Markers In Primary Breast Cancer Research Articles

Comment on: Prognostic role of preoperative circulating systemic inflammatory response markers in primary breast cancer: meta-analysis.

Comment on: Prognostic role of preoperative circulating systemic inflammatory response markers in primary breast cancer: meta-analysis.

Clinical, Pathological, and Molecular Features of Breast Carcinoma Cutaneous Metastasis.

Simple SummaryMetastasis is the last stage in the development of cancer and usually results in mortality. Cutaneous metastases (CMs) account for 2% of all skin malignancies. Nearly 70% of CMs in women originate from breast cancer (BC). Since CM is usually associated with poor prognosis, the development of management strategies for these patients remains an important clinical challenge. Identifying molecular markers in primary BC that predict CMs and determining the molecular differences between primary tumors and their metastases is of great interest for designing new therapeutic approaches.Cutaneous metastases (CMs) account for 2% of all skin malignancies, and nearly 70% of CMs in women originate from breast cancer (BC). CMs are usually associated with poor prognosis, are difficult to treat, and can pose diagnostic problems, such as in histopathological diagnosis when occurring long after development of the primary tumor. In addition, the molecular differences between the primary tumors and their CMs, and between CMs and metastases in other organs, are not well defined. Here, we review the main clinical, pathological, and molecular characteristics of breast cancer CMs. Identifying molecular markers in primary BC that predict CM and can be used to determine the molecular differences between primary tumors and their metastases is of great interest for the design of new therapeutic approaches.

Androgen receptor in breast cancer: A wolf in sheep's clothing? A lesson from prostate cancer.

The possibility that a receptor for androgen is expressed in Breast Cancer (BC) is fascinating given that the tumor is predominantly estrogen-dependent. The androgen receptor (AR) is emerging as a new marker and a potential new therapeutic target in the treatment of BC patients. The recent availability of selective AR inhibitors (e.g. bicalutamide, enzalutamide, apalutamide) approved for the treatment of prostate cancer has opened up the possibility to use them in BC patients whose tumors express AR. However, AR appears to have various functions according to the BC subtype, e.g. ER-positive or triple negative BC and the patient prognosis is different on the basis of the presence or absence of estrogen and progesterone receptors. Moreover, a different AR expression was seen according to the various ethnicities. Of note, in population at low economical income, the availability of anti-AR compounds at low cost could open the possibility to treat AR-positive triple negative BC that are highly present in these populations. Up to now, AR detection is not routinely performed in BC. The standardization of AR detection methods could render AR an easily detectable marker in primary BC and metastatic samples. Nevertheless, the overall concordance of 60% of AR expression in primary tumor and metastasis implies that a clinician who need the AR value to give anti-AR therapy should have the data on both the tumor materials. Following the comprehensive studies on prostate cancer the possibility to test AR on liquid biopsies suggest the use of this biomarker for a real-time disease monitoring. Finally, considering the possibility to treat patients with immune checkpoint inhibitors there is the need to know the relation between microenvironment and AR in BC.

BRCA1 AND microRNAs 7, 10B, 205ab, 218ª expression as prognostic markers in primary breast cancers – a retrospective cohort study

BRCA1 AND microRNAs 7, 10B, 205ab, 218ª expression as prognostic markers in primary breast cancers – a retrospective cohort study

Abstract P2-02-04: Distinct clinical and biological values of subpopulations of circulating tumor cells (CTCs) in primary breast cancer

Abstract Background: CTCs represent a heterogeneous population of cells with different phenotypes and biological values. Epithelial to mesenchymal transition (EMT) gives rise to cells with stem cell-like properties with increased resistance to chemotherapy that may be under detected by currently approved assays. The aim of this study was to characterize CTCs based on the expression of epithelial and mesenchymal markers in primary breast cancer (BC) and to correlate them with patients'/tumor characteristics. Methods: This prospective translational study included 422 patients with primary BC enrolled from March 2012 to February 2015. Blood for CTC detection was drawn before surgery (422 patients), before 1st cycle (95 patients) and before 2nd cycle (53 patients) of adjuvant therapy. Isolated peripheral blood mononuclear cells (PBMC) were depleted of cells of hematopoietic origin (CD45+) using RossetteSep kit (StemCell Technologies) negative selection with anti-CD45 antibody. RNA extracted from CD45-depleted (CD45) PBMC was interrogated for expression of EMT-inducing transcription factors (TWIST1, SNAIL1, SLUG, ZEB1) and epithelial (CK19) gene transcripts by quantitative reverse transcription-PCR. Expressions of gene transcripts in CD45- PBMC from patients were compared to those of CD45- PBMC of 60 healthy donors. Results: Totally, CTCs were detected in 116/422 (27.5%) patients before surgery, in 21/95 (22.1%) patients after surgery and before 1st cycle and in 19/53 (35.8%) of patients before 2nd cycle of adjuvant therapy. Before surgery, CTCs exhibited only epithelial markers in 38 (9.0%) patients, only EMT markers in 68 (16.1%) of patients, while in 10 (2.4%) patients CTCs with both epithelial and EMT markers were detected. Epithelial CTCs were more often detected before surgery compared to after surgery (11.4% vs. 2.1%; p = 0.003), while mesenchymal CTCs were more often detected after the 1st cycle of chemotherapy as opposed to detection before surgery (30.2% vs. 18.2%; p = 0.05). Patients with N2-3 disease had more often detectable CTCs compared to patients with N0-1 disease (41.4% vs. 24.9%, p = 0.01) and this was mainly driven by mesenchymal CTCs (31.0% for N2-3 vs. 16.0% for N0-1; p = 0.007). Similarly, patients that lacked p53 expression (wild type TP53) in primary tumor had more often CTCs with EMT phenotype opposite to patients with p53 expression (p = 0.02). Presence of epithelial CTCs was significantly associated with lower absolute lymphocyte (p = 0.02) and neutrophil (p = 0.02) counts in peripheral blood. Conclusions: Our results support the concept of CTCs phenotypic heterogeneity in breast cancer patients. These results support the role of EMT in cancer pathogenesis and suggest that CTCs with EMT phenotype are involved in tumor dissemination while their increase after chemotherapy might be a mechanism of treatment resistance. Moreover, these data suggest inverse relationship between immune cells and epithelial CTCs which stress the role of immune cells in tumor dissemination. Citation Format: Mego M, Jurisova S, Karaba M, Minarik G, Benca J, Sedlackova T, Manasova D, Malejcikova M, Sieberova G, Macuch J, Gronesova P, Sufliarsky J, Pindak D, Cristofanilli M, Reuben JN, Mardiak J. Distinct clinical and biological values of subpopulations of circulating tumor cells (CTCs) in primary breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-04.

Abstract P3-06-34: Platelet predominant breast cancer is a new predictor for pathological complete response to neoadjuvant chemotherapy

Abstract Background and Objective: Platelets, the smallest anucleate hematopoietic cells, are increasingly recognized as the key regulator of tumor progression and metastasis in breast cancer. They contribute significantly to hematogenous metastasis, which increases tumor invasiveness, by protecting tumor cells from shear stress or immune surveillance and facilitating their extravasation to distant sites. Additionally, recent studies have demonstrated direct signaling between platelets and tumor cells during epithelial–mesenchymal transition (EMT) in the blood stream. However, the existence of platelets in primary breast cancer and its relationship with clinicopathological factors and pathological response to chemotherapy remain poorly understood. This study aimed to investigate (1) the presence of platelet infiltration in tumor tissue, (2) relationships between platelets and clinicopathological features, and (3) the association of platelets with pathological complete response (pCR) to neoadjuvant chemotherapy and EMT markers in primary breast cancer. Methods: We retrospectively analyzed the pre-chemotherapeutic biopsy specimens from 100 breast cancer patients who underwent surgical resection after anthracycline/taxane-based neoadjuvant chemotherapy at Kanazawa University Hospital. Platelet subsets (glycoprotein Ib [CD42b]) and the expression of EMT markers (E-cadherin, vimentin, and β-catenin) were evaluated by immunohistochemistry and correlated with pCR after neoadjuvant chemotherapy. Platelet-predominant breast cancer (PPBC) was defined as the presence of ≥10% of anucleate cells positively stained for CD42b in direct contact with tumor cells. pCR was defined as the absence of residual invasive tumor cells in breast and lymph nodes (ypT0/is, ypN0). Results: PPBC was observed in 48 patients (48%). The prevalence of PPBC was significantly higher in human epidermal growth factor receptor 2 (HER2)-negative patients (HER2 [+] vs. HER2 [-]; 65% vs. 90%, p = 0.008). There was no significant association between CD42b expression and stage, nuclear grade, histology, and estrogen receptor status. PPBC patients had a significantly decreased pCR rate (6/48, 12.5%) compared to non-PPBC patients (33/51, 64.7%; p < 0.001). Tumor cells surrounded by platelets exhibited EMT-like morphological changes, EMT marker expression, nuclear-staining of β-catenin, loss of E-cadherin expression, and expression of vimentin. Conclusions: Our results suggested the presence of tumor-associated platelets in breast cancer as a new predictor of response to neoadjuvant chemotherapy. Platelets might facilitate EMT at the primary site by potentiating tumor cell adhesion. Platelet-tumor interaction might be a new therapeutic strategy for breast cancer. If our finding is validated in further investigations, it might serve as the basis for novel therapeutic approaches of combining conventional chemotherapy with antiplatelet therapy. Our data also identified new predictive parameters to stratify patients with an increased chance of response to anthracycline/taxane-based neoadjuvant chemotherapy. Citation Format: Satoko Ishikawa, Masafumi Inokuchi, Hironori Hayashi, Katsunobu Oyama, Hisatoshi Nakagawara, Tomoharu Miyashita, Hidehiro Tajima, Hiroyuki Takamura, Itasu Ninomiya, Hirohisa Kitagawa, Sachio Fushida, Takashi Fujimura, Tetsuo Ohta. Platelet predominant breast cancer is a new predictor for pathological complete response to neoadjuvant chemotherapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-34.

Cyclooxygenase-2 expression in non-metastatic triple-negative breast cancer patients.

Triple-negative breast cancer (TNBC) is characterised by lack of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER)2/neu gene amplification. TNBC patients typically present at a younger age, with a larger average tumor size, higher grade and higher rates of lymph node positivity compared to patients with ER/PR-positive tumors. Cyclooxygenase (COX)-2 regulates the production of prostaglandins and is overexpressed in a variety of solid tumors. In breast cancer, the overexpression of COX-2 is associated with indicators of poor prognosis, such as lymph node metastasis, poor differentiation and large tumor size. Since both TNBC status and COX-2 overexpression are known poor prognostic markers in primary breast cancer, we hypothesized that the COX-2 protein is overexpressed in the primary tumors of TNBC patients. The purpose of this study was to determine whether there exists an association between TNBC status and COX-2 protein overexpression in primary breast cancer. We prospectively evaluated COX-2 expression levels in primary tumor samples obtained from 125 patients with stage I-III breast cancer treated between February, 2005 and October, 2007. Information on clinicopathological factors was obtained from a prospective database. Baseline tumor characteristics and patient demographics were compared between TNBC and non-TNBC patients using the Chi-square and Fisher's exact tests. In total, 60.8% of the patients were classified as having ER-positive tumors, 51.2% were PR-positive, 14.4% had HER-2/neu amplification and 28.0% were classified as TNBC. COX-2 overexpression was found in 33.0% of the patients. TNBC was associated with COX-2 overexpression (P=0.009), PR expression (P=0.048) and high tumor grade (P=0.001). After adjusting for age, menopausal status, body mass index (BMI), lymph node status and neoadjuvant chemotherapy (NACT), TNBC was an independent predictor of COX-2 overexpression (P=0.01). In conclusion, the association between TNBC and COX-2 overexpression in operable breast cancer supports further investigation into COX-2-targeted therapy for patients with TNBC.

Trefoil factor 1 as a predictive factor of bone metastases in breast cancer.

11022 Background: Patients with breast cancer frequently develop bone metastases, which are responsible for high morbidity and reduced quality of life. The early identification of patients with a high probability of relapsing in this site could be used to select candidates for tailored therapy with bone-specific drugs such as bisphosphonates or RANK-L inhibitors. We aimed to identify a pattern of tissue markers in primary breast cancer that could predict bone metastatization. Methods: Expression of different markers was retrospectively analyzed in frozen breast cancer tissue samples from 90 patients comprising 30 cases with no evidence of disease (NEDP), 30 with bone metastases (BMP) and 30 with visceral metastases (VMP). Eight transcripts were analyzed by Quantitative Real time PCR: trefoil factor 1(TFF1), bone sialoprotein (IBSP), heparanase (HPSE), secreted protein acidic and rich in cysteine (SPARC), connective tissue growth factoe (CTGF), B2 microglobulin (B2M) and receptor activator of Nf-kB (RANK). Immunohistochemistry of TFF1 was performed on a part of the case series. Results: Marker expression analysis in the 3 different subgroups showed at least twofold higher median values of all markers in NEDP and VMP subgroups than in BMP. In particular, TFF1, B2M and CXCR4 levels showed statistically significant values. Median TFF1 value in BMP patients was 430.64 compared to 115.83 and 32.79 in VMP and NEDP, respectively (p=0.0043). Considering markers as dichotomous variables, TFF1 expression in BMP reached 59% compared to 21% and 23 % in NEDP and VMP, respectively (p=0.0022). Univariate analysis confirmed that TFF1 predicted the relapse and also the site of relapse. Immunohistochemistry data on TFF1 revealed that this protein was expressed only by cancer cells. Furthermore, the accuracy of the marker did not change at RNA or protein level, thus excluding a post transcriptional control of the RNA. Conclusions: In this preliminary study we identified a gene expression pattern in primary breast cancer that can identify patients destined to relapse to the bone. In particular, TFF1 would seem to be a suitable marker for bone metastatization and a possible target for the development of new drugs.

Abstract P3-12-09: The risk of brain metastases according to expression of selected immunohistochemical markers in primary breast cancers

Abstract Background: About 10–30% of breast cancer patients will develop brain metastases. In untreated patients with brain metastases the median survival is 1–2 months, and in those undergoing palliative radiotherapy — 3–6 months. The mechanism of brain metastases remains largely unknown. The identification of molecular markers might help in selecting high risk patients, and enable active surveillance, prevention and early treatment. The aim of this study was to analyze predictive value of expression of selected tumor proteins for the risk of brain metastases in breast cancer patients. Material and methods: This study included 198 advanced breast cancer patients treated between 2001 and 2007 in 11 oncology centers in Poland, including 96 woman with and 102 without overt brain metastases, respectively. The median age at diagnosis in these two groups was 52 and 60 years, respectively, with 52% and 32% of patients being premenopausal. Stage at diagnosis was similar in both groups and ductal carcinoma was a dominant histological type (76% and 86% of cases, respectively). Immunohistochemistry was performed on formalin-fixed paraffin embedded microarray cores derived from the primary tumor. Expression analysis included ER, PR, HER2, Ki67, CK5/6, EGFR, HER3, CXCR4, RAD51, E-cadherin, and claudin 3 and 4. Cox regression model was used to estimate the relative risk of brain metastases. Results: Expression of HER2, CK5/6, EGFR, RAD51 (both cytoplasmatic and nuclear staining), CXCR4 (cytoplasmatic staining) and Ki67 ≥14%, as well as ER or PR negativity was associated with increased risk of brain metastases in the univariate analysis (Table 1). Of those, Ki67 ≥14% (HR 2.76 [95%CI 1.70–4.48]; p < 0.001), cytoplasmatic expression of double strand DNA repair gene RAD51 (HR 1.87 [95%CI 1.14–3.08]; p = 0.014) and ER negativity (HR 1.72 [95%CI 0.36–0.94]; p = 0.029) were found to be significantly related to the risk of brain relapse in the multivariate analysis. Four molecular profiles composed of the latter three markers were created, of which a profile including ER, Ki67 and RAD51 was associated with the highest risk of brain metastases (HR 4.43 [95%CI 2.69–7.27]; p < 0.001). Molecular subtype analysis showed the highest risk of BM in the ER/PR/ HER2-negative (triple negative) subset (HR 1.21 [95%CI 1.11–1.32]; p < 0.001). Conclusion: Expression of proteins related to high tumor proliferation, DNA repair and ER negativity is associated with increased risk of brain metastases in breast cancer patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-12-09.

Stem cell-related markers in primary breast cancers and associated metastatic lesions

AbstractIt has been reported previously that: (1) normal-breast epithelial cells that are CD24−/44+ express higher levels of stem/progenitor cell-associated genes; (2) cancer cells that have undergone epithelial to mesenchymal transition display CD24−/44+ cell-surface expression, a marker for breast cancer stem cells; (3) loss of E-cadherin is a preliminary step in epithelial to mesenchymal transition; and (4) vimentin is a marker of mesenchymal phenotype. We hypothesized that stem cell subpopulations would be more frequent in metastatic than in primary tumors. Therefore we assessed by immunohistochemical analysis, tissue microarrays containing tissue from primary and associated metastatic breast cancers for expression of CD24, CD44, E-cadherin and vimentin to evaluate candidate cancer-initiating cell populations in breast cancer subtypes and metastatic lesions. The occurrence of CD24−/44+ and CD24+/44− cells did not differ in primary vs matched lymph node or distant and locoregional metastatic lesions; E-cadherin expression was decreased in primary vs lymph node metastases (P=0.018) but not decreased in distant and locoregional metastases relative to primary tumor, whereas vimentin, was more frequently expressed in lymph node and distant and locoregional metastases (P=0.013, P=0.004) than in matched primary cancers. Thus, the frequency of CD24−/44+ cells does not differ in metastases relative to the primary breast cancer but differs by tumor stage and subtype.

Clinical Usefulness of Cancer Markers in Primary Breast Cancer

The aim of this study was to investigate the diagnostic power of CA 549, MSA and CA 15-3 in identifying breast cancer. The study included 232 patients of which 56 were healthy, 43 had benign breast cancer and 191 with other growths. The results were obtained using a specific immunoassay and using producers' cut offs. The following sensitivity and specificity of markers were found: CA 549 (sen.: 40%/spec.: 90%), MSA (sen.: 22%/spec.: 96%), and CA 15-3 (sen.: 33%/spec.: 86%). Ideal cut offs were defined with ROC curves. A significant correlation was found between CA 549, MSA, and CA 15-3. The combination of markers does not improve the clinical usefulness to identify only breast cancer. Serum tumor markers are abnormally elevated in patients with breast cancer. CA 549, MSA, CA 15-3 are useful clinical markers, good indicators of disease extent, and may have important prognostic value. This study demonstrates the role of the tumor markers in breast cancer.

Abstract 5167: A new panel of tissue markers for the prediction of bone metastatization from primary breast cancer

Abstract Background: It is known that numerous molecular markers associated with proliferation, invasion and adhesion processes, growth factors, chemokines and their receptors, and molecules with bone-like phenotype are involved in metastasization to bone. Only cancer cells with specific molecular profiles are capable of reaching and colonizing bone tissue. The early prediction of relapse in this site could be used to select candidates for tailored therapy with bone-specific drugs such as bisphosphonates or RANK-L inhibitors. In the present study we aimed to identify primary tumor markers capable of predicting relapse to bone. Material and methods: The expression of different markers was retrospectively analyzed in frozen breast cancer tissue samples from 40 patients comprising 15 cases with no evidence of disease (NEDP), 15 with bone metastases (BMP), and 10 with visceral metastases (VMP). Twelve transcripts were analyzed by Quantitative Real time PCR: trefoil factor 1(TFF1), DKK1, bone sialoprotein (IBSP), heparanase (HPSE), osteopontin (SPP1), Agr2, SPARC, CTGF, COMP, follistatin (FST), osteoprotegerin and RANK. The predictive accuracy of each marker was calculated using receiver operating characteristic (ROC) curves. Results: The analysis of marker expression in the 3 different subgroups showed twofold higher median values of COMP and HPSE in BMP with respect to either NEDP or VMP (no significant difference). Furthermore, TFF1 median value was about sevenfold higher in BMP than in the other 2 subgroups (p=0.05). The area under the curve (AUC) was 0.73 for TFF1 and 0.62 for COMP and HPSE. In the dichotomous variable analysis, the most important result was observed for TFF1; in particular, TFF1 positivity was observed in 67% of cases in the BMP subgroup compared to only 21% and 20 % in NEDP and VMP, respectively. The combination analysis of TFF1 and other markers showed that positivity to at least one of the three markers, TFF1, DKK1, or IBSP, was observed in 80% of cases for the BMP group, and in only in 21% and 20% of cases in NEDP and VMP subgroups, respectively (p=0.041). Conclusions: The results from this preliminary study suggest that Real-Time PCR analysis of gene expression and, in particular, the coexpression of some markers in primary breast cancer could identify patients destined to bone relapse who could benefit from adjuvant treatment with bone-specific drugs. 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 5167. doi:10.1158/1538-7445.AM2011-5167

Abstract P1-13-04: Immunohistochemical Evaluation of RANK/RANKL/OPG Axis and CXCR4 in Primary Breast Cancer and Their Role in Bone Metastasization

Abstract Background: The RANK/RANKL/OPG axis plays an important role in the bone metastasization process. CXCL12 is overexpressed in bone and, like its receptor, CXCR4, is a determinant of organ tropism. The objective of the present study was to evaluate whether the expression of these markers in primary breast cancer can predict the onset of bone metastases. Methods: Marker expression was evaluated by immunohistochemical staining in paraffin-embedded sections of primary breast cancers from 40 individuals: 10 patients (median age 64 years, range 48-78) showed no evidence of disease (NED — control group) at a median of 9.8 (range 6.9-11.5) years, while 30 (median age 67 years, range 42-87) had relapsed. In the latter group, 10 (median age 66 years, range 42-87) had visceral metastases (VM — control group) and 20 (median age 69 years, range 42-87) had bone metastases (BM — case group), both confirmed radiologically. The 3 patient subgroups (NED, visceral lesions, bone lesions) were matched for age classes (≥60 years, > 60 years). The study design was reviewed and approved by the local ethics committee. Results: In the overall series, 17.5% of tumors were positive for RANK, 22.5% for OPG and 25% for CXCR4. None of the patients expressed RANKL. RANK and OPG resulted equally expressed in 20% of NED patients, while CXCR4 was expressed in only 10% of this group. OPG was expressed in 20% of VM patients, whereas RANK or CXCR4 were not detected. RANK and OPG positivity was present in 25% of BM patients, while CXCR4 was expressed in 45% of cases. CXCR4 was the only marker with a significantly higher frequency of expression in bone metastases than in visceral lesions (p=0.013). Taking into consideration all the patients without bone involvement (NED plus VM patients), it was observed that CXCR4 expression, alone and in combination with RANK, significantly discriminated between BM patients and the control group (p=0.008 and P<0.001, respectively). ER-positivity and HER2-negative status were observed in 80% and 95% of BM patients, respectively, but neither discriminated between cases and controls. Conclusions: In our study, ER-positivity and HER2- negativity identified a high percentage of bone relapse patients, but also highlighted patients who did not have bone involvement. RANK and CXCR4 expression, on the other hand, would appear to be a more accurate predictor of bone relapse. Such evidence could help to identify patients with a high probability of developing bone metastases, which can be treated with bisphosphonates or other bone-targeted therapies, such as denosumab, in an attempt to prevent distant metastases. Our results are currently being validated in a larger series of breast cancer patients. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-13-04.

560 BRCA1 protein expression correlates with cancer stem cell markers in primary breast cancer

Tumor immunology fundamentals suggest immunological surveillance has the ability to recognize malignant cells and kill them before a tumor develops. However, cancer cells employ evasion mechanisms whereby the immune system may be actively suppressed or even tolerized to the tumor. Recently cancer stem cells were linked to tumor initiation and formation. However, no reports have addressed whether these cells participate in a tumor’s ability to evade immune surveillance. Recently the glycoprotein CD200, expressed within the innate immune system and other tissues and cells, was shown to be involved in tolerance. Here we describe CD200 co-expression with stem cell markers found on prostate, breast, brain, and colon cancers. This is the first report describing an immunomodulatory molecule on epithelial cancer stem cells. This important finding suggests a mechanism by which a tumor might evades immune system detection.

Accuracy of RANK/OPG and CXCR4 compared to hormone receptors in predicting bone metastases in patients with breast cancer.

e11507 Background: The RANK/RANK-L/OPG system is an important part of the bone metastasization process. CXCL12 is overexpressed in bone and like its receptor, CXCR4, is a determinant of organ tropism. The objective of the present study was to evaluate whether the expression of these markers in primary breast cancer can predict the onset of bone metastases. Methods: Marker expression was evaluated by immunohistochemical staining in paraffin-embedded sections of primary breast cancers from 40 individuals: 10 patients (median age 64 years, range 48-78) were disease-free (DFP) at a median of 9.8 (range 6.9-11.5) years, while 30 (median age 67 years, range 42-87) had relapsed. Within the latter subgroup, 10 (median age 66 years, range 42-87) had visceral metastases (VMP), and 20 (median age 69 years, range 42-87) had bone metastases (BMP). Results: In the overall series, 68% of tumors were ER-positive and 45% were PgR-positive. Moreover, 17.5% of tumors were positive for RANK, 22.5% for OPG and 25% for CXCR4. No significant associations were observed among the different markers. Analyzing the different patient subgroups, it was seen that RANK and OPG were equally expressed in 20% of DFP and in 25% of BMP. CXCR4 was expressed in only 10% of DFP but in 45% of BMP. Moreover, 20% of VMP expressed OPG but none showed RANK or CXCR4 expression. None of the patients expressed RANKL. The highest sensitivity in predicting bone metastasis was observed for ER positivity (80%), which, however, showed low specificity (30%), followed by triple positive RANK, CXCR4 and OPG (75%). Moreover, the highest specificity was observed for CXCR4 (90%) followed by RANK or OPG (80%). Conclusions: Our results indicate that, despite high sensitivity, ER-positive status is burdened by low specificity. Conversely, RANK, OPG, and CXCR4 expression would appear to be a more accurate predictor of bone relapse. The validation of these results is ongoing in a larger series of patients with breast cancer. No significant financial relationships to disclose.

Numb protein expression correlates with a basal-like phenotype and cancer stem cell markers in primary breast cancer

Decreased expression of Numb, resulting in activation of the proto-oncogene Notch1 and reduction in the tumor suppressor p53, has been demonstrated in mammary carcinomas. The aim of this study was to investigate the relationship between Numb protein expression and clinicopathological characteristics, tumor biological subtypes and putative cancer stem cell markers in a well-characterized cohort of primary human breast cancers. Immunohistochemistry was performed on tissue microarrays of primary invasive breast tumors using a polyclonal anti-Numb primary antibody. Of the 241 tumors evaluated, 50 (21%) displayed deficient or reduced Numb immunoreactivity. Retained Numb expression was significantly correlated to estrogen (ER) and progesterone receptor (PR) positivity (P < 0.001 and P = 0.004, respectively). Interestingly, we found that a higher percentage of the tumors with deficient or reduced Numb expression belonged to the triple-negative (ER-/PR-/HER2-) subgroup compared to tumors with retained Numb expression (P = 0.004). Transcriptional profiling of a subset of these tumors linked NOTCH1 and BIRC5, both downstream targets of Numb, to the triple-negative subgroup in an inverse manner. Typically, subgroups characterized by the low expression of Numb expressed higher levels of NOTCH1 and BIRC5 (encoding survivin). We also found deficient expression of Numb in a significantly higher proportion of BRCA1 dependent tumors, which are usually triple-negative, compared to sporadic tumors. The expression of Numb in 14 breast cancer cell lines correlated similarly to their respective molecular subtypes. We further established an inverse correlation between the Numb expression levels and the CD44+/CD24- cancer stem cell phenotype (P = 0.05) in primary tumors. Finally, decreased Numb expression was associated with poorer distant disease-free survival (P = 0.01). Taken together, our results indicate that loss of Numb expression is a marker of tumor aggressiveness, potentially linked to BRCA1 status and a cancer stem cell phenotype in primary breast cancer.

Identification of biomarkers associated with metastastic breast cancer to bone by immunohistochemistry on tissue microarrays.

Abstract Abstract #2065 Background: Bone is the most common site of breast cancer metastasis. Efforts have been made to understand the complex molecular mechanisms underlying this. It involves invasion, proliferation at metastatic sites, and activity of cytokines (IL1,6,8,11, TNF, and TGFß), chemokines, their receptors (stromal-derived-factor1 and CXC chemokine[CXCR4, also called fusin, involved in HIV tropism]), and matrix metalloproteinases (MMP). Microarray analyses of primary tumors with different metastatic sites have distinct gene expression patterns indicating tumor heterogeneity. Both lung and bone metastasis gene signatures include CXCR4, and MMP1, whereas bone metastasis gene signature also includes IL11. PTHrP, activated by TGFß signaling, also mediate metastasis. A subpopulation of breast cancer cells, expressing CD44 with stem cell properties, was found to promote breast tumor invasion and metastases. Trefoil factor1 (TFF1), a proangiogenic factor, involved in invasion of kidney and colonic cancer cells, has a high correlation with bone metastasis, is expressed under estrogen control, and found in a subset of ER+ breast cancers. New prognostic markers are needed to identify patients at high risk of bone metastases to individualize therapy. This study's aim was, for the first time, to evaluate the expression of selected biomarkers associated with tumor relapse to bone, by immunohistochemistry (IHC) on tissue microarrays (TMA).&amp;#x2028; Materials and Methods: 16 cases of metastastic breast cancer to bone (MBC-B) were retrieved from MWH tumor registry and a TMA with 3-fold redundancy was created (TMA1), consisting of cores with a diameter of 0.6 mm. To study the expression of these markers in primary breast cancers (PBC), a second TMA (TMA2) was created for 63 breast cancers. IHC was performed for selected markers (TFF1,CXCR4, MMP1, PTHrP, CD44, FGFR3, and IL11) on both TMAs. Clinical and other pathologic parameters were available. All patients had received standard chemotherapy/hormonal treatment.&amp;#x2028; Results: In the MBC-B group, 14 cases were infiltrating ductal and 2 cases were infiltrating lobular (average tumor size 2.6cm;range 0.4-8.0cm). 50% (8/16) had axillary lymph node metastases. 94% (15/16) were ER and PR positive and 19% (3/16) were HER2 positive. 44% (7/16) were rapid bone metastasis progressors (&amp;lt;1y) and 56% (9/16) were slow progressors (&amp;gt;1y). Currently, no statistical difference in the expression of markers in MBC-B vs PBC was seen (100% for CXCR4, MMP1, PTHrP, FGFR3, IL11). However, a high expression of ER, CD44, and TFF1 is seen in MBC-B. Ongoing studies are evaluating whether these markers are independent prognostic markers for bone metastases and tumor specific survival.&amp;#x2028; &amp;#x2028; Discussion: We found a high level of expression of CXCR4, MMP1, PTHrP, FGFR3, and IL11 which signifies their importance in both PBC and MBC-B. Overall, expression of ER, CD44, and TFF1 potentially may serve as prognostic markers in identifying, at an early stage, patients at risk of bone metastases. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2065.

Serum HER-2 concentration in patients with primary breast cancer

Objective: To evaluate whether serum HER-2/neu (HER-2) concentration is a valid index of HER-2 status in women with primary breast cancer, and to establish a normal reference range for serum...

BM micrometastases and circulating tumor cells in breast cancer patients: where have we been, where are we now and where does the future lie?

Over the past 15 years early tumor cell dissemination has been detected in patients with breast cancer using sensitive immunocytochemical and molecular assays based on the use of MAb and PCR, respectively. Clinical studies involving more than 4,000 breast cancer patients have now demonstrated that the presence of disseminated tumor cells in BM identified with immuncytochemical assays at primary diagnosis is a strong and independent prognostic factor. The published studies for the detection of disseminated tumor cells in BM fulfill the highest level of evidence as prognostic markers in primary breast cancer. In addition, various assays for the detection of circulating tumor cells in the peripheral blood have been developed recently and some studies suggest a potential clinical relevance of this parameter as a prognostic and predictive factor. Comparative analyzes indicate that the prognostic information derived from BM and blood screening seems to be complementary and not redundant. Advanced methods for molecular characterization of single tumor cells and the surrounding environment have been developed lately, and this approach allows new insights into the metastatic cascade and characterization of targets for therapeutic approaches. Taken together, these findings provide the basis for the implementation of disseminated tumor cells in BM or blood as markers for stratification and assessment of therapies in prospective clinical trials. The valuable information derived from these trials should help to improve future treatment of breast cancer patients.

Tumor tissue levels of tissue inhibitor of metalloproteinase-1 as a prognostic marker in primary breast cancer.

In the present study, we investigated the association between tumor tissue levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and prognosis in patients with primary breast cancer and analyzed whether TIMP-1 may be useful as a prognostic marker in combination with urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1). In cytosolic extracts of 2984 primary breast tumors, total levels of TIMP-1 were determined using an established, validated ELISA. Levels of uPA and PAI-1 have previously been determined in the extracts. Univariate survival analysis showed a significant relationship between higher levels of TIMP-1 (continuous log-transformed variable) and poor prognosis [recurrence-free survival (RFS), overall survival (OS); P < 0.001]. Performing isotonic regression analysis, we identified a cut point to classify tumors as TIMP-1-low or TIMP-1-high. Using this cut point, high levels of TIMP-1 were significantly associated with shorter survival in univariate analysis, both in the total patient group (RFS, OS; P < 0.001), in the node-negative subgroup (RFS, hazard ratio = 1.28, P = 0.006), and in the node-positive subgroup (RFS, hazard ratio = 1.43, P < 0.001). In multivariate analysis, including uPA and PAI-1, TIMP-1 was significantly associated with shorter RFS, both when included as a continuous log-transformed (P = 0.03) and as a dichotomized variable (P = 0.002). This study validates previous findings that tumor tissue levels of TIMP-1 are associated with prognosis in patients with primary breast cancer. It confirms that TIMP-1 may be useful as a prognostic marker in combination with uPA/PAI-1 and adds substantial positive information on the use of TIMP-1 as a prognostic marker in breast cancer.