Supplementary Table S1 from Carcinoembryonic Antigen Cell Adhesion Molecule 6 Predicts Breast Cancer Recurrence following Adjuvant Tamoxifen

Abstract The potential role of the androgen receptor (AR) as a predictive or prognostic factor in breast cancer remains unclear. We aimed to determine the prognostic significance of AR in a cohort of breast carcinomas with long-term follow-up and to critically appraise this in the context of existing literature. Four hundred and eight cases of invasive breast cancer were incorporated into tissue microarrays (TMAs). All received tamoxifen and comprised 108 cases which relapsed and 300 cases which did not. Mean follow-up time for the former was 84 months (range 1–142, SD 38.8) and for the latter was 77 months (range 11–229, SD 49.7). TMAs were immunohistochemically stained with AR and scored as a continuous variable and using the Allred score. AR expression was significantly associated with grade, recurrence on tamoxifen, non-breast cancer death estrogen receptor alpha (ERα) and progesterone receptor (PR). AR correlated significantly with better overall survival (OS) and disease-free survival (DFS) using an Allred cut-off of 4 (log rank=0.0053 and 0.0044, respectively), and 20% positive tumor cells (log rank=0.0027 and 0.0059, respectively). AR expression was additionally associated with a reduced risk of recurrence following endocrine therapy. In summary, AR positive breast tumors have better OS and DFS and are less likely to recur following endocrine treatment.

Objective: Postoperative wound complications after excisional surgery for primary breast cancer can result in patients requiring additional treatments and delay adjuvant therapy and are associated with worse prognoses. We investigated factors that might predispose patients to wound complications.Background: A number of patient characteristics have been associated with wound complications, but there is currently no quantitative measure of the risk of their occurrence. Our hypothesis was that wound complications are related, in part, to the immune status of patients.Methods: We recruited patients undergoing surgery for primary breast cancer and determined their circulating levels of various immune cells shortly before and after surgery as a measure of immune status.Results: One hundred seventeen patients were recruited; 16 (13.7%) developed wound complications. The following patient and tumor characteristics were associated with higher wound complication rates: diabetes (P = 0.02); larger tumors (T2/3 vs T1; P = 0.02); metastatic axillary nodes (P = 0.006). With respect to immune status, no significant differences in preoperative levels of circulating immune cells were detected between patients who developed wound complications and those who did not. However, patients who developed complications showed greater reductions in lymphocyte levels 4 hours postoperatively than those who did not (P < 0.001). Multivariate analyses demonstrated that falls in lymphocyte levels of greater than 20% or 50% 4 hours postoperatively acted as a significant and independent predictor of wound complications (P < 0.005 and P < 0.0001, respectively).Conclusions: Perioperative changes in lymphocyte levels could provide a practical predictive marker for wound complications on which selective antibiotic prophylaxis could be based.

Surgery and anaesthesia result in a variety of metabolic and endocrine responses, which result in a generalised state of immunosuppression in the immediate post-operative period. Surgery induced immunosuppression has been implicated in the development of post-operative septic complications and tumour metastasis formation. In addition the effectiveness of many treatments in the adjuvant setting is dependent on a functioning immune system. By understanding the mechanisms contributing to surgery-induced immunosuppression, surgeons may undertake strategies to minimise its effect and reduce potential short-term and long-term consequences to patients.

Estrogen receptor (ER) action is modulated by posttranslational modifications. Although ERalpha phosphorylation correlates with patient outcome, ERbeta is similarly phosphorylated but its significance in breast cancer has not been addressed. We investigated whether ERbeta that is phosphorylated at serine 105 (S105-ERbeta) is expressed in breast cancer and assessed potential clinical implications of this phosphorylation. Following antibody validation, S105-ERbeta expression was studied in tissue microarrays comprising 108 tamoxifen-resistant and 351 tamoxifen-sensitive cases and analyzed against clinical data. S105-ERbeta regulation in vitro was assessed by Western blot, flow cytometry, and immunofluorescence. Nuclear S105-ERbeta was observed in breast carcinoma and was associated with better survival (Allred score > or =3), even in tamoxifen-resistant cases, and additionally correlated with ERbeta1 and ERbeta2 expression. Distinct S105-ERbeta nuclear speckles were seen in some higher grade tumors. S105-ERbeta levels increased in MCF-7 cells in response to 17beta-estradiol, the ERbeta-specific agonist diarylpropionitrile, and the partial ERbeta-agonist genistein. S105-ERbeta nuclear speckles were also seen in MCF-7 cells and markedly increased in size and number at 24 hours following 17beta-estradiol and, in particular diarylpropionitrile, treatment. These speckles were coexpressed with ERbeta1 and ERbeta2. Presence of S105-ERbeta in breast cancer and association with improved survival, even in endocrine resistant breast tumors suggest S105-ERbeta might be a useful additional prognostic marker in this disease.

Involvement of an intramammary lymph node with metastatic breast cancer is an uncommon clinical or radiological presentation. Previously reported series of patients are small in number and the clinical advice is unclear. We identified 100 patients on our pathology database with intramammary lymph nodes in association with a primary breast cancer. Ten were identified pre-operatively on breast imaging and 90 were first discovered on pathological assessment of excised breast tissue. Twenty one contained metastasis. Factors that predicted for intramammary node metastasis were increasing age (p = 0.017), lymphovascular invasion (p = 0.002) and grade of tumour (p = 0.012). The presence of metastasis within the intramammary lymph node was associated with a poorer disease free survival (p = 0.007) and reduced overall survival (p = 0.035). Sixty seven percent of patients with intramammary node metastasis had further axillary metastases. One patient had an intramammary node metastasis but uninvolved axillary sentinel node. She presented 19 months later with an axillary nodal recurrence. The presence of intramammary lymph node metastasis is associated with poorer outcome in breast cancer patients. Pre-operative detection of intramammary lymph node metastasis is helpful to guide breast and axillary surgeries. Intramammary lymph node metastasis predicts strongly for axillary metastatic disease and axillary node clearance is recommended.

Oestrogen receptors (ERs) are critical regulators of the behaviour of many cancers. Despite this, the roles and regulation of one of the two known ERs – ERβ– are poorly understood. This is partly because analyses have been confused by discrepancies between ERβ expression at mRNA and proteins levels, and because ERβ is expressed as several functionally distinct isoforms. We investigated human ERβ 5′ untranslated regions (UTRs) and their influences on ERβ expression and function. We demonstrate that two alternative ERβ 5′UTRs have potent and differential influences on expression acting at the level of translation. We show that their influences are modulated by cellular context and in carcinogenesis, and demonstrate the contributions of both upstream open reading frames and RNA secondary structure. These regulatory mechanisms offer explanations for the non‐concordance of ERβ mRNA and protein. Importantly, we also demonstrate that 5′UTRs allow the first reported mechanisms for differential regulation of the expression of the ERβ isoforms 1, 2 and 5, and thereby have critical influences on ERβ function.

Abstract Abstract #604 Background: Estrogen receptor (ER) expression is a key determinant of breast tumour behaviour, although the role of ERβ, unlike the well-studied ERα, remains uncertain. This is partly because analyses have been confused by discrepancies between ERβ mRNA and protein levels. We have identified 3 alternative 5'-untranslated regions (5'UTRs) for ERβ: UTRa (including upstream exon 0K), UTRa long (UTRa containing an additional 5' sequence) and UTRb (including upstream exon 0N) from the literature and EST databases. We demonstrate that these alternative 5'UTRs allow differential post-transcriptional regulation of ERβ expression, which may be responsible for non-concordance of mRNA and protein and could provide an important level of modulation of ER activity. Methods and Results: Semi-quantitative PCR was used to show that ERβ 5'UTRs are differentially expressed between breast normal and tumour tissues. We investigated the properties of these 5'UTRs using established reporter assays, in which each 5'UTR was cloned upstream of a GFP reporter. A panel of breast cell lines were transiently transfected with either an unmodified GFP reporter as a control (this was identical to experimental vectors except for its non-specialised 5'UTR), or with equal copy numbers of specific 5'UTR reporters. Effects of each 5'UTR on translation were assessed by measurement of relative GFP protein and mRNA expression from each plasmid using flow cytometry and qPCR. Our results are the first to show that each 5'UTR has a profound and differential influence on mRNA translational efficiency. We also investigated the sequence determinants of these effects by mutating the AUG start codon of each upstream open reading frame (uORF) within these 5'UTRs to UUG or AUC by site-directed mutagenesis. We determined that the presence of uORFs partly mediates inhibition of translation. In addition, using gene-specific primers for cDNA synthesis we enriched for ERβ1, ERβ2 and ERβ5; isoforms of ERβ that are particularly relevant in breast cancer. Interrogation of the 5' ends by qPCR revealed that each 5'UTR is preferentially associated with mRNA variants containing different 3' splicing patterns, which code for different biological functions. Furthermore, using a 424-case breast cancer tissue microarray (TMA), we identified a positive correlation between the expression of ERβ1 and ERβ2, and eIF4E; a basal translation factor that enhances the translation of mRNAs with highly structured 5'UTRs. Finally, an over-expression of eIF4E de-repressed translation of GFP in cells transfected with the UTRb-specific GFP reporter. This indicates that secondary structure within UTRb also mediates inhibition of translation and suggests a role for cross-talk between 5'UTRs and cellular factors in defining ERβ expression. Discussion: Post-transcriptional regulation plays an important role in determining the level of ERβ protein expression and may therefore have an influence on overall ER activity. This may have important implications on our understanding of breast cancer biology and treatment. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 604.

Neoadjuvant chemotherapy (NACT) is a useful approach in the treatment of many breast cancers. One of the main advantages of NACT is the possibility of breast conservation surgery in patients who would otherwise require a mastectomy. Most literature on NACT focuses on the effectiveness of different chemotherapy regimen and subsequent mastectomy rates. There is little guidance in the literature on aspects of individual patient management and decision making during NACT. This paper considers practical management advice where NACT is considered and adopted.

Abstract Introduction:Ultrasound combined with fine needle aspiration cytology is effective in pre-operative staging of the axilla in breast cancer. Accurate pre-operative diagnosis of lymph node metastases allows for a one stage axillary operation and may also influence decisions regarding neo-adjuvant chemotherapy and breast reconstruction. The aim of this study is to identify pathological and patient factors that influence the accuracy of ultrasound and FNAC in determining the status of the axilla pre-operatively.Methods:Three hundred patients with primary operable invasive breast cancer had an axillary ultrasound pre-operatively. If the ultrasound was normal the patient was offered a sentinel node biopsy. If it identified equivocal or pathological nodes a fine needle aspirate cytology was performed. If the cytology was malignant then an axillary node clearance was performed.Results:Ultrasound combined with FNAC correctly determined the status of the axilla pre-operatively in 78% of cases. Sensitivity for the detection of metastases was 32% with 100% specificity. Factors affecting the accuracy of ultrasound and FNAC were the pathological size of tumour (p<0.05) and the size of the metastases (p<0.05). Age of the patient, pathological grade and type of tumour did not significantly affect the accuracy of the procedure.Conclusion:Ultrasound combined with fine needle aspirate cytology can be used effectively to determine the status of the axilla pre-operatively. All patients regardless of age or their particular tumour characteristics should be offered this procedure pre-operatively. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5020.

Increased eukaryotic translation initiation factor 4E (eIF4E) expression occurs in many cancers, and makes fundamental contributions to carcinogenesis by stimulating the expression of cancer-related genes at post-transcriptional levels. This key role is highlighted by the facts that eIF4E levels can predict prognosis, and that eIF4E is an established therapeutic target. However, eIF4E activity is a complex function of expression levels and phosphorylation statuses of eIF4E and eIF4E-binding proteins (4E-BPs). Our hypothesis was that the combined analyses of these pathway components would allow insights into eIF4E activity and its influence on cancer. We have determined expression levels of eIF4E, 4E-BP1, 4E-BP2 and phosphorylated 4E-BP1 within 424 breast tumours, and have carried out analyses to combine these and relate the product to patient survival, in order to estimate eIF4E activity. We show that this analysis gives greater prognostic insights than that of eIF4E alone. We show that eIF4E and 4E-BP expression are positively associated, and that 4E-BP2 has a stronger influence on cancer behaviour than 4E-BP1. Finally, we examine eIF4E, estimated eIF4E activity, and phosphorylated 4E-BP1 as potential predictive biomarkers for eIF4E-targeted therapies, and show that each determines selection of different patient groups. We conclude that eIF4E's influence on cancer survival is modulated substantially by 4E-BPs, and that combined pathway analyses can estimate functional eIF4E.

Abstract Abstract #1009 Introduction Intramammary lymph nodes are defined as lymph nodes surrounded by breast tissue. They are a potential site of regional spread for breast cancer and metastases in this node is reported in up to 9.8% of operable breast cancers. Despite this, the existence of the intramammary node is not widely appreciated and the clinical significance of intramammary nodal metastases remains uncertain. The aim of this study is to determine the prognostic relevance of intramammary lymph node metastases and how it impacts on current breast cancer treatment modalities. Methods All patients with cytologically or histologically proven intramammary nodes were identified from the pathology database at the Leeds Teaching Hospitals. A total of 102 specimens were identified in 100 patients with a primary breast malignancy over a 13 year period (1994-2007). Clinical data including information on recurrences and death was obtained from patient records. Results One hundred patients were identified on our pathology database to have intramammary lymph nodes in association with a primary breast cancer. Ten were identified pre-operatively on breast imaging and 90 were first discovered on pathological assessment of excised breast tissue. Twenty one contained metastases. Factors that predicted for intramammary node metastases were lymphovascular invasion (p=0.002) and grade of tumour (p=0.012). The presence of metastases within the intramammary lymph node was associated with a poorer disease free survival (p=0.0072) and reduced overall survival (p=0.0128) at a mean follow up of 34.7 months. Sixty seven percent of patients with intramammary node metastases had further axillary metastases. In no case was the intramammary node identified as the sentinel node. One patient had an intramammary node metastases but benign axillary sentinel node. An axillary node clearance was not performed and she presented 19 months later with an axillary nodal recurrence. Conclusion The presence of intramammary lymph node metastases is associated with poorer outcome in breast cancer patients. Pre-operative detection of intramammary lymph node metastases is helpful to guide breast and axillary surgery. With the use of lymphoscintigraphy intramammary nodes will be increasingly identified as the sentinel node. Intramammary lymph node metastases predicts strongly for axillary metastatic disease and axillary node clearance should be recommended even when it is associated with a benign axillary sentinel node biopsy. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1009.

Abstract IntroductionWe have previously demonstrated an association between post-operative wound complications and systemic breast cancer recurrence (p<0.0001), Murthy et al (2007) Br J Cancer 97, 1211-7. The aim of this study was to examine the potential role of the immune system in establishing this association, and therefore whether immune factors might be used to predict either wound complications or cancer recurrences.MethodsPatients with primary operable breast cancer were prospectively recruited to the study. Serial blood investigations were performed pre-operatively, peri-operatively and post-operatively. Absolute numbers of various lymphocyte cell populations were measured using multi-colour flow-cytometry including CD45+lymphocytes, CD19+ B lymphocytes, CD3+ T lymphocytes, CD4+ helper T cells, CD8+ cytotoxic T cells and CD56+ NK cells. We also measured the levels of the NK cytotoxicity receptors NKp30 and NKp46 on the NK cell population.ResultsOne hundred and nine patients were recruited to the study and there was a wound complication rate of 13.4%. Absolute numbers of CD3+ T lymphocytes, CD4+ helper T cells, CD8+ cytotoxic T cells and CD56+ NK cells were significantly lower 4 hours post-operatively compared to pre-operative levels (p<0.05), although levels had typically recovered after 24 hours. However, NKp30 expression remained significantly reduced at 24 hours (p<0.05). Mastectomy patients had a significantly greater fall in T lymphocyte numbers than those having breast conserving surgery (p<0.05). Patients who went on to develop wound complications post-operatively had a significantly greater fall in their CD4+ helper T cells at 4 hours post-operatively, than those patients who did not go on to develop wound complications (p<0.05).ConclusionsBreast cancer surgery results in severe disruption to the immune system, with dramatic changes in levels of immune regulatory blood cells populations. Changes are predominantly immuno-suppressive. The greater the immune disruption as a result of surgery, the more likely the patient is to develop a wound complication. We believe that this peri-operative immuno-suppression may also provide a window of opportunity for the successful dissemination of tumour cells post-operatively thereby increasing the risk of future metastases; we are maintaining follow up on this patient cohort in order to test correlations between peri-operative immuno-suppression and systemic recurrences. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4132.

Abstract Abstract #1019 Introduction Sentinel lymph node biopsy is now standard practice in axillary staging in breast cancer. It is associated with less morbidity than an axillary node clearance. A drawback of sentinel node biopsy is the need for a second surgical procedure if the sentinel node shows metastases. Clinical examination of the axilla has been the standard pre-operative assessment but this has been shown to be unreliable. More recently ultrasound has been used to identify axillary metastases pre-operatively. The aim of our study is to assess the role of pre-operative ultrasound and fine needle aspirate cytology in refining the selection of patients for whom sentinel node biopsy is appropriate. Methods Three hundred patients with primary operable invasive breast cancer had axillary ultrasound preoperatively. If the ultrasound was normal the patient was offered a sentinel node procedure. If it identified equivocal nodes a fine needle cytology was performed. If the cytology was benign a sentinel node biopsy was performed. If the cytology was malignant then an axillary node clearance was performed. In cases where pathological nodes were identified on imaging, cytology was performed to confirm malignancy. An axillary node clearance was then performed. Results. Eighty three percent of our patients (n=249) had a normal axillary ultrasound pre-operatively. Of these 74.5% had a benign sentinel node biopsy. Sixty two percent of patients with equivocal nodes on pre-operative imaging had metastases on final histology (n=19). Of these 63% (n=12) were correctly identified pre-operatively with fine needle aspirate cytology of the equivocal node and had an axillary node clearance performed form the outset. Ultrasound identified pathological nodes in 7% of our patients (n=20). Malignancy was confirmed on cytology and an axillary node clearance was performed. Using ultrasound and fine needle aspirate cytology to assess the axilla pre-operatively sentinel node biopsy was performed in 265 patients (88%). Of these, 74.8% had a benign final histology. Thirty two patients had a pre-operative diagnosis of nodal metastases and had an axillary node clearance. All 32 had lymph node metastases on final histology. Factors that predicted for a malignant sentinel node were lymphovascular invasion (p<0.001), tumour grade (p<0.008) and size of tumour (p<0.001). Conclusion Eighty eight percent of our patients had a sentinel lymph node biopsy to stage the axilla. Pre-operative ultrasound combined with cytology correctly determined the status of the node in 78% of cases. Our rate of second axillary operations was reduced by 32%. Importantly all patients diagnosed with node metastases pre-operatively and advised to have an axillary node clearance did have metastases on final histology. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1019.

The discovery of a second oestrogen receptor, ER beta, was a subject of much interest, as this suggested a means to improve the prognostic stratification of invasive breast cancer, better predict response to endocrine therapy, develop new chemotherapeutic/chemopreventative drugs and perhaps prevent inappropriate treatment. However, this has not proved to be straightforward with the discovery of five ER beta isoforms and numerous exon deletion variants. This review sets out to identify the present state of knowledge regarding the clinicopathological role of ER beta isoforms and discusses possible reasons for conflicting results arising from recent research findings.

Abstract Abstract #5045 Background: The factors leading to breast cancer recurrence are incompletely understood. We carried out a retrospective study of outcome for 1065 breast cancer patients for which we examined factors correlating with cancer recurrence. We found that infection of wounds after surgery for primary disease positively correlated with cancer recurrence (Murthy et al, BJC 2007). Patients with wound complications were 3 fold more likely to have systemic recurrences than those without (p<0.0001). The aim of our study is to determine mechanisms responsible for this correlation. Our approach is based on two possible theories. First, patients may have an underlying immune dysfunction that predisposes them to developing both wound complications and also recurrence. Secondly, factors released at sites of wound complications may have direct influences on the remaining occult tumour cells, thereby increasing the likelihood of metastases. Methods: Patients with primary operable breast cancer were recruited prospectively. Blood was collected from patients pre-operatively, 4 and 16h post-operatively and again at 2 weeks, 3 and 6 months post-operatively. A variety of investigations were carried out on each sample to establish the immune status of the patient at that time point, and to identify potential mediators of cross talk between the immune system, the wound and any occult tumour cells. These investigations include: a) Full blood count; b) Immune cell phenotyping (absolute numbers/frequency of B, T lymphocytes and Natural Killer (NK) cell sub-types using multi-colour flow-cytometry); c) Cytokine profiling (using fluid phase cytometric multiplex immunoassays for 27 critical cytokines). Patients were followed up post-operatively for wound complications. Results: In our cohort of 57 patients, those who underwent a mastectomy were four times more likely to develop a post-operative wound infection than those who had breast conserving surgery (p=0.022). There was no significant difference in pre-operative absolute numbers of B and T lymphocytes or NK cells between the two groups. The percentage drop in absolute numbers of NK cells at 4 hours post-operatively was greater in the mastectomy group than in patients having breast conserving surgery (p=0.008). Mastectomy patients also dropped their CD45 counts by a significantly greater percentage (p=0.024). No significant difference was noted in the CD3, CD4, CD8 and CD19 counts between the two groups. Patients who developed wound infections showed peaks in levels of either Interleukin 5 or Interleukin 6. Conclusion Disruption of immune mechanisms as a result of increased surgical stresses in mastectomy patients contributes to a higher rate of wound complications. This may also provide a window of opportunity for the dissemination of tumour cells with their latent potential for reactivation and the development of metastases. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5045.

Background Previous epidemiological studies have investigated the relationship between individual nutrients such as vitamin D and vitamin B 12 and mammographic density, a strong marker of breast cancer risk [1], with varied results.There has been limited research on overall dietary patterns and most studies have focused on adult dietary patterns [2].We examine prospective data to determine whether dietary patterns from childhood to adult life affect mammographic density.Methods The Medical Research Council National Survey of Health and Development is a national representative sample of 2,815 men and 2,547 women followed since their birth in March 1946 [3].A wealth of medical and social data has been collected in over 25 follow-ups by home visits, medical examinations and postal questionnaires.Dietary intakes at age 4 years were determined by 24-hour recalls and in adulthood (ages 36, 43 years) by 5-day food records.Copies of the mammograms (two views for each breast) taken when the women were closest to age 50 years were obtained from the relevant NHS centres.A total of 1,319 women were followed up since birth in 1946 for whom a mammogram at age 50 years was retrieved, and the percentage mammographic density was measured using the computer-assisted threshold method for all 1,161 women.Breast cancer incidence for the whole cohort is being ascertained through the National Health Service Central Register.Statistical analysis Reduced rank regression analysis, a relatively new approach to dietary pattern analysis, is being used to identify dietary patterns associated with mammographic density [4].This approach identifies patterns in food intake that are predictive of an intermediate outcome of the disease process, such as mammographic density, and subsequently examines the relationship between the identified dietary patterns and breast cancer risk. ResultsPreliminary analyses so far suggest that variations in dietary patterns in adulthood might explain more than 10% of the variation in percentage mammographic density at age 50 years (age 36 years: 13%; age 43 years: 14%), with variations in patterns in childhood explaining slightly less.Further work is being carried out on the characteristics of these dietary patterns and their effects on percentage mammographic density and its two components (that is, absolute areas of dense and nondense tissues) and on breast cancer risk, after adjusting for socioeconomic status, anthropometric variables and reproductive factors. ConclusionThe present study will provide for the first time information on the relationship between dietary patterns across the life course and mammographic density, and will help to clarify the pathways through which diet may affect breast cancer risk.

Circulating tumour cells (CTCs) have been of considerable interest for many years. The rarity of these cells presents the main challenge associated with their analysis. Current detection methods use antibody and nucleic acid techniques and are sensitive for CTC detection but limited in their utility by the occurrence of false-positive results. Despite this, there are a number of clinical studies which show that the presence of CTCs is an important prognostic indicator, particularly in the metastatic setting. Current efforts to phenotype CTCs may provide a valuable insight into the metastatic process and may also allow the development of specific CTC-targeted treatment strategies in the future.

Abstract Purpose: Tamoxifen remains therapy of choice for premenopausal estrogen receptor α–positive breast cancer. However, resistance and recurrence are serious problems. Our previous work indicated that carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) was significantly up-regulated in tamoxifen-resistant (TAMr) MCF-7 derivatives. The aim of this study was to determine the functional role of CEACAM6 in endocrine-resistant breast cancer and to retrospectively test whether it was predictive of resistance in a large cohort of breast cancers with long-term follow-up. Experimental Design: siRNA silencing of CEACAM6 was done in TAMr cells and effects on clonogenicity and endocrine sensitivity were determined. CEACAM6 immunohistochemistry was done on a tissue microarray comprising 108 relapsed primary human breast cancers and 243 tamoxifen-sensitive controls. Results: siRNA-mediated silencing of CEACAM6 reduced both clonogenicity and anchorage-dependent and anchorage-independent growth of TAMr cells. Importantly, CEACAM6 silencing restored sensitivity of TAMr cells to 4-hydroxytamoxifen and proliferative response to 17β-estradiol. Immunohistochemistry showed significantly more CEACAM expression in the relapsed group compared with nonrelapsed controls [35 of 108 (33.3%) and 32 of 243 (13.2%), respectively; odds ratio, 3.16 (95% confidence interval, 1.83-5.47); P < 0.0001]. Additionally, we derived an outcome predictor model based on CEACAM expression that restratified patients in the Nottingham prognostic index intermediate-risk group into either higher-risk or lower-risk group. Conclusions: Our data support an important role for CEACAM6 in endocrine resistance, which can serve as a powerful predictor of future recurrence.