostic significance of lymphatic invasion in lymph node-positive breast carcinoma: findings from a large case series with long-term follow-up using immunohistochemical endothelial marker

The poor prognostic significance of lymphatic invasion (LI) in breast carcinoma (BC) as a whole and in lymph node (LN)-negative patients in particular has been recognized in several studies; however, its prognostic role in LN-positive patients is still questionable. Aim of the current study was to assess prognostic role of LI in LN-positive BC specimens. Sections from non-selected 557 LN-positive BC specimens were stained with antibody to podoplanin/D2-40. LI was identified and correlated with clinicopathological features and patients' outcome. Twenty-year overall survival (OS), disease-free interval (DFI), and development of distant metastasis (DM) or recurrence were known for all patients. LI was detected in 262/557 (47%) of specimens ranging from 1 to 350 lesion per tumor section. Its presence was associated with higher grade tumors (P<0.0001), negative hormonal receptors (P<0.0001), high HER-2 expression (P=0.006), and with increased number of positive LNs (P=0.019). In the whole LN-positive BC, presence of LI was a poor prognostic factor for OS, DFI, and development of DM both in univariate and in multivariate analysis. In further stratification of patients, LI was associated with poorer prognosis in patients with single positive LN and not in patients with >1 positive LN. In T1N1 stage, LI was highly associated with poor OS (P=0.002), DFI (P<0.0001), and DM (P<0.0001). In T2N1 patients, LI was associated only with poorer DFI (P=0.037) but not with death or DM. In the two former patient groups, LI lost significance in multivariate analysis. In conclusion, LI is a poor prognostic factor in LN-positive BC particularly for patients having single positive LN. LI therefore would add further prognostic significance when considered in treatment in those patients. We recommend incorporation of LI in breast carcinoma staging and in prognostic indices.

Adjuvant chemotherapy may contribute to an increased risk for metabolic syndrome in patients with breast cancer.

Cytotoxic treatment may cause weight gain and important alterations in the metabolic status of breast cancer (BC) patients. The aim of this study was to investigate the changes in metabolic and anthropometric parameters of patients with BC who received adjuvant chemotherapy. All consecutive women treated with adjuvant TAC (docetaxel 75 mg/m(2), doxorubicine 50 mg/m(2), cyclophosphamide 500 mg/m(2)) chemotherapy for node-positive breast carcinoma at our Institution between 2008 and 2010 were included. Among 104 patients, 84 of them were stage II and 20 of them were stage III. When we compared the measurements between 1(st) and 6(th) adjuvant chemotherapy, we observed statistically significant increases in weight and serum triglyceride levels, and decreases in high density lipoprotein, apolipoprotein A-1, transferrin, albumin and prealbumin levels. An elevation of follicle stimulating hormone, luteinizing hormone together with the decrease of estradiol was detected. Waist-to-hip ratio has also increased significantly. In subgroup analyses, we observed dramatic changes in body mass index in pre-menopausal women whereas no significant change was seen in the post-menopausal group. Adjuvant chemotherapy may contribute to an increased risk for metabolic syndrome in patients with BC and these changes are more profound in pre-menopausal patients.

Lymph Node Ratio May Be Supplementary to TNM Nodal Classification in Node‐positive Breast Carcinoma Based on the Results of 2,151 Patients

The aim of the present study was to determine how lymph node ratio (LNR; the ratio of the number of metastatic lymph nodes to the number of removed lymph nodes) can supplement the TNM nodal classification in breast carcinoma. We retrospectively reviewed the file records of 2,151 patients. Lymph node ratio-based low- (LNR ≤ 0.20), intermediate- (LNR 0.21-0.65), and high-risk (LNR > 0.65) patient groups had significantly different disease-free survival (DFS) (P < 0.001). The DFS of patients with N1, N2, and N3 disease was significantly different (P < 0.001). When LNR and TNM nodal groupings were included together in the Cox analysis, both groupings had independent prognostic significance (P < 0.001 and P < 0.001, respectively). The most significant LNR threshold value separating patients in low-risk and high-risk groups in terms of disease recurrence was 0.20 for N1 disease (P < 0.001), 0.35 for N2 disease (P < 0.001), and 0.90 for N3 disease (P < 0.001). Lymph node ratio and TNM nodal groupings show no superiority over each other in categorizing patients with node-positive breast carcinoma into prognostic groups of low-, intermediate-, and high-risk. However, LNR grouping may supplement TNM nodal classification by categorizing patients within each TNM nodal group into low-risk and high-risk groups with significantly different survival.

Abstract P6-07-11: Is the prognosis of lymphotropic invasive micropapillary carcinoma worse than invasive ductal carcinoma?: A population-based study of 645 patients

Abstract Purpose: Invasive micropapillary carcinoma (IMPC) is an uncommon distinct variant of breast carcinoma and is associated with an increased risk for regional lymph node metastases. Therefore, IMPC is considered to have an unfavorable prognosis when compared to invasive ductal carcinoma (IDC). The prognostic factors for IMPC are not well characterized due to the relative scarcity of cases reported in the literature. Methods: We analyzed the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) database to evaluate prognostic factors of a population of 645 breast IMPC patients and 300,060 breast IDC patients reported between 2001 and 2008. Using univariate and multivariate analyses, hazard ratios (HR) were calculated for disease-specific (DSS) and overall survival (OS) for these patients using parameters such as patient age at diagnosis, histological grade, ER status, PR status, tumor size, and degree of lymph node positivity. Subset analysis of high grade, lymph node-positive patients was performed to compare DSS and OS between IMPC and IDC. Results: The 5-year DSS and OS for IMPC patients were 92.1% and 84.6% compared to 5-year DSS and OS of 88.5% and 80.2% for IDC patients. At presentation, TNM staging of IMPC cases was similar to IDC except for a higher percentage of LN metastases (52.4% in IMPC vs. 34.7% in IDC). Of those with known estrogen receptor (ER) status, 84.2% of IMPC cases were ER-positive, which was associated with better DSS (Hazard Ratio (HR) 0.36, p &amp;lt; 0.002) and OS (HR 0.62, p = 0.072). Patients with four or more positive lymph nodes had worse DSS (HR 7.1, p &amp;lt; 0.0001) and OS (HR 3.2, p &amp;lt; 0.0001) than node-negative patients, but those with one to three positive lymph nodes had similar DSS (HR 1.04, p = 0.96) and OS (HR 0.99, p = 0.97) as node-negative patients. In the subset of patients with high grade, node-positive breast carcinoma, patients with micropapillary histology had better DSS (p &amp;lt; 0.03) and a trend towards better OS (p = 0.12) than high grade, node-positive invasive ductal carcinoma. This subset of IMPC patients also had a higher percentage of ER-positive tumors (77%) compared to IDC patients (56%). Conclusions: While IMPC has a high propensity for lymph node metastasis, it has a disease-specific and overall prognosis that compares favorably to IDC. The higher percentage of hormone-receptor positivity may account for this survival advantage, even in high grade, node-positive disease. Therefore, estrogen-receptor-negativity or having four or more positive lymph nodes at presentation may potentially serve as prognostic markers for IMPC patients. In this study population, patients with one to three positive lymph nodes have DSS and OS similar to node-negative patients. Additional clinical studies are warranted to further investigate this observation. This is the largest study of IMPC to date, and these findings help our understanding of this uncommon histological variant of breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-07-11.

279P - Progesteron Receptor Status in Determining the Prognosis of Estrogen Receptor Positive / HER2-Negative Breast Carcinoma

ABSTRACT Background The aim of this retrospective study was to determine whether progesteron receptor (PgR) status have an influence on the prognosis of estrogen receptor positive (ER+)/HER2-negative breast carcinoma (BC). Methods We retrospectively reviewed the medical files of 1680 operable BC patients (pts) diagnosed between 1996 and 2011 and 456 of whom ER,PgR and HER2 status known were included in this study. Patients were categorized into 2 groups; as group A (ER + /PgR-/HER2-negative) and group B (ER + /PgR + /HER2-negative). Twenty one percent (97 pts) of the pts were in group A. Results Median follow up was 33.5 (0-177) months. Median age was 54 (21-90) years. Sixty-one percent (278) of the pts had node-positive BC. Sixty percent (276) of the pts were postmenopausal. Eighty percent (365) of the pts received adjuvant chemotherapy (ACT). Adjuvant hormonotherapy (AHT) was recommended to nearly all patients (mostly tamoxifen). Pts in group A had significantly higher lymph node positive disease as compared to group B (%70 vs 59%, p = .046). Although there was no statistically significant difference between two groups for the first site of recurrence (mostly bone and soft tissue, p = .51), the number of recurrence and mortality events in group B were proportionally less than group A [27.8% and 21.2% for group B and 42.7% and 32.2% for group A;p = .005 and p = .004, respectively). In the node-positive subgroup, an important difference for relative risk RR of BC recurrence between groups A and B was found (34/68 vs 75/207, RR 1.3, CI:1.02-1.86, p = .034). But, the mortality risk was similar for lymph node-positive pts in groups A and B (16/63 vs 50/201,RR 1.0, CI:0.62-1.66,p = 0.93). Although DFS for group B was shorter, it was not statistically significant (5-year DFS rate 45% vs 35% p = .24). However, 5-year overall survival (OS) was significantly longer for pts in group B than group A (78% vs 67%,p = .043). Conclusions PgR status has a great influence on the prognosis of BC pts especially negative PgR may have a significant negative influence on the prognosis of node-positive ER + /HER2- BC pts. Disclosure All authors have declared no conflicts of interest.

Lysine 394 is a novel Rad6B-induced ubiquitination site on beta-catenin

The ubiquitin conjugating enzyme Rad6B is overexpressed in breast cancer and induces β-catenin transcriptional activation and stabilization via K63-linked polyubiquitination. Here we identify β-catenin and Rad6B interacting regions, identify potential Rad6B ubiquitination sites in β-catenin, and characterize their breast cancer tissue expression. β-catenin and Rad6B colocalize in breast carcinoma and coimmunoprecipitate from MDA-MB-231 cells. Pull-down assays using GST-β-catenin and His-Rad6B deletion mutants identified amino acids 131–181 and 50–116, respectively, as necessary for their interaction. Ubiquitination assays using β-catenin deletion mutants mapped Rad6B-induced ubiquitination within β-catenin 181–422 encompassing Armadillo repeats 2–7. Lysine to arginine mutations within repeats 5–7 identified K394 as the major Rad6B ubiquitination site in vitro and in vivo, and confirmed by Rad6B ubiquitination of a β-catenin peptide encompassing K394. Ubiquitination of wild type- but not K394R-β-catenin was decreased by Rad6B silencing. Compared to wild type-, K312R-, K335R-, K345R-, or K354R-β-catenin, K394R mutation caused ~50% drop in TOP/Flash activity in Wnt-silent MCF-7 cells. Consistent with these data, expression of Rad6B, itself a β-catenin/TCF transcriptional target, was also reduced in K394R-β-catenin transfected cells. Steady-state K394R-β-catenin levels are decreased compared to wild type-β-catenin. The decreased expression is not due to proteasomal degradation as treatment with MG132 failed to rescue its levels. Lymph node-positive breast carcinomas express higher levels of Rad6 protein and Rad6 activity, and K63-linked ubiquitinated β-catenin than reduction mammoplasties. These data suggest that K394 is a novel site of β-catenin ubiquitination that may be important for the stability and activity of β-catenin in breast cancer.

Differential protein expression in primary breast cancer and matched axillary node metastasis

Axillary lymph node (ALN) metastasis is a key step of tumor progression in breast cancer and is associated with an unfavorable prognosis. However, the mechanisms of this process are not well understood. Proteomic technologies have led to identification of specific protein markers and a better understanding of the cellular processes. To explore this, differential protein expression was analyzed between node-positive breast carcinoma and node-negative breast carcinoma (11 samples) and between primary breast carcinoma and matched metastatic ALN (five pairs) using a combination of 2D-SDS-PAGE and LC-MC/MS. Of the total 678 protein spots, 19 proteins were up-regulated and 3 proteins were down-regulated in node-positive breast carcinomas compared to node-negative breast carcinomas. Four up-regulated proteins were identified, namely annexin 5, carbonic anhydrase I, peroxiredoxin 6 and proteasome α2 subunit. For proteins altered in metastatic ALN compared to primary tumors, 6 of 14 up-regulated proteins were identified: heat shock 70 kDa protein 5, protein disulfide isomerase, prolyl 4-hydroxylase β subunit precursor, lactate dehydrogenaseB, triosephosphate isomerase 1 and β-tubulin and 5 of 23 down-regulated proteins were identified including 90 kDa heat shock protein, chain A apo-human serum transferrin, chain A α1-antitrypsin, enolase 1 and macrophage migration inhibitory factor. Immunohistochemistry showed stronger immunostaining for β-tubulin in metastatic ALN compared to primary breast tumor. All of the identified proteins function in various processes involved in cell survival and growth. Our results suggest that these processes are critical for tumor progression and metastasis and the proteins identified could be candidate markers of clinical usefulness.

Factors predicting non-sentinel lymph node involvement in sentinel node positive breast carcinoma

In routine practice, axillary lymph node dissection is performed in early invasive breast cancer patients with positive sentinel node biopsy. However, sentinel node is the only involved axillary node in 40-70% of patients, and determining factors that predict axillary non-sentinel node involvement will therefore prevent unnecessary axillary lymph node dissection and decrease morbidity. In this study, 119 invasive breast cancer patients with sentinel node metastasis who underwent axillary lymph node dissection between 1998-2009 at our institution were studied. Primary tumor characteristics and features of the metastatic tumors in sentinel nodes, such as microanatomic location, size of metastasis, and the ratio of metastatic tumor area to the total sentinel node area were evaluated. Student's t-test and multivariate logistic regression were used for statistical analysis. The mean age of the patients was 50.7 years (28-80). Forty-three patients (36%) had invasive ductal and 25 patients (21%) had invasive lobular carcinoma. Most of the patients had either pT1 (44%) or pT2 (54%) tumors. Fifty-four patients (45%) had no further positive nodes in the axilla. The metastatic deposits in the sentinel node were subcapsular in 16 patients (13%). The percent area of sentinel node occupied by tumor (p < 0.001), number of sentinel nodes (p=0.041), and microanatomic location of metastatic tumor (p=0.002) were significantly associated with non-sentinel node metastasis in univariate analysis. The percent area of sentinel node occupied by tumor (p < 0.001) and number of sentinel nodes (p=0.033) remained significantly associated with non-sentinel node involvement in multivariate analysis. In patients with invasive breast cancer and positive sentinel node, area percent of sentinel node occupied by tumor, and the number of sentinel nodes removed are independently predictive of non-sentinel node involvement.

Predictors of 4 or More Positive Axillary Nodes in Patients with Node-positive T1-2 Breast Carcinoma: The Indications for Adjuvant Irradiation of the Level III Axilla and Supraclavicular Fossa

Purpose: We evaluate the predictors of 4 or more involved axillary nodes in patients with node-positive T1-2 breast carcinoma to select a group of patients who are indicated for adjuvant irradiation of the level III axilla and supraclavicular fossa (SCF). Methods: We analyzed 286 patients with positive axillary nodes and who were without distant metastases and who underwent breast conserving surgery and axillary lymph node dissection or modified radical mastectomy. We investigated the relationship between the patients and the tumor factors and 4 or more positive axillary nodes. Results: On the multivariate logistic-regression analysis, an increased tumor size (p=0.002), the presence of lymphovascular space invasion (LVSI) (p<0.001) and a palpable mass p<0.001) were positively associated with involvement of 4 or more axillary lymph nodes. In our study, 86.1% of the patients with all the unfavorable factors had involvement of 4 or more nodal metastases. Conclusion: Our data suggest that for patients with node-positive T1-2 breast cancer, the presence of 4 or more involved nodes is frequently observed for the patients with an increased tumor size, the presence of LVSI and a palpable mass at the time of diagnosis, and we recommend that they undergo irradiation of the high axilla and SCF for adjuvant care, if they do not undergo complete axillary dissection.

Survival Benefit with Radiation Therapy in Node-Positive Breast Carcinoma Patients

Postoperative radiation therapy (RT) has been the subject of discussion, especially in patients with one to three positive lymph nodes (< or = 3 pN+) in the axillary dissection. The authors investigated whether postoperative RT provides a survival benefit for pT1-2 pN+ breast cancer patients. Patients included were selected from the SEER database (NCI--Surveillance, Epidemiology and End Results, release 2000; n = 24,410) and the UZ Brussel database (1984-2002; n = 1,011) according to the following criteria: women aged 25-95, no previous cancer, unilateral pT1-pT2 breast tumors, total mastectomy (ME) or breast-conserving surgery (BCS), postoperative RT, and an axillary dissection showing at least one pathologic lymph node. The overall survival (OS) of patients in the SEER and UZ Brussel databases who received postoperative RT was identical. However, patients in the SEER database who did not receive RT had a significantly worse outcome (p < 0.0001). After ME or BCS, all patients (SEER and UZ Brussel) who had > or = 4 pN+ and received RT had comparable outcomes after 15 years. The 15-year OS in the subgroup with ME and < or = 3 pN+ nodes was 57.0% and 46.6% (p = 0.0004) with RT (UZ Brussel) and without RT (SEER), respectively. For BCS and < or = 3 pN+, the same significant difference in OS at 15 years was seen: 63.8% after RT (UZ Brussel) and 60.4% without RT (SEER; p = 0.0029). RT provides a survival benefit in patients with < or = 3 or > or = 4 pN+; the indication for postoperative RT should therefore be adapted in future consensus meetings.