Biomarkers In Metastatic Breast Cancer Research Articles

Abstract P2-05-18: Deregulation of the Abl interactor 1 signaling as a potential biomarker for metastatic breast cancer.

Abstract Withdrawn by Author Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-05-18.

A targeted glycoproteomic approach identifies cadherin-5 as a novel biomarker of metastatic breast cancer

Aberrant glycosylation has long been recognised as a hallmark of cancer, and is increasingly being exploited in biomarker discovery studies. Helix pomatia agglutinin (HPA) is known to bind aberrant glycans associated with metastatic breast cancer, and was used here to isolate glycoproteins from pooled breast cancer serum samples of (i) patients with recurrent breast cancer and (ii) patients with no sign of recurrence 5years after diagnosis of their primary tumour. Pregnancy zone protein, the polymeric immunoglobulin receptor and cadherin-5 emerged as potential markers of metastasis following proteomic identification of HPA binding glycoproteins. ELISAs were developed to verify these findings, and to assess protein glycosylation, in individual patient sera. The cadherin-5 ELISA discriminated serum samples of patients with recurrent breast cancer from those with no sign of recurrence, and analysis of cadherin-5 glycosylation by HPA also showed a significant difference between the two sample groups. The targeted glycoproteomic and validatory approach developed here has shown that when taking into account both the protein levels and HPA binding, serum cadherin-5 discriminated patients with recurrent breast cancer from those with no sign of recurrence with 90% specificity.

Abstract 695: Significant loss of nuclear expression of Actinin-4 in metastatic breast carcinoma and lymph nodes: A novel biomarker for metastatic breast carcinoma

Abstract Background: Actinin-4 is an actin cross-linked protein. The expression and subcellular distribution of actinin-4 (ACTN4) has been associated with multiple roles in tumorigenicity and cancer metastasis. In the present study, we developed a novel antibody against human actinin-4 and investigated the clinical utility of actinin-4 as a biomarker in metastatic breast cancer. Methods and findings: We generated a novel monoclonal antibody (AB-Actinin-4) using hybridoma technology and native ACTN4 protein as an immunogen. The antibody specifically recognized both native and denatured human Actinin-4 protein and displayed high affinity as assessed by ELISA, immunohistochemistry assay, western blot and flow cytometry. Using this antibody and ELISA as well as immunohistochemical techniques, we examined plasma levels and tissue expression of actinin-4 in healthy controls’ and breast cancer patients’ samples. 50 plasma samples from healthy controls and breast cancer patients were analyzed for the presence of soluble actinin-4. ELISA results showed significantly decreased actinin-4 levels in plasma samples from patients with breast carcinomas compared to healthy control plasma. A total of 156 cases, including 70 normal, 36 non-metastatic breast carcinoma, 50 breast carcinoma tissues with regional metastasis, and 50 matched metastatic lymph nodes were examined. The immunoreactivity of actinin-4 was located mainly in nucleus and cytoplasm of cells. To assess the difference between groups, the staining intensity and intracellular distribution were compared and the intensity in nucleus and cytoplasm was separately scored as 0, 1+, 2+ and 3+. Grades 0 and 1+ were defined as negative and 2+ and 3+ as positive. Significant differences between the groups were found in nuclear staining. 92% of normal and 89% of non-metastatic breast carcinoma samples displayed positive nuclear staining. In contrast, only 29% of metastatic lymph nodes and 51% of metastatic breast cancer tissues showed strong nuclear staining. Conclusion: Actinin-4 demonstrated significantly decreased expression in plasma from breast cancer patients. Metastasis leads to significant loss of the nuclear expression of Actinin-4 in breast cancer tissues, in comparison to normal and non-metastatic breast carcinoma tissues. Our data suggests that Actinin-4 might have significant impact in breast cancer metastasis. Actinin-4 may serve as a reliable biomarker for the diagnosis and prognosis of metastatic breast cancer as well as could be a therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 695. doi:1538-7445.AM2012-695

Body fluid MMP-2 as a putative biomarker in metastatic breast cancer

In the present study, we investigated the role of matrix metalloproteinase (MMP)-2 and -9 as novel biomarkers in the body fluid of patients with metastatic breast cancer. We measured the expression of MMPs in 37 samples of body fluid (10 peritoneal and 27 pleural fluids) from metastatic breast cancer patients between 2000 and 2009. Zymography and ELISA assays were used to determine the cut-off level and to quantify MMP expression from a positive control, HT-1080 conditioned media. MMP expression in patient samples was measured with ELISA and compared with other clinical parameters. Ascitic carcinoembryonic antigen (CEA) and pleural CEA were measured in patient samples with a chemiluminescent enzyme immunoassay. Body fluid cytology had a positivity of 45% (9/20) for pleural fluid and 28.6% (2/7) for ascites. However, MMP-2 had a positivity of 85.2% (23/27) in 27 pleural fluid samples and 100% (10/10) in ascitic fluid with cut-off levels of 8.6 and 0.14 ng/ml for MMP-2 and -9, respectively. When body fluid CEA and MMP-2 were combined, the positivity improved to 96% in pleural fluid and 100% in ascites. MMP-2 expression in body fluid did not show any significant differences, but MMP-9 expression was lower in ascites than in pleural fluids (p<0.005). Our results suggest that MMP-2 expression in body fluid be used as an additive diagnostic marker for metastatic breast cancer patients.

Clinical validity of circulating tumor cell (CTC) enumeration in 841 subjects with metastatic breast cancer (MBC).

10592 Background: CTCs are associated with poor clinical outcomes in MBC. Multiple technologies have been evaluated for CTC capture, enumeration, and characterization, but only one (CellSearch; Veridex) has been evaluated independently by different investigators with serial assessments and longitudinal follow-up in a defined prospective clinical trial. We combined these datasets to further validate the prognostic and predictive significance of this biomarker in MBC. Methods: Six studies were identified in the peer-reviewed published literature; all used the same CTC decision point (>=5 CTC/7.5 mL blood) and clinical endpoints (PFS, OS) in patients with progressive MBC. CTC enumeration was performed with the FDA cleared immunomagnetic/immunofluorescence assay (CellSearch). Study teams contributed blinded data to create a dataset of 841 subjects with CTC results linked to clinicopathologic variables and survival. Analyses were defined prior to data collection and conducted in subsets where pertinent data were available. Results: >=5 CTCs predicted for worse PFS at baseline (HR 1.32; CI 1.13, 1.55) and 2-5 (HR 2.16; CI 1.67, 2.78), 6-8 (HR 2.75; CI 2.05, 3.70), 9-12 (HR 2.71; CI 2.08, 3.52), 13-20 (HR 2.64; CI 2.03, 3.42), and >=21 wks (HR 1.88; CI 1.45, 2.42) from initiating new therapy (tx). OS results were also significant. The predictive value of CTC level was not affected by type (chemotx, endocrine tx, biologic tx) or line (first, second, >=third) of tx, presence/absence of bone metastases, or presence/absence of disease limited to bone. Multivariate Cox regression analysis was performed with the baseline CTC level adjusting for clinicopathologic variables to predict PFS and OS; some factors maintained an association, but CTC level was the strongest predictor of outcomes. Conclusions: This analysis substantiates the prognostic and predictive utility of CTCs assessed before and during tx for MBC. Persistently elevated CTC levels are highly associated with treatment failure irrespective of clinicopathologic variables, disease subtype, and type or line or tx. Serial CTC assessments should be used as a surrogate marker for patient outcomes to assist in selecting appropriate treatment regimens in MBC.

Elevated Circulating Myeloid Derived Suppressor Cells (MDSC) Are Associated with Inferior Overall Survival (OS) and Correlate with Circulating Tumor Cells (CTC) in Patients with Metastatic Breast Cancer.

Abstract Background: MDSC acumulation is an important mechanism of tumor escape from immune surveillance and cancer progression. Pre-clinical studies have shown a correlation between tumor progression and MDSC. While we and others have shown that circulating MDSC correlate with clinical stage, to date there are few clinical studies that have explored the correlation of MDSC with metastatic tumor burden or as an adverse prognostic factor. Consequently, the goal of this study was to determine whether levels of MDSC in patients with stage IV breast cancer correlate with tumor burden using CTC and Swenerton Scores (SS) as surrogates for metastatic disease burden, and if circulating MDSC levels were predictive of overall survival.Methods: Flow cytometric analysis was performed on peripheral whole blood from 25 patients with stage IV breast cancer prior to initiation of therapy and at defined intervals during therapy. The MDSC population was defined as lineage-, CD33+, HLA-DR-, CD11b+. CTC (CellSearch®) and MDSC were drawn simultaneously and SS were calculated using a standardized scoring system previously defined and published in the literature. A generalized estimating equation regression model was created and fitted individually for each predictor. OS was defined as time of study enrollment to date of death.Results: Using a generalized estimating equation regression model the percentage of MDSC in peripheral blood was found to have a close correlation with CTC (Pearson correlation 0.62, p=0.0001) and a coefficient of 0.29 (0.14, 0.43 95%CI). The correlation between percent MDSC and SS was not significant (Pearson 0.31, p=0.12). Interestingly, no significant correlation between CTC and SS was noted either. MDSC levels were also found to predict OS in patients with the highest percentages at the first visit having significantly decreased median overall survival [6.8 vs. 19.6 months; p=0.05]Similarly, high MDSC levels at the final blood draw was also found to correlate with significantly shorter median OS [p=0.023].Conclusions. MDSC correlated well with CTC but not SS. High circulating MDSC levels were associated with significantly decreased overall survival in patients with metastatic breast cancer. MDSC appear to be a promising prognostic immunologic biomarker in metastatic breast cancer. Further studies are needed to better define the role of MDSCs in breast cancer progression and may be a useful therapeutic target to enhance the effectiveness of immune based therapies in breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4135.

Circulating tumor cells (CTC): Assessment of treatment efficacy in metastatic breast cancer (MBC)

10535 Background: Preliminary data in MBC suggest that ≥5 CTC/7.5 mL blood is associated with worse progression free survival (PFS) and overall survival (OS), and that the persistence of ≥5 CTC/7.5 mL blood after the initiation of therapy predicts for treatment failure (NEJM 2004. 351:781.). We are conducting a prospective clinical research study to validate the prognostic and predictive significance of this serum biomarker in MBC. Methods: Serial CTC levels are obtained in patients starting a new systemic treatment regimen for progressive, radiographically measurable MBC. 10 mL samples of peripheral blood are collected before the start of treatment and then at 3–4 week intervals. All subjects are followed prospectively for PFS and OS, and they are offered the opportunity to continue CTC testing upon disease progression. CTC enumeration is performed on a 7.5 mL blood volume using the CellSearch technology (Veridex, LLC; Warren, NJ). Epithelial cells are immunomagnetically separated and fluorescently labeled, and nucleated (DAPI+) cells with the EpCAM+, cytokeratin 8/18/19+, and CD45- phenotype are counted as CTC. Clinical outcomes are based on radiographic studies and physical examination in accordance with RECIST criteria. Results: 46 of 100 subjects have been accrued, and 33 have completed at least one radiographic staging evaluation with a median follow up of 7 mos (range 2–18 mos). Treatment for the 33 evaluable patients includes chemotherapy (27%), endocrine therapy (46%), and combination therapy with a biologic agent (27%). At baseline, 85% (28/33) had at least 1 CTC/7.5 mL (range 1–78), and 27% (9/33) had ≥5 CTC/7.5 mL. Median PFS was 2.57 months and 6.77 months for subjects with ≥5 vs &lt;5 CTC/7.5 mL at baseline, respectively (p=0.02). Conclusions: The current data validate the observation that baseline CTC levels correlate with PFS in patients with MBC and measurable disease. Patient accrual and data analysis are ongoing to confirm that persistent CTC levels ≥5/7.5 mL correlate with a lack of treatment efficacy and therefore are a reliable surrogate marker of disease responsiveness and PFS. [Table: see text]