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EGFr Research Studies
MN/CAIX Research Studies
Ras p21 Research Studies
Serum HER-2/neu Research Studies
TIMP-1 Research Studies
VEGF165 Research Studies
uPA and PAI-1 Research Studies

EGFr Research Studies 1 Allen CL, Chapman CJ, Hitch A, et al. Serum EGFR and HER-2 levels in metastatic breast cancer patients receiving gefitinib therapy. Breast Cancer Res Treat, 2006. 100(suppl 1): Abstract No. 2034 ABSTRACT Introduction: Epidermal growth factor receptor (EGFR) and HER-2/neu are highly homologous members of the cerbB receptor family. Both are pivotal in cell signalling pathways and breast carcinogenesis. HER-2 gene amplification is seen in 20-30% of breast cancers and it is linked to a more aggressive disease state and a poorer prognosis. Serum HER-2/neu levels are routinely measured to monitor Herceptin therapy. EGFR is a target for Gefitinib, a current anti-EGFR therapy. However the value of serum EGFR in predicting therapeutic response and survival in breast cancer is unknown. Aims: To investigate the independent and additive roles of serum EGFR and HER-2/neu levels in a panel of metastatic breast cancer patients, and their role in predicting therapeutic response and overall patient survival. Patients and Methods: Serial serum samples were taken from 32 metastatic breast cancer patients receiving 250-500mg/day Gefitinib (Iressa, Astra-Zeneca, UK), in a phase II trial. All patients were categorised according to UICC criteria and divided into 2 cohorts, clinical benefit (CB=9) (complete response + partial response + stable disease >6 months) or progressive disease (PD=23) (progression in <6 months). Serum EGFR levels were determined using a commercial immunoassay (Oncogene Science Bayer corporation) and HER-2/neu levels were determined using the ADVIA Centaur automated HER-2/neu immunoassay. Statistical analysis was carried out using a paired samples t-test. 2 Hamer PJ, Jarosz DE, Leitzel K, et al. Serum EGFr and HER-2/neu Predicts Poor Survival in Metastatic Breast Cancer. AACC Annual Meeting Abstracts, 2006. Clin Chem Vol 52(6) Suppl,pg A168: Abstract No. E11 ABSTRACT Background: The epidermal growth factor receptor (EGFR/ErbB1) is one of four members of the ErbB family of receptor tyrosine kinases, which also includes HER-2/neu/erbB2, HER-3/ErbB3 and HER-4/ErbB4. EGFR is overexpressed in primary breast cancer and, in some reports, has been shown to be a poor prognostic factor. We have previously reported that metastatic breast cancer (MBC) patients with elevated pretreatment serum HER-2/neu have decreased response to first-line hormone therapy and shortened survival. Methods: Pretreatment serum EGFR levels were determined in samples obtained from a first-line phase III letrozole-tamoxifen trial, as well as in samples obtained from healthy postmenopausal controls using an EGFR ELISA from Oncogene Science/Bayer HealthCare, Diagnostics Division. Results: Serum EGFR from 117 healthy post-menopausal female controls measured 64.1 + 13.3 ng/ml (mean + SD)(Range 39.5-117.1 ng/ml). Using a cutpoint of 44.1 ng/ml as determined from the control group (5% non-parametric method), 62/521 MBC patients (11%) had decreased serum EGFR levels. Patients with decreased serum EGFR had no significant difference in objective response rate (ORR), clinical benefit rate (CBR), time to progression (TTP), or time to treatment failure (TTF) to first-line hormone therapy, but did have significantly reduced survival compared to patients with normal serum EGFR levels (median survival 22.8 vs. 30.4 mos.)(p=0.006). Of note, combined serum EGFR and HER-2/neu analysis identified a subgroup of patients with decreased serum EGFR and normal serum HER-2/neu (n=38 /521, 7.3%) that had significantly reduced survival compared to patients with normal serum levels of both EGFR and HER-2/neu (median survival 22.9 vs. 39.9 mos.)(p=0.007). In multivariate analysis, decreased serum EGFR level remained a significant independent prognostic factor for decreased survival (hazard ratio HR=1.59, p=0.005). Other significant variables for decreased survival included the presence of liver metastasis (HR=2.31, p=<0.0001) and elevated serum HER-2/neu (HR=1.80, p=<0.0001), while positive hormone receptor status lowered the risk of mortality (HR=0.78, p=0.03). Conclusions: In metastatic breast cancer patients, decreased serum EGFR/normal serum HER-2/neu levels predicted shorter survival compared to normal levels of serum EGFR/HER-2/neu. The survival of the patients with decreased serum EGFR/normal serum HER-2/neu levels was similar to that of patients with elevated serum HER-2/neu levels. This patient subgroup deserves further study to assess response to and selection for anti-EGFR-directed therapies. 3 Muller V, Witzel I, Pantel K, et al. Prognostic and predictive impact of soluble epidermal growth factor receptor (sEGFR) protein in the serum of patients treated with chemotherapy for metastatic breast cancer. Anticancer Res. 2006 Mar-Apr;26(2B):1479-87. ABSTRACT BACKGROUND: A soluble fragment of the epidermal growth factor receptor (EGFR) extracellular domain (sEGFR) can be detected in the serum of cancer patients, but the role of sEGFR is still unclear. MATERIALS AND METHODS: Blood samples from patients receiving chemotherapy for metastatic breast cancer were collected before (n = 101) and after 3 courses of therapy (n=39). Levels of sEGFR and serum HER-2/neu extracellular domain (ECD) were determined by standardized ELISA. RESULTS: A higher percentage of cancer patients (15%) showed sEGFR values below 45ng/mL compared with control subjects (3%, p<0.001). Patients with sEGFR levels below 45 ng/mL showed a trend towards shorter overall survival (median 11.7 versus 15.4 months, p=0.08), which was more pronounced in patients with estrogen receptor-positive primary tumors (median 9.6 versus 15.4 months, p=0.022) Patients with low sEGFR and elevated serum HER-2/neu ECD (>15 ng/mL) also showed a shorter overall survival than those with normal values for both parameters (7.1 versus 15.4 months, p=0.03). Again, this difference was higher in patients with estrogen receptor-positive tumors (4.6 versus 15.4 month, p<0.0001). During treatment, a decrease of sEGFR levels occurred in 74.4% of the patients (p=0.014). CONCLUSION: Low sEGFR levels in patients with metastatic breast cancer are associated with a shorter overall survival, particularly in patients with estrogen receptor-positive tumors. Chemotherapy frequently induces a decrease of sEGFR. The combined, determination of sEGFR and serum HER-2/neu ECD also delivers relevant information. These findings suggest that the sEGFR status in metastatic breast cancer could be of clinical relevance. 4 Souder C, Leitzel K, Ali S, et al. Serum epidermal growth factor receptor/HER-2 predicts poor survival in patients with metastatic breast cancer. Cancer. 2006 Nov 15;107(10):2337-45. ABSTRACT BACKGROUND: Epidermal growth factor receptor (EGFR, HER-1, and erbB1) is overexpressed in primary breast cancer and had been identified as a poor prognostic factor. METHODS: Pretreatment serum EGFR levels were quantified by using an enzyme-linked immunoadsorbent assay in a Phase III first-line trial of letrozole and tamoxifen and were correlated with patient outcomes. RESULTS: Serum EGFR levels in a control group of 117 healthy, postmenopausal women measured 64.1 +/- 13.3 ng/mL (mean +/- standard deviation). Using a cutoff EGFR level of 44.1 ng/mL from the control group (5% nonparametric method), 53 of 535 patients (10%) had decreased serum levels of EGFR. Patients with decreased serum EGFR had no significant difference in objective response rate (ORR), clinical benefit rate (CBR), time to progression (TTP), or time to treatment failure (TTF); however, they did have significantly reduced survival compared with patients who had normal serum EGFR levels (median survival, 23.3 months vs. 30.9 months; P = .007). A combined analysis of pretreatment serum EGFR and HER-2 yielded no additional predictive information for ORR, CBR, TTP, or TTF compared to serum HER-2 alone. However, in the current analysis, a subgroup of patients who had decreased serum EGFR and normal serum HER-2 was identified (n = 39 of 535 patients; 7.3%) that had significantly reduced survival compared with patients who had normal serum levels of both EGFR and HER-2 (median survival, 23.5 months vs. 37.1 months; P = .005). In multivariate analysis, a decreased serum EGFR level remained a significant independent prognostic factor for decreased survival (hazards ratio, 1.58; P = .007). CONCLUSIONS: In patients who had metastatic breast cancer, decreased serum EGFR/normal serum HER-2 predicted shorter survival compared with patients who had normal levels of serum EGFR/HER-2. This patient subgroup deserves further study to assess their response to and selection for anti-EGFR-directed therapies. 5 Witzel I, Thomssen C, Krenkel S, et al. Clinical utility of determination of HER-2/neu and EGFR fragments in serum of patients with metastatic breast cancer. Int J Biol Markers. 2006 Jul-Sep;21(3):131-40. ABSTRACT INTRODUCTION: The growth factor receptors EGFR and HER-2/neu are targets for new treatment strategies and are of potential use as prognostic and predictive factors. However, the optimal method of determination in order to obtain clinically relevant information remains a source of controversy. METHODS: HER-2/neu and EGFR expression was examined by immunohistochemistry in primary tumors of patients with breast cancer. In addition, serum was tested for the extracellular domains of HER-2/neu (HER-2/neu ECD) and EGFR (sEGFR) before initiation of therapy for metastatic disease (n=76). The course of disease from the time of metastasis with regard to these parameters was evaluated by univariate and multivariate analyses. RESULTS: HER-2/neu ECD levels at the time of metastatic disease were correlated with HER-2/neu expression determined by immunohistochemistry from primary tumors (p=0.001). No correlation was observed between expression of EGFR in primary tumors and sEGFR serum levels. HER-2/neu ECD and sEGFR levels at the onset of metastatic disease did not show a significant impact on overall survival. CONCLUSIONS: Determination of HER-2/neu ECD levels in the serum measured by ELISA at the onset of metastatic disease could offer an alternative to immunohistochemistry of the primary tumor since serum levels are correlated with protein expression in primary tumors. In contrast, no such correlation was observed for EGFR. 6 Ali SM, Leitzel K, Marx III JH, et al. Serial serum EGFR and decreased survival in metastatic breast cancer. AACR Meeting Abstracts, 2005. Proc Amer Assoc Cancer Res, Volume 46: Abstract No. 3143 ABSTRACT Background: The epidermal growth factor receptor (EGFR, erbB1) has been reported to be overexpressed in breast cancer, and in some reports is a significant prognostic factor based on immunohistochemistry of the primary tumor. We previously reported that 71/265 metastatic breast cancer patients (26.8 %) had a decreased pretreatment serum EGFR level and decreased response and survival in a phase III second-line hormone therapy trial (Proc. ASCO 21:436a, Abstract 1743, 2002). Methods: The Oncogene Science/Bayer Diagnostics EGFR ELISA was used to quantify serial serum EGFR from 71 metastatic breast cancer patients who had decreased pretreatment serum EGFR associated with decreased response and survival to hormone therapy. These patients were selected from 265 post-menopausal metastatic breast cancer patients enrolled in a multicenter, randomized, double-blind, phase III second-line hormone therapy trial (fadrozole vs. megestrol acetate). The pretreatment and 1 month post-treatment serial serum EGFR profiles were correlated with subsequent patient response and survival to second-line hormone therapy. Serum EGFR from a control group of 59 healthy females was also measured. Results: In the healthy control group, mean serum EGFR was significantly higher in post-menopausal females (89.87±16.40 ng/mL, n=13) compared to pre-menopausal females (79.54±9.87 ng/mL, n=46) (p=0.006). Using a cutpoint of 57.07 ng/mL (mean - 2SD) derived from the post-menopausal female control group, 71/265 patients (26.8%) had a decreased pretreatment serum EGFR level, compared to 0/13 controls. Serial serum samples were available from 57 of the 71 patients with decreased pretreatment serum EGFR. When defining a serial serum EGFR change of greater than 20% difference from the pretreatment serum EGFR level as a significant change, 29/57 patients (50.9%) had an increasing serum EGFR level at 1 month post treatment, whereas 28 patients (49.1%) had either a decreasing (6 patients) or unchanged serial EGFR level (22 patients). When comparing the decreasing or unchanged serial serum EGFR patient group with the increasing serial EGFR patient group, there was no significant difference in objective response rate (ORR, p= .70), clinical benefit rate (CBR, p= .70), and time to progression (TTP, p= .46) to second-line hormone therapy. However, there was a trend for shorter overall survival in the patient group who had decreasing or unchanged serial serum EGFR (p=.058)(median survival 15.6 mo. vs. 28.1 mo.).Conclusions: Metastatic breast cancer patients with decreasing or unchanged serial serum EGFR profile after second-line hormone therapy have a shorter overall survival. Monitoring serum EGFR level may have potential for evaluation and/or selection of patients for anti-EGFR-directed therapies. 7 Leitzel K, Souder C, Ali SM, et al. Serum EGFR/HER-2 combination predicts poor survival in metastatic breast cancer. J Clin Oncol, ASCO Annual Meeting Proceedings, 2005. Vol 23, No. 16S, Part I of II (June 1 Supplement),Abstract No. 9613 ABSTRACT Background: Epidermal growth factor receptor (EGFR, erbB1) is overexpressed in primary breast cancer and in some reports is a poor prognostic factor. We previously reported that metastatic breast cancer patients with elevated pretreatment serum HER-2/neu have decreased response and survival. (JCO 21: 1967, 2003). Methods: Pretreatment serum EGFR was quantified in the 1st line phase III letrozole-tamoxifen trial and in controls using the Oncogene Science/Bayer Diagnostics EGFR ELISA. Results: Serum EGFR from 117 healthy post-menopausal female controls measured 64.1 + 13.3 ng/ml (mean + SD)(Range 39.5-117.1 ng/ml). Using a cutpoint of 44.1 ng/ml from the control group (5% non-parametric method), 62/542 patients (11 %) had decreased serum EGFR. Patients with decreased serum EGFR had no significant difference in objective response rate (ORR), clinical benefit rate (CBR), time to progression (TTP), or time to treatment failure (TTF) to 1st line hormone therapy, but did have significantly reduced survival compared to patients with normal serum EGFR levels (median survival 22.8 vs. 30.4 mos.)(P= .006). Combined analysis of pretreatment serum EGFR and HER-2 yielded no additional predictive information for ORR, CBR, TTP, and TTF compared to serum HER-2 alone. However, combined serum EGFR and HER-2 analysis identified a subgroup of patients with decreased serum EGFR and normal serum HER-2 (n = 43/542, 8%) that had significantly reduced survival compared to patients with normal serum levels of both EGFR and HER-2 (median survival 23.5 vs. 37.1 mos.)(P= .007). In multivariate analysis, decreased serum EGFR level remained a significant independent prognostic factor for decreased survival (hazard ratio HR = 1.59, P= .005). Other significant variables for decreased survival included the presence of liver metastasis (HR = 2.31, P <.0001) and elevated serum HER-2 (HR = 1.80, P= <.0001), while positive hormone receptor status lowered the risk of mortality (HR = .78, P= .03). Conclusions: In metastatic breast cancer patients, decreased serum EGFR/normal serum HER-2 predicted for a shorter survival - similar to elevated serum HER-2. This patient subgroup deserves further study to assess response to anti-EGFR-directed therapies. 8 Gregorc V, Ceresoli GL, Floriani I, et al. Effects of gefitinib on serum epidermal growth factor receptor and HER2 in patients with advanced non-small cell lung cancer. Clin Cancer Res. 2004 Sep 15;10(18 Pt 1):6006-12. ABSTRACT PURPOSE: The aim of this study was to assess serum extracellular binding domains of epidermal growth factor receptor (EGFR) and HER2 as surrogate markers of Gefitinib (Iressa, ZD1839, AstraZeneca, London, United Kingdom) activity in patients with non-small cell lung cancer. EXPERIMENTAL DESIGN: Serum EGFR and HER2 levels were monitored in blood samples taken within 1 week of starting Gefitinib at day 28 and at every computed tomography scan evaluation. EGFR and HER-2 were assayed in duplicate using commercial sandwich enzyme-linked immunosorbent assay kits (Oncogene Science Bayer Corporation, Cambridge, UK). A logistic regression analysis was performed to evaluate: (1) the relationship between best overall tumor response and basal EGFR and HER2 levels, and (2) the association between best overall tumor response and the differences of EGFR and HER2 levels obtained at the best overall tumor response and at baseline. RESULTS: Forty-six pretreated patients were evaluated, including F/M:11/35, Eastern Cooperative Oncology Group performance status 0-1/2:39/7, IIIB/IV:11/35, and adenocarcinoma/nonadenocarcinoma 29/17. Five partial responses (11%) and 14 stable disease responses (30%) were observed. Median pretreatment EGFR and HER2 were 83.3 ng/ml and 13.7 ng/ml. For baseline EGFR and HER2, the odds ratio of progression was 0.95 [95% confidence interval (CI), 0.91-0.98; P=0.01] and 0.87 (95% CI, 0.74-1.03; P=0.11), respectively. The difference between the best overall tumor response and basal EGFR value was predictive for response with a 6% increase in the odds of progression for an increase of 1 ng/ml (odds ratio, 1.06; 95% CI, 1.01-1.11; P=0.009) and for progression-free survival with a hazard ratio of 1.03 (95% CI, 1.01-1.04; P=0.003). CONCLUSION: Modifications of EGFR serum values during treatment seem to reflect Gefitinib activity. 9 Baron AT, Cora EM, Lafky JM, et. al. Soluble Epidermal Growth Factor Receptor (sEGFR/sErbB1) as a Potential Risk, Screening, and Diagnostic Serum Biomarker of Epithelial Ovarian Cancer. Cancer Epidemiol Biomarkers Prev. 2003 Feb; 12(2): 103-13. ABSTRACT: Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancies in the United States, for which risk assessment, screening, and diagnostic tests are needed. We have shown previously that women with stage III/IV EOC have lower serum p110 sEGFR/sErbB1 (Soluble Epidermal Growth Factor Receptor) concentrations than healthy women. Here, we show that serum p110 sEGFR/sErbB1 is the product of a 3-kb EGFR/ERBB1 alternate transcript. We report that serum sEGFR concentrations in stage I/II and stage III/IV EOC patients are significantly lower than in healthy women, and that serum sEGFR concentrations are not associated with disease stage or tumor grade. Logistic regression models show that: (a) lower serum sEGFR concentrations are associated significantly with a greater risk of EOC; (b) the risk associated with lower serum sEGFR concentrations is reduced by older age or menopause; and (c) age- or menopausal status-specific cutoff values for sEGFR concentration are appropriate. Receiver operating characteristic curves indicate that: (a) serum sEGFR concentrations are more effective in discerning stage III/IV than stage I/II EOC cases from healthy women; and (b) sEGFR concentrations have an 89% probability of correctly discerning EOC patients from healthy women when accounting for effect modification by age. By maintaining a test specificity of approximately 95% across strata of age or menopausal status with appropriate cutoff values, we observe that sEGFR concentrations are most useful for detecting stage I/II (sensitivity: 64-67%) and stage III/IV (sensitivity: 75-81%) EOC in young, premenopausal women. We conclude that serum sEGFR concentrations warrant additional investigation in the risk assessment, early detection, and/or diagnosis of EOC. 10 Krocker J, Eggemann H, Elling D, et al. Randomized trial comparing dose-intense, biweekly epirubicin (E) followed by paclitaxel (P) to 3-weekly epirucibin (E) plus cyclophosphamide (C) followed by paclitaxel (P) for adjuvant therapy of nodal-positive breast cancer: Serum results of HER-2/neu, EGFR and CA27.29. ASCO Annual Meeting Proceedings, 2003. Proc Am Soc Clin Oncol 22, Abstract No. 105 ABSTRACT: In a previous study in premenopausal patients, we demonstrated that ovarian ablation leads to a statistically significant upregulation of HER-2/neu, using HER-2/neu in serum as surrogate marker. We retested this concept in an ongoing multicenter trial for nodal positive (1-3 LN) breast cancer patients. Material and Methods: Patients either receive conventional ECP (arm A; E 90 mg/m2 plus C 600 mg/m2 x4 q3w, followed by P 175 mg/m2 x4, q3w) or dose-dense EP plus G-CSF (arm B; E 120 mg/m2 x4, q2w, followed by P: 175 mg/m2 x4, q2w). Serum is collected at baseline, end of chemotherapy and relapse. HER-2/neu and EGFR in serum are measured prospectively using ELISA assays (Bayer Diagnostics/Oncogene Science, Tarrytown/Cambridge, USA) with reference ranges of >15 ng/ml and 52-76 ng/ml, respectively. The tumor marker CA27.29 (Bayer Diagnostics, Tarrytown, USA) with a cut-off > 30 U/ml is evaluated in parallel. Results: Longitudinal serum pairs (baseline and end of chemotherapy) are currently available from 154 patients (81 in arm A; 73 in tarm B). The concentrations of all biochemical markers are displayed in Table 1. Serum HER-2/neu concentrations increased from 9.3 ng/ml at baseline to 11.4 ng/ml at the end of chemotherapy in both treatment arms (p < 0.05) while the concentrations of EGFR did not change over time. Starting with normal concentrations, CA 27.29 levels increased over 30 U/ml in approximately half of the patients. Conclusions: Serum HER-2/neu increases statistically significantly during adjuvant chemotherapy as result of regulatory mechanisms at the HER-2/neu gene while serum EGFR does not change over time. The increase of serum HER-2/neu derives from overal HER-2/neu expression in epithelial organs like bladder, lungs or pancreas and does not indicate relapse of breast cancer. 11 Maihle NJ, Baron AT, Barrette BA, et al. EGF/ErbB receptor family in ovarian cancer. Cancer Treat Res. 2002; 107: 247-58. ABSTRACT: In summary, the EGF/ErbB family of receptor tyrosine kinases has been shown to play a key role in normal ovarian follicle development, and cell growth regulation of the ovarian surface epithelium. Disregulation of these normal growth regulatory pathways, including overexpression and/or mutation of EGFR/ErbB receptor family members, as well as elements of their downstream signalling pathways, have been shown to contribute to the etiology and progression of epithelial ovarian cancer. It is, therefore, not surprising that these gene products, and their related soluble receptor isoforms may have clinical utility as tumor and/or serum biomarkers of disease activity. Moreover, since several of these soluble receptor isoforms have potent growth inhibitory activity, and are naturally occurring in the circulation, they are ideal candidates for the development of novel therapeutics for the treatment of ovarian cancer patients. 12 Baron AT, Lafky JM, Suman VJ, et al. A preliminary study of serum concentrations of soluble epidermal growth factor receptor (sErbB1), gonadotropins, and steroid hormones in healthy men and women. Cancer Epidemiol Biomarkers Prev. 2001 Nov; 10(11): 1175-85. ABSTRACT: Soluble ErbB (sErbB) growth factor receptors are being investigated as cancer biomarkers. Gonadotropic and steroid hormones have been shown to modulate the expression of ERBB family members in vivo. Accordingly, the range of sErbB1 values and their relationship to gonadotropic and steroid hormones need to be established in healthy subjects to provide a baseline for future clinical studies. We assayed sera from healthy men and women to determine p110 sErbB1 concentrations by acridinium-linked immunosorbent assay (ALISA). Follicle-stimulating hormone (FSH), estradiol, and testosterone concentrations were measured using the ACS:180 Immunoassay Analyzer. Luteinizing hormone (LH) and progesterone concentrations were quantified using the Access Immunoassay System. Unadjusted for age, p110 sErbB1 concentrations in healthy men and women do not differ significantly. However, sErbB1 concentrations show a strong age-gender interaction, increasing with age in men but decreasing with age in women. Consequently, sErbB1 concentrations are significantly higher in premenopausal women compared with either postmenopausal women or age-matched men and in age-matched men compared with postmenopausal women. Serum sErbB1 concentrations show significant negative associations with both FSH and LH concentrations in healthy women and a significant positive association with FSH concentrations in healthy men. Univariate linear regression models show that these respective gonadotropic hormones and age are independent predictors of sErbB1 concentrations in men and women. Multivariate models show that when age and FSH and LH concentrations are mutually adjusted for each other, they account for 22% of the variability observed in sErbB1 concentrations in healthy women. These data support the hypothesis that gonadotropic and steroid hormones may modulate ERBB1 expression in vivo and suggest that age- and gonadotropin-adjusted sErbB1 concentrations may be of clinical utility. Furthermore, these data demonstrate that gender, age, menstrual cycle phase, menopausal status, and exogenous hormone use must be considered when using serum p110 sErbB1 concentrations as cancer biomarkers. 13 Baron AT, Lafky JM, Connolly DC, et al. A sandwich type acridinium-linked immunosorbent assay (ALISA) detects soluble ErbB1 (sErbB1) in normal human sera. J Immunol Methods. 1998 Oct 1; 219(1-2): 23-43. ABSTRACT: The epidermal growth factor receptor (ErbB1) is overexpressed in various human tumor-derived cell lines and neoplasms, where it is believed that receptor dysregulation plays a role in oncogenic transformation and tumor progression. In addition to the ErbB1 holoreceptor, numerous studies demonstrate that cells synthesize soluble or secreted forms of ErbB1, i.e., sErbB1. Overexpression of ErbB1 in a variety of tumors has led us to hypothesize that sErbB levels also may be altered during oncogenesis, tumor progression, and/or metastasis; and that these molecules may be useful tumor biomarkers. To address this hypothesis we have developed an acridinium-linked immunosorbent assay (ALISA) specific for the extracellular domain of ErbB1 that can be used to quantify the levels of sErbB1 molecules in body fluids and conditioned culture media. This assay can also detect full-length ErbB1 in cell and tissue extracts. Our ALISA is characterized by high sensitivity (intra-assay LLD < 1 fmol/ml), a broad linear range (approximately 1 to 4000 fmol/ml), and good reproducibility (CVs < 10%). Specificity experiments show that this ALISA detects p170 ErbB1 and soluble forms of ErbB1 that embody extracellular subdomains I through IV, but not forms of sErbB1 lacking subdomain IV. Our ALISA does not detect full-length ErbB2, ErbB3, or ErbB4; or p105 soluble ErbB2. We report that serum sErbB1 levels of healthy women (median = 3716 fmol/ml), ranging in age from 43 to 76 years, differ significantly from those of healthy men (median = 24,512 fmol/ml), ranging in age from 25 to 79 years. Additional analyses do not indicate that serum sErbB1 levels change with age in either healthy men or women. Immunoprecipitation experiments show that monoclonal antibodies specific for extracellular epitopes of ErbB1 completely neutralize the detection of sErbB1 in normal human sera by ALISA. Finally, we show by immunoprecipitation and Western immunoblot analyses with monoclonal antibodies specific for the extracellular domain of ErbB1 that normal human female and male sera contain a approximately 110-kDa protein. We conclude that our ALISA is measuring the relative levels of this p110 sErbB1 analog in normal human sera. Our ALISA, therefore, should be useful for measuring the levels of ErbB1 and sErbB1 molecules in tumor biopsy specimens and body fluids, respectively, and for determining whether sErbB1, like ErbB1, is a useful tumor biomarker.     MN/CAIX Research Studies 1 Sivendran S, Leitzel K, Ali SM, et al. Elevated serum MN (carbonic anhydrase IX) levels in metastatic breast cancer patient serum. Proceedings for the American Association for Cancer Research, 2007. Abstract No. 2666 ABSTRACT Background: The transmembrane protein MN (carbonic anhydrase IX) catalyzes the hydration of carbon dioxide to carbonic acid and decreases pH. Normal expression of MN protein is restricted to primarily gastric, intestinal and liver mucosa, however in cancer cells up-regulation of MN gene expression occurs under hypoxic conditions and is believed to be involved in sensing and maintaining the acidic environment of hypoxic cells, particularly hypoxic regions within tumors. Significant levels of MN protein have been detected in a variety of cancers including kidney, cervix, lung, bladder, colon, breast, liver, gall bladder, and pancreas. Breast cancer patients whose tumors had higher levels of MN have been reported to have a poorer prognosis. Methods: Pretreatment serum MN / CAIX levels were determined from 147 breast cancer patients enrolled in a phase III 2nd-line hormone therapy trial comparing letrozole vs. megace, and in 50 healthy control subjects (25 post-menopausal treated with hormone replacement therapy and 25 post menopausal women not treated with HRT). The MN/CAIX ELISA from Oncogene Science / Bayer HealthCare, Cambridge MA, was used for determination of serum MN / CAIX levels. Results: Serum MN / CAIX levels from the 50 healthy post-menopausal female controls had a mean + SD of 244 + 131 pg/ml (range 87-667 pg/ml). Serum MN / CAIX levels were not significantly different between post-menopausal groups treated with (n=25 subjects) or without (n=25 subjects) hormone replacement therapy. The upper limit of normal was defined as the mean + 2 SD (506.5 pg/ml). Using this cutpoint, 3 of 50 control subjects (6 %) had elevated serum MN / CAIX levels. Pretreatment serum MN / CAIX levels were elevated above the upper limit of normal in 32 of 147 patients (32%) (range 62- 1439 pg/ml) enrolled in a phase III 2nd-line hormone therapy trial (p = 0.01 vs. control group, Fisher’s Exact). Serum MN / CAIX levels were also significantly elevated in the metastatic breast cancer patient group compared to the control group using Wilcoxon rank sum statistical analysis (p<0.0001). Conclusions: Pretreatment serum MN / CAIX levels were elevated in 32 % of metastatic breast cancer patients compared to healthy post-menopausal female control serum. Serum MN / CAIX level deserves further study to determine its predictive and prognostic biomarker potential in metastatic breast cancer patients.     Ras p21 Research Studies 1 Nelli E, Leitzel K, Ali S, et al. Elevated serum ras levels in patients with hematologic malignancies. AACR Meeting Abstracts, 2006. Proc Amer Assoc Cancer Res, Volume 47: Abstract No. 1144 ABSTRACT Background: Ras is a GDP/GTP binding G protein that acts as a molecular switch converting signals from the cell membrane to the nucleus to regulate cell proliferation, differentiation, and protein synthesis. Activation of ras oncogenes has been identified in a variety of cancers, including 30% of acute myelogenous leukemia (AML) patients. The purpose of our study was to evaluate serum ras levels in hematologic cancer patients. Methods: A novel ras p21 ELISA (Oncogene Science/Bayer Diagnostics) employing two monoclonal antibodies was utilized to quantify ras levels in serum obtained from patients with various hematologic malignancies including AML, acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia (CLL). A control group of 48 healthy subjects was also used to establish a cutoff for the upper limit of normal for serum ras levels. Fisher’s exact test was used to evaluate differences in the frequency of elevated serum ras levels in control vs. patient groups. Results: The median serum ras level in the 48 healthy control subjects was 125 ng/ml, with a 5% to 95% range of 50-422 ng/ml. The upper limit of normal for serum ras was defined as 422 ng/ml, as determined using the 95th percentile method. Patients were compared to controls. In the total leukemia patient group, 16 of 52 patients (30.8 %) had elevated serum ras levels (p= 0.001). In patient subgroups, 6/24 (25%, p= 0.014) AML patients, 3/7 (42.9%, p= 0.012) ALL patients, and 6 /13 (46%, p= 0.001) CML patients had significantly elevated serum ras levels. Only 1/8 (12.5 %, p= 0.376) CLL patients had an elevated serum ras level. Conclusions: The results of this study indicate that leukemia patients have significantly elevated serum ras levels when compared to healthy controls. Serum ras should be evaluated as a potential biomarker in larger leukemia trials, especially for response to treatment with inhibitors of the ras signaling pathway. 2 Nelli EM, Leitzel K, Ali SM, et al. Elevated serum ras level predicts decreased survival in patients with hematologic malignancies. J Clin Oncol, ASCO Annual Meeting Proceedings Part I, 2006. Vol 24, No. 18S (June 20 Supplement), Abstract# 6562 ABSTRACT Background: Ras is a GDP/GTP binding G protein that acts as a molecular switch converting signals from the cell membrane to the nucleus to regulate cell proliferation, differentiation, and protein synthesis. Activation of ras oncogenes has been identified in a variety of cancers, including 30% of acute myelogenous leukemia patients. The purpose of our study was to evaluate serum ras levels and correlate with survival in hematologic cancer patients. Methods: A novel ras p21 ELISA (Oncogene Science/Bayer Diagnostics, Cambridge, MA) employing two monoclonal antibodies reactive with H, K, and N ras was utilized to quantify total ras levels in serum obtained from patients with various hematologic malignancies including acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia (CLL). Results: The total leukemia patient group consisted of 52 patients. At the 75th percentile serum ras cutpoint (524 pg/ml) 11/52 patients were defined as elevated for serum ras. From this patient group, 38 patients had clinical followup available and were included in the Kaplan-Meier analysis of overall survival. Patients with elevated serum ras (>524 pg/ml) had significantly shorter overall survival compared to those without (median OS 205 vs. 677 days) (p= 0.04). In a multivariate analysis including serum ras level and type of leukemia, serum ras level remained a significant independent variable for shorter overall survival (p=0.004). Within leukemia subtypes 2/18 AML, 4/9 CML, 3/7 ALL, and 0/4 CLL patients had elevated serum ras levels. Conclusions: Leukemia patients with elevated serum ras levels had a significantly shorter overall survival. Serum ras should be evaluated as a potential biomarker in larger leukemia trials, especially for response to treatment with inhibitors of the ras signaling pathway.     Serum HER-2/neu Research Studies 1 Mazouni C, Hall A, Broglio K, et al. Kinetics of serum HER-2/neu changes in patients with HER- 2-positive primary breast cancer after initiation of primary chemotherapy. Cancer. 2007 Feb 1;109(3):496-501. ABSTRACT BACKGROUND: The purpose of the study was to determine the utility of quantitation of the extracellular domain (ECD) of the HER-2/neu receptor in the serum for predicting response to treatment in patients with primary breast cancer receiving neoadjuvant therapy. METHODS: HER-2/neu ECD was measured in sera obtained from 39 patients with HER-2-amplified stage II-III primary breast cancer undergoing neoadjuvant chemotherapy. Patients were randomly assigned to either 4 cycles of paclitaxel followed by 4 cycles of fluorouracil, epirubicin, and cyclophosphamide (FEC) (n = 10) or to the same chemotherapy with simultaneous weekly trastuzumab for 24 weeks (n = 29). Changes in HER-2 ECD were monitored with the Bayer HER-2/neu assay over 6 months and correlated with pathological response to treatment. RESULTS: Before initiation of chemotherapy, 28.2% of patients had elevated concentration of the HER-2 ECD (>15 ng/mL). The median baseline serum HER-2 ECD concentration was 13.6 ng/mL (mean +/- SD, 20.3 +/- 35.5 ng/mL). A decrease in the median HER-2 ECD levels from baseline to Week 3 and from baseline to Week 6 of chemotherapy was seen regardless of treatment regimen. No significant difference in baseline HER-2 ECD levels was observed between the groups who achieved pathological complete response (pCR) and the group with residual disease (P = .41). However, a 9% drop from Week 3 to Week 6 after initial chemotherapy was predictive of pCR (P = .04). CONCLUSION: A decrease in serum HER-2 ECD levels early during treatment was associated with pathological response in patients receiving primary chemotherapy, particularly trastuzumab-based regimens. Serum HER-2 ECD levels may serve to monitor neoadjuvant therapy in HER-2-positive primary breast cancer. 2 Ali SM, Brault D, Brown-Shimer S, et al. Serum HER-2/neu decline predicts improved response to trastuzumab-based therapy in patients with normal and elevated baseline serum HER-2/neu levels. Breast Cancer Res Treat, 2006. 100(suppl 1): Abstract No. 2030 ABSTRACT Background: Trastuzumab (Herceptin) monotherapy has a 34 % objective response rate (ORR) in patients with HER-2/neu IHC 3+ or FISH-positive first-line metastatic breast cancer (C. Vogel et al, JCO 20:719-726, 2002). Predicting response and survival to trastuzumab-based therapy is an unsolved problem. The HER-2/neu extracellular domain (ECD) is released after cleavage by the ADAM metalloproteinases, and the remaining membrane-bound internal domain is constitutively activated. Trastuzumab inhibits cleavage of the HER-2/neu ECD. Material and Methods: A pooled analysis of 7 trials of first-line trastuzumab therapy (with or without chemotherapy) with serial serum HER-2/neu levels were included. The FDA-approved HER-2/neu ELISA (Oncogene Science/Bayer HealthCare) was used to determine serum HER-2/neu levels. A pretreatment and post-treatment serum (16-120 days) from 307 patients was available. 236 patients had data on overall survival. Kaplan Meier Life table analysis was performed to compare duration of response (DRP), time to progression (TTP), and overall survival (OS). Results: The median decrease in serum HER-2/neu levels for all patients was 31.0% (Range: 98% decrease to 239% increase). Patients with > 20% decrease in HER-2/neu levels had significantly higher ORR, and longer DRP, TTP and OS, regardless of baseline serum HER-2/neu level. Conclusion: Patients with < 20% decrease in serum HER-2/neu levels have decreased benefit from trastuzumab therapy. Patients who do not have a significant decrease in serum HER-2/neu levels should be considered for additional HER-2/neu-targeted therapies. 3 Ali SM, Esteva FJ, Fornier M, et al. Serum HER-2/neu change predicts clinical outcome to trastuzumab-based therapy. J Clin Oncol, ASCO Annual Meeting Proceedings Part I, 2006. Vol 24, No. 18S (June 20 Supplement), Abstract No. 500 ABSTRACT Background: Trastuzumab monotherapy has a 34% objective response rate (ORR) in patients with HER-2/neu IHC 3+ or FISH-positive first-line metastatic breast cancer (C. Vogel et al, JCO 20:719-726, 2002). Predicting response and survival to trastuzumab-based therapy is an unsolved problem. The HER-2/neu extracellular domain (ECD) is released after cleavage by the ADAM metalloproteinases, and the remaining membrane-bound internal domain is constitutively activated. Trastuzumab inhibits cleavage of the HER-2/neu ECD. Methods: A pooled analysis of 7 trials of first-line trastuzumab therapy (with or without chemotherapy) with serial serum HER-2/neu levels were included. The FDA-approved HER-2/neu ELISA (Oncogene Science/Bayer HealthCare) was used to determine serum HER-2/neu levels. A pretreatment and post-treatment serum (16-120 days) from 307 patients was available. 236 patients had data on overall survival. Kaplan Meier Life table analysis was performed to compare duration of response (DRP), time to progression (TTP), and overall survival (OS). Results: The median decrease in serum HER-2/neu levels for all patients was 31.0% (Range: 98% decrease to 239% increase). Patients with > 20% decrease in HER-2/neu levels had a significantly higher objective response rate (ORR, complete + partial response) and longer DRP, TTP and OS. The results were similar regardless of the timing of the second serum draw (< 30 days vs. > 30 days) after the start of trastazumab. Conclusion: Patients with < 20% decrease in serum HER-2/neu levels have decreased benefit from trastuzumab therapy. Patients who do not have a significant decrease in serum HER-2/neu levels should be considered for additional HER-2/neu-targeted therapies. 4 Ali SM, Esteva FJ, Fornier M, et al. Serum HER-2/neu Decline Predicts Improved Response to Trastuzumab-based Therapy (Serum HER-2/neu Study Group). Ann Oncol, ESMO Proceedings, 2006. 17 (Supplement 9): ix69 Abstract No. 1390 ABSTRACT Background: Trastuzumab (Herceptin) monotherapy has a 34% objective response rate (ORR) in patients with HER-2/neu IHC 3+ or FISH-positive first-line metastatic breast cancer (C. Vogel et al, JCO 20:719-726, 2002). Predicting response and survival to trastuzumab-based therapy is an unsolved problem. The HER-2/neu extracellular domain (ECD) is released after cleavage by the ADAM metalloproteinases, and the remaining membrane-bound internal domain is constitutively activated. Trastuzumab inhibits cleavage of the HER-2/neu ECD. Material and methods: A pooled analysis of 7 trials of first-line trastuzumab therapy (with or without chemotherapy) with serial serum HER-2/neu levels were included. The FDA-approved HER-2/neu ELISA (Oncogene Science/Bayer HealthCare) was used to determine serum HER-2/neu levels. A pretreatment and post-treatment serum (16–120 days) from 307 patients was available. 236 patients had data on overall survival. Kaplan Meier Life table analysis was performed to compare duration of response (DRP), time to progression (TTP), and overall survival (OS). Results: The median decrease in serum HER-2/neu levels for all patients was 31.0% (Range: 98% decrease to 239% increase). Patients with > 20% decrease in HER-2/neu levels had significantly higher ORR, and longer DRP, TTP and OS, regardless of the timing of the 2nd blood collection between 16 to 120 days post-treatment. Conclusion: Patients with < 20% decrease in serum HER-2/neu levels have decreased benefit from trastuzumab therapy. Patients who do not have a significant decrease in serum HER-2/neu levels should be considered for additional HER-2/neu-targeted therapies. 5 Allen CL, Chapman CJ, Hitch A, et al. Serum EGFR and HER-2 levels in metastatic breast cancer patients receiving gefitinib therapy. Breast Cancer Res Treat, 2006. 100(suppl 1): Abstract No. 2034 ABSTRACT Introduction: Epidermal growth factor receptor (EGFR) and HER-2/neu are highly homologous members of the cerbB receptor family. Both are pivotal in cell signalling pathways and breast carcinogenesis. HER-2 gene amplification is seen in 20-30% of breast cancers and it is linked to a more aggressive disease state and a poorer prognosis. Serum HER-2/neu levels are routinely measured to monitor Herceptin therapy. EGFR is a target for Gefitinib, a current anti-EGFR therapy. However the value of serum EGFR in predicting therapeutic response and survival in breast cancer is unknown. Aims: To investigate the independent and additive roles of serum EGFR and HER-2/neu levels in a panel of metastatic breast cancer patients, and their role in predicting therapeutic response and overall patient survival. Patients and Methods: Serial serum samples were taken from 32 metastatic breast cancer patients receiving 250-500mg/day Gefitinib (Iressa, Astra-Zeneca, UK), in a phase II trial. All patients were categorised according to UICC criteria and divided into 2 cohorts, clinical benefit (CB=9) (complete response + partial response + stable disease >6 months) or progressive disease (PD=23) (progression in <6 months). Serum EGFR levels were determined using a commercial immunoassay (Oncogene Science Bayer corporation) and HER-2/neu levels were determined using the ADVIA Centaur automated HER-2/neu immunoassay. Statistical analysis was carried out using a paired samples t-test. 6 Bramwell VHC, Doig GS, Tuck AB, et al. Serial measurement of serum extracellular domain of HER2 (ECD/HER2) has prognostic value in metastatic breast cancer. Breast Cancer Res Treat, 2006. 100(suppl 1): Abstract No. 1005 ABSTRACT Background: In a prospective study of 158 women with metastatic breast cancer, our group has shown that serial measurement in plasma of osteopontin (OPN), a novel tumor marker, is strongly associated with poor survival (Bramwell V et al, Clin Cancer Res 2006, 12:3337). As several studies have suggested that serial measurement of serum ECD/HER2 also has prognostic value in metastatic breast cancer, we present data on this marker analysed in the same patient population. Materials and Methods: Blood samples were taken at every clinic visit from the time of diagnosis of metastases until death, for 158 women with advanced breast cancer. Serum ECD/HER2 was measured using a commercially available kit. Multivariate time-dependent survival analyses were conducted using models that right censored patient outcomes 3, 6 and 12 months after last ECD/HER2 measurement. Results: ECD/HER2 was measured in 1378 samples (median, 9 per patient). Baseline data for ECD/HER2 were available on 137 women. Median level was 10.2 ng/ml (range 4.1 40.4), with 34 (25%) patients having elevated levels (upper limit of normal 15 ng/ml). Higher baseline levels of ECD/HER2 were correlated with poor survival (p = 0.001). Data on serial measurements of ECD/HER2 after baseline were available for 156 women, and any subsequent increases were associated with worse survival at 3, 6 and 12 mos after the last measurement (p = 0.025, RR 1.57, at 12 mos). Exploratory regression models examining ECD/HER2 increases of 3, 5, 12, 15 ng/ml showed that ECD/HER2 increases of 12 ng/ml had the strongest association with survival (p = 0.0001, RR 5.89, at 12 mos). Conclusions: Serial measurement of both OPN and ECD/HER2 have prognostic value for survival in this cohort of women with metastatic breast cancer. We are further exploring their relative value in monitoring survival and response to treatment. 7 Dinocca S, Blot E, Laberge-Le-Couteulx S, et al. Serum level of HER2 extra cellular domain (ECD) is a significant prognostic factor for survival in metastatic breast cancer without HER2 overexpression on primary tumor. Breast Cancer Res Treat, 2006. 100(suppl 1): Abstract No. 1035 ABSTRACT Introduction: This study evaluated the prognostic value of serum HER2 ECD level at the time of metastasis in breast cancer patients having a primary tumour without HER2 overexpression/amplification. Patients and Methods: One hundred forty-five patients treated for metastatic breast cancer between January 1999 and February 2005 at the Centre Rgional de Lutte Contre le Cancer de Haute Normandie were included in this retrospective study. Inclusion required available pre-treatment frozen serum at the time of metastasis and an initial tumour tissue without HER2 overexpression/amplification (0 or 1+ by immunohistochemistry). Serum HER2 ECD level was quantified using an ELISA test (Oncogene Science, Cambridge, MA). Normal HER2 ECD level was 15g/l as specified by the manufacturer. Results: Twenty-six out of 145 (18%) patients without HER2 overexpression/amplification in primary tumour had elevated serum HER2 ECD levels before treatment. In univariate analysis, the median survival for patients with elevated serum HER2 ECD level was significantly shorter (14.5 months) than for patients with normal serum HER2 ECD level (35.5 months) (p=0.001). Kaplan-Meier survival curves showed that elevated serum HER2 ECD level was statistically linked with survival (Figure). In multivariate analysis, delay 24 months between initial tumour and metastasis (p<0.001), visceral site of metastases (p=0.001) and elevated pre-therapeutic serum HER2 ECD level (p=0.03) were significantly linked with a worst prognosis. Conclusion: These results need further confirmation in prospective and larger series. If confirmed, it could suggest a potential interest for the use of trastuzumab in metastatic breast cancer with elevated serum HER2 ECD level even without HER2 overpression/amplification in the primary tumour. 8 Fehm T, Lane N, Solomayer E, et al. Reassessment of HER2 status in HER2 negative or HER2 unknown breast cancer patients with recurrent metastatic disease by analyzing serum HER2 and HER2 status of circulating tumor cells. Breast Cancer Res Treat, 2006. 100(suppl 1): Abstract No. 1004 ABSTRACT Introduction: Several studies have indicated that HER2 status of HER2 negative patients can change during course of disease. Therefore, the HER2 status should be reassessed at the time of disease progression to optimize treatment decisions. Since metastatic tissue may not be available, serum HER2 status can be analyzed. A second method is the evaluation of HER2 status in circulating tumor cells (CTCs). Therefore, the aim of this study was to determine the serum HER2 status of serum and corresponding CTCs in patients initially HER2 negative or of unknown HER2 status with metastatic disease. Methods: Blood samples were obtained from 77 metastatic breast cancer patients with negative (n=47) or unknown (n=30) HER2 status. Serum HER2 was determined using the commercial HER-2/neu ELISA-kit (Oncogene Science, MA, USA). 15 ng/ml was used as cut-off. CTCs were detected by a slide based assay using ferrofluid/immunomagnetic enrichment and characterized by pheno- and genotyping. Alternatively, a commercial kit (Adnagen, Langenhagen, Germany) based on RT-PCR was used for CTC detection and characterization. Results: 77 metastatic breast cancer patients were included in the analysis. 19 of 77 (25%) metastatic patients had elevated serum HER2 levels. CTCs could be detected in 20 of 67 patients. 8 of these (40%) patients had CTCs with HER2 amplification. There was concordance between HER2 status of CTCs and serum HER2 in 15 of 20 patients (75%) (Table 1). In 5 patients non concordant results were obtained. Three patients with elevated serum HER2 levels had HER2 negative CTCs whereas 2 patients with HER2 amplified CTCs had normal serum HER2 levels. Conclusions: Our study confirms that a subset of HER2 negative patients develop elevated serum HER2 levels and HER2 positive CTCs associated with metastatic disease. However, nonconcordant results were obtained in 25% of patients using both methods. Similar nonconcordance occurs in primary breast carcinomas. Therefore, we hypothesize that different mechanisms may account for shedding HER2 versus its expression. Hence, correlating clinical responses to Herceptin based on each method should be further studied to help determine when such treatment should be given. 9 Gomez HL, Chavez MA, Doval DC, et al. Updated biomarker results from a phase II randomized study of lapatinib as first-line treatment for patients with ErbB2-amplified advanced or metastatic breast cancer. Breast Cancer Res Treat, 2006. 100(suppl 1): Abstract No. 1090 ABSTRACT Background: Lapatinib is a reversible, oral small molecule inhibitor of ErbB1 (EGFR) and ErbB2 (HER-2/neu) tyrosine kinase activity. Recent research has shown that elevated pretreatment serum ErbB2 ECD levels are associated with favorable response to trastuzumab-based therapies and that ErbB2 ECD levels decline in patients who respond to treatment. The primary objective of this study was to evaluate the response rate of two lapatinib dosing regimens as first-line treatment for patients with ErbB2-amplified advanced or metastatic breast cancer (MBC) documented by FISH. Target enrollment of 138 pts was reached in January 2006. Results reported herein pertain to IHC analysis of ErbB1, ER/PR, IGF-1R and serum ErbB2 ECD levels for these patients. Material and methods: Eligible patients were randomized (1:1, open label) to lapatinib 1500mg as a single daily dose (QD) or lapatinib 500mg twice daily (BID). Tumor tissue was obtained from the patients most recent biopsy and was centrally analyzed for biomarker activity via IHC. Sequential evaluation of serum ErbB2 ECD levels was performed at baseline, every 4 weeks and at the end of therapy. Results: IHC analysis of tumor tissue indicated 105 patients (76%) were ER-/PR-. Over one-third of patients (47/119, 39%) tested 3+ for IHC analysis of IGF-1R. Most subjects (95/123, 77%) were ErbB1 negative, with IHC scores of 0 (74, 60%) or 1+ (21, 17%). The median serum ErbB2 ECD level at baseline was 38.6 ng/mL, and decreased by 17% and 37% at Week 4 and Week 8, respectively. Conclusion: Previous interim analysis results suggested an association of response to an IGF1-R score of 3+, and no association of response to ErbB1 and ER/PR status. Data on additional biomarkers and correlation of biomarker levels to clinical activity will be presented. 10 Gori S, Ludovini V, Mosconi A, et al. Prognostic value of serum HER-2 in early stage breast cancer (BC) patients. Breast Cancer Res Treat, 2006. 100(suppl 1): Abstract No. 2035 ABSTRACT Background: HER2 is amplified and/or overexpressed in approximately 20-30% of invasive BC and is associated with poor prognosis. It is also a predictive marker of response to trastuzumab. The extracellular domain of the HER2 protein (sHER2) is frequently cleaved and released into the circulation where it can be detected by ELISA in up to 45% of advanced BC. We evaluated HER2 expression in paired serum and tissue samples of operable BC pts to analyze 1) the correlation between sHER2 and HER2 tumor status 2) their relationship with clinical-pathological parameters and 3) their impact on the outcome. Patients and Methods: 188 consecutive stage I-III BC pts were included in this study from May 2000 to July 2005. sHER2 was measured by ELISA (manual Kit Oncogene Science Diagnostics and automated version ADVIA Centaur, Bayer Diagnostics) before the local treatment. Tumor tissue was analyzed by IHC with CB11 antibody and scored with Dako Hercept-test. HER2 amplification was determined using the VentanaTM FISH assay in patients with 2+ by IHC. Chi-squared test was used to evaluate the association between HER2 and patients clinical-pathological features. Survival outcomes were analyzed using Coxs model. Results: Median age was 56.6 years; 122 pts received adjuvant chemotherapy, 54 endocrine therapy and 83 both. Forty-three pts (23%) had HER2 overexpression/amplification in tumor tissue and 25 pts (13%) had sHER2 levels 15ng/ml (cut-off level) with a concordance of 85%. Both high sHER2 levels and HER2 tumor expression were associated with high histological grade (p=.02 and p<.0001 respectively) and negativity of ER (p=.0035and p<.0001) and PgR (p=.0056 and p=. 002). At a median follow-up of 2.4 years we observed 8 deaths and 19 relapses. At univariate analysis high sHER2 levels (evaluated as continuous variable) were significantly correlated with shorter DFS (p=0.0002) even if at multivariate analysis high sHER2 levels, after adjustment for stage and ER status, were associated with a shorter DFS with borderline significance (p=0.06). Discussion: Our preliminary data indicate that measurement of sHER-2 at diagnosis appears to be a useful tool for identifying high-risk BC pts and helping with treatment decisions. 11 Hamer PJ, Carney WP, Gaur VK, et al. Elevated serum levels of TIMP-1 and HER-2/neu in metastatic breast cancer patients (MBC) may be valuable in guiding targeted therapies. AACR Meeting Abstracts, 2006. Proc Amer Assoc Cancer Res, Volume 47: Abstract No. 3589 ABSTRACT: The search for new biomarkers in various cancers is becoming a vital area of cancer research. The use of more than one marker may provide additional information compared to any single marker. The use of circulating sera markers allows the researcher to collect samples in a less invasive fashion and to monitor the progress of the patients disease. We measured the circulating levels of HER-2/neu and tissue inhibitor of metalloproteinase-1 (TIMP-1) in pretreatment samples from 600 post-menopausal metastatic breast cancer (MBC) patients. These patients were part of a trial examining the efficacy of first-line treatment comparing letrozole to tamoxifen. A panel of 50 serum samples from normal, healthy post-menopausal women was analyzed in order to establish a cutoff value for the TIMP-1 marker. Cutoff value, calculated as the mean + 2 standard deviations (SD) for the normal, healthy population, was determined to be 454 ng/mL for TIMP-1. For serum HER-2/neu, the well-established cutoff value of 15 ng/mL was used. These cutoff values were then used to determine the percentage of MBC patients that had elevated levels of TIMP-1 and serum HER-2/neu. This study found a sizeable percentage of patients whose pretreatment serum showed elevations of one or both of the circulating markers. Specifically, there were 26% of the patients who showed an elevation of TIMP-1 levels and 31% showed elevated serum HER-2/neu levels. More specifically, of the 187 samples that were elevated for serum HER-2/neu, 68 (36%) were elevated for TIMP-1. Of the 416 samples that showed normal levels of serum HER-2/neu, 85 (20%) were elevated for TIMP-1. Therefore, 11% of the total samples showed elevations of both HER-2/neu and TIMP-1. In a previous study (ECCO 2001), we presented data showing that in MBC patients treated with second-line hormone therapy, 34% had elevated uPA serum levels. This increase of uPA seen in pretreatment serum samples was statistically significant for TTP and OS. Markers such as uPA and TIMP-1 that are associated with tissue degradation during metastasis may be valuable for selecting patients for new classes of protease inhibitor therapies. In addition, the use of serum markers as diagnostic tests, which allow monitoring of the patient’s response may allow for a more efficacious use of targeted cancer therapies. Elevation of both uPA and the inhibitor TIMP-1 imply that proteolytic systems have become activated, which could contribute to an increased metastatic potential. Since estrogen receptor-positive (ER+) MBC patients derive greater benefit from letrozole than tamoxifen, and HER-2/neu positive patients benefit from the anti-HER-2/neu therapy trastuzumab, it may be valuable to select ER+ patients with elevated uPA and TIMP-1 levels to receive combination therapy with letrozole and anti-TIMP-1 and uPA-targeted therapy. 12 Hamer PJ, Jarosz DE, Leitzel K, et al. Serum EGFr and HER-2/neu Predicts Poor Survival in Metastatic Breast Cancer. AACC Annual Meeting Abstracts, 2006. Clin Chem Vol 52(6) Suppl,pg A168: Abstract No. E11 ABSTRACT Background: The epidermal growth factor receptor (EGFR/ErbB1) is one of four members of the ErbB family of receptor tyrosine kinases, which also includes HER-2/neu/erbB2, HER-3/ErbB3 and HER-4/ErbB4. EGFR is overexpressed in primary breast cancer and, in some reports, has been shown to be a poor prognostic factor. We have previously reported that metastatic breast cancer (MBC) patients with elevated pretreatment serum HER-2/neu have decreased response to first-line hormone therapy and shortened survival. Methods: Pretreatment serum EGFR levels were determined in samples obtained from a first-line phase III letrozole-tamoxifen trial, as well as in samples obtained from healthy postmenopausal controls using an EGFR ELISA from Oncogene Science/Bayer HealthCare, Diagnostics Division. Results: Serum EGFR from 117 healthy post-menopausal female controls measured 64.1 + 13.3 ng/ml (mean + SD)(Range 39.5-117.1 ng/ml). Using a cutpoint of 44.1 ng/ml as determined from the control group (5% non-parametric method), 62/521 MBC patients (11%) had decreased serum EGFR levels. Patients with decreased serum EGFR had no significant difference in objective response rate (ORR), clinical benefit rate (CBR), time to progression (TTP), or time to treatment failure (TTF) to first-line hormone therapy, but did have significantly reduced survival compared to patients with normal serum EGFR levels (median survival 22.8 vs. 30.4 mos.)(p=0.006). Of note, combined serum EGFR and HER-2/neu analysis identified a subgroup of patients with decreased serum EGFR and normal serum HER-2/neu (n=38 /521, 7.3%) that had significantly reduced survival compared to patients with normal serum levels of both EGFR and HER-2/neu (median survival 22.9 vs. 39.9 mos.)(p=0.007). In multivariate analysis, decreased serum EGFR level remained a significant independent prognostic factor for decreased survival (hazard ratio HR=1.59, p=0.005). Other significant variables for decreased survival included the presence of liver metastasis (HR=2.31, p=<0.0001) and elevated serum HER-2/neu (HR=1.80, p=<0.0001), while positive hormone receptor status lowered the risk of mortality (HR=0.78, p=0.03). Conclusions: In metastatic breast cancer patients, decreased serum EGFR/normal serum HER-2/neu levels predicted shorter survival compared to normal levels of serum EGFR/HER-2/neu. The survival of the patients with decreased serum EGFR/normal serum HER-2/neu levels was similar to that of patients with elevated serum HER-2/neu levels. This patient subgroup deserves further study to assess response to and selection for anti-EGFR-directed therapies. 13 Kashiwaba M, Inaba T, Wakabayashi G, et al. Serum HER2/nue extra-cellualar domain level predicts clinical response in the patients treated with sequential trastuzumab monotherapy and combination with chemotherapy. Breast Cancer Res Treat, 2006. 100(suppl 1): Abstract No.1008 ABSTRACT Background: Trastuzumab has >30% objective response rate in monotherapy and >70% in combination with chemotherapy in the patient with HER2/nue IHC 3+ or FISH+ at the first line therapy for metastatic breast cancer. Serum HER2/neu extracellular domain (HER2-ECD) is cleavage by the ADAM metalloproteinases. Previous trials suggested serum HER2-ECD level might be a predictive marker of clinical response. But the role of serum HER2-ECD level in serial treatments with trastuzumab and chemotherapy remains uncertain. Methods: Serial serum HER2-ECD levels ware measured at the point of initial day of treatment and evaluated day as treatment failure. Serum sampling was continued as long as trastuzumab was used. The new HER2/neu ELISA(Bayer Healthcare) was used to determine comparing with the results measured by ErbB-2 EIA(Nichirei). Results: Patients with >20% decrease in HER2-ECD level measured by HER2/neu ELISA had a significantly higher response rate and longer time to progression even in serial treatments. Conversely ErbB-2 EIA only showed good correlation with initial therapy but following HER2-ECD serum levels were not corresponded with clinical responses. Results: The HER2-ECD level is a significant predictive marker in the treatment with trastuzumab. This might select the patients who has higher response with trastuzumab or should be treated another therapy. 14 Kong SY, Kang JH, Kwon Y, et al. Serum HER-2 concentration in patients with primary breast cancer. J Clin Pathol, 2006. 59(4):373-6 ABSTRACT 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 HER-2 concentration in Korean women. METHODS: Serum HER-2 concentration was measured and immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) carried out on tissue samples from 86 consecutive female patients. The results of the three datasets were compared. The cut off value of HER-2 concentration was determined from the mean +2SD values derived from the serum of 93 healthy volunteers. RESULTS: The IHC and FISH data were significantly correlated (p<0.01), but neither showed significant correlation with the serum HER-2 data. The cut off value of serum HER-2 was 10.2 microg/l, and the serum HER-2 concentration of patients with primary breast cancer ranged from 5.0 to 17.5 microg/l. Only five patients had a serum HER-2 value above the cut off value. CONCLUSIONS: Serum HER-2 concentration cannot be substituted for IHC or FISH to evaluate HER-2 status, nor can it be used as a diagnostic tumour marker in primary breast cancer, considering the low prevalence of serum HER-2 elevation. 15 Kong SY, Nam BH, Lee KS, et al. Predicting Tissue HER2 Status Using Serum HER2 Levels in Patients with Metastatic Breast Cancer. Clin Chem. 2006 Jun 15 ABSTRACT BACKGROUND: Immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) are reliable ways to identify amplification of the HER-2/neu (HER2, symbol ERBB2) gene, but each technique requires a high-quality tissue sample, which may not be available. We investigated whether serum concentrations of the HER2 extracellular domain (ECD) can be used as an alternative to tissue HER2 status in metastatic breast cancer, and we defined an optimal decision level of serum HER2 for prediction of tissue HER2 status. METHODS: We determined HER2 expression in 195 metastatic breast cancer patients by IHC and performed FISH analysis on tumors for which IHC staining was graded as 2+. We measured serum HER2 by immunoassay and used receiver operating characteristics (ROC) curve analysis to determine optimal serum HER2 ECD concentrations for differentiation between positive and negative HER2 status. RESULTS: IHC results were 0/1+ for 30 (15%) of the patients, 2+ for 89 (46%), and 3+ for 76 (39%). FISH revealed HER2 amplification in 19 (21%) of the IHC 2+ tumors. Mean (SE) serum HER2 ECD was 22.2 (5.1) microg/L in the tissue HER2-negative group, significantly lower than the concentration of 363 (96) microg/L in the tissue HER2-positive group (P <0.0001). ROC curve analysis showed 95% specificity and 62% sensitivity for tissue HER2 positivity at 37 microg/L of serum HER2. CONCLUSION: To use serum HER2 concentration as an alternative to direct determination of tissue HER2 status, we suggest 37 microg/L as a cutoff for predicting positive tissue HER2 with 95% specificity. Sensitivity, however, is low. 16 Mazouni C, Hall A, Anderson K, et al. An early decrease in serum HER-2/neu levels after initiation of primary chemotherapy for HER-2 positive breast cancer indicates response to treatment. Breast Cancer Res Treat, 2006. 100(suppl 1): Abstract No. 1039 ABSTRACT Introduction: To determine the utility of detection of the extracellular domain of the Her-2/neu receptor in the serum of patients with primary breast cancer receiving neoadjuvant chemotherapy. Materials and Methods: Her-2/neu extracellular domain (ECD) was measured in sera obtained from 39 patients with stage II-III primary breast cancer, HER2-amplified undergoing neoadjuvant chemotherapy. Patients were randomly assigned to either four cycles of paclitaxel followed by four cycles of fluorouracil, epirubicin, and cyclophosphamide (FEC) (n=10) or to the same chemotherapy with simultaneous weekly trastuzumab for 24 weeks (n=29). Changes in HER-2/neu were monitored with the Bayers HER-2/neu assay over 6 months (baseline, week 3, 6 and 24) and correlated with pathological response to treatment. The comparisons between serum HER-2/neu baseline and serial throughout treatment concentrations were made with the Wilcoxon-signed ranks test. Results: The mean serum HER-2/neu baseline concentration was 20.3 ng/mL with a median of 13.6 ng/mL. In 28.2% of patients, elevated concentration of the HER-2/neu ECD (> 15 ng/mL) was detected before initiation of chemotherapy (8.3-232.8ng/mL). There was no correlation between HER-2 FISH amplification and serum HER-2/neu levels (p=0.42). A decrease of the median HER-2/neu levels after week 3 and week 6 of chemotherapy was seen in the group with pathological complete response (p= 0.003), whereas no significant change in HER-2/neu levels was observed in the group with residual disease (p=0.11). A decrease in the median HER-2/neu levels after week 6 of chemotherapy was only seen in the group of patient who received trastuzumab (p=0.007). No significant difference in baseline HER-2/neu ECD levels was observed between the groups who achieved pathological complete response and the group with residual disease (p=0.41). Conclusion: A decrease in serum Her-2/neu level indicates response to treatment in patients receiving primary chemotherapy and serve to monitor trastuzumab-based regimens. 17 Muller V, Witzel I, Pantel K, et al. Prognostic and predictive impact of soluble epidermal growth factor receptor (sEGFR) protein in the serum of patients treated with chemotherapy for metastatic breast cancer. Anticancer Res. 2006 Mar-Apr;26(2B):1479-87. ABSTRACT BACKGROUND: A soluble fragment of the epidermal growth factor receptor (EGFR) extracellular domain (sEGFR) can be detected in the serum of cancer patients, but the role of sEGFR is still unclear. MATERIALS AND METHODS: Blood samples from patients receiving chemotherapy for metastatic breast cancer were collected before (n = 101) and after 3 courses of therapy (n=39). Levels of sEGFR and serum HER-2/neu extracellular domain (ECD) were determined by standardized ELISA. RESULTS: A higher percentage of cancer patients (15%) showed sEGFR values below 45ng/mL compared with control subjects (3%, p<0.001). Patients with sEGFR levels below 45 ng/mL showed a trend towards shorter overall survival (median 11.7 versus 15.4 months, p=0.08), which was more pronounced in patients with estrogen receptor-positive primary tumors (median 9.6 versus 15.4 months, p=0.022) Patients with low sEGFR and elevated serum HER-2/neu ECD (>15 ng/mL) also showed a shorter overall survival than those with normal values for both parameters (7.1 versus 15.4 months, p=0.03). Again, this difference was higher in patients with estrogen receptor-positive tumors (4.6 versus 15.4 month, p<0.0001). During treatment, a decrease of sEGFR levels occurred in 74.4% of the patients (p=0.014). CONCLUSION: Low sEGFR levels in patients with metastatic breast cancer are associated with a shorter overall survival, particularly in patients with estrogen receptor-positive tumors. Chemotherapy frequently induces a decrease of sEGFR. The combined, determination of sEGFR and serum HER-2/neu ECD also delivers relevant information. These findings suggest that the sEGFR status in metastatic breast cancer could be of clinical relevance. 18 Okegawa T, Kinjo M, Nutahara K, et al. Pretreatment serum level of HER2/nue as a prognostic factor in metastatic prostate cancer patients about to undergo endocrine therapy. Int J Urol. 2006 Sep;13(9):1197-201. ABSTRACT BACKGROUND: Overexpression of the HER2 receptor protein and amplification of the HER2 gene has been implicated in tumor development and progression, and has been associated with a poor prognosis in several types of cancer. The aim of this study was to evaluate whether pretreatment serum HER2 levels can be used to predict biochemical recurrence-free survival in prostate cancer patients about to undergo endocrine therapy. METHODS: The study population consisted of 379 untreated patients with histologically diagnosed prostate cancer: 197 with T2N0M0, 93 with T3N0M0, 19 with TxN1Mx, and 70 with TxNxM1. Serum HER2 levels were assessed in the prostate cancer patients prior to treatment as well as in a control group of 100 patients with histologically confirmed non-cancer. Biochemical recurrence-free curves for the patients were investigated separately using the Kaplan-Meier method. RESULTS: The mean level of HER2 in serum was significantly higher in prostate cancer patients than non-prostate cancer patients (P = 0.006). Also, the serum HER2 level was significantly higher in bone metastatic cancer patients (14.3 +/- 6.3 ng/mL) than in non-metastatic patients (T2: 11.9 +/- 2.3 ng/mL, P = 0.003; T3: 12.2 +/- 2.8 ng/mL, P = 0.011). The metastatic patients were divided into those with low and high HER2 levels using a cutoff value of 12.6 ng/mL based on receiver-operating characteristic curves. The biochemical recurrence-free rate was significantly poorer in patients with a high HER2 level (P = 0.0078, log-rank test). Multivariate Cox logistic regression analysis demonstrated that the pretreatment serum HER2 value (P = 0.022), serum prostate-specific antigen value (P = 0.018), and extent of disease score (P = 0.027) were independent predictors of recurrence. CONCLUSIONS: The pretreatment serum HER2 level may be a useful independent prognostic factor that is associated with a high risk of biochemical recurrence in metastatic prostate cancer patients about to undergo endocrine therapy. 19 Quaranta M, Daniele A, Coviello M, et al. c-erbB-2 protein level in tissue and sera of breast cancer patients: a possibly useful clinical correlation. Tumori. 2006 Jul-Aug;92(4):311-7. ABSTRACT AIMS AND BACKGROUND: The aims of this study were to assess the clinical utility of circulating preoperative HER-2 extracellular domain p105 detected by enzyme immunoassay (ELISA), to compare the tissue expression of HER-2/neu determined by immunohistochemistry (IHC), to correlate prognostic factors including tumor size, nodal involvement, and hormone receptor status, and to analyze the prognostic significance of the marker in relation to clinical outcome as measured by disease-free and overall survival. METHODS: In this study, we enrolled 108 consecutive patients with breast carcinoma, and obtained serum samples and frozen tumor tissues. We compared them with 57 women with fibroadenoma and 63 healthy women as controls. RESULTS: Univariate ANOVA analysis showed no relationship between HER-2/neu in tissue and serum. Preoperative serum levels of p105 were significantly higher in breast cancer patients than in women with benign disease or healthy women. Concerning the correlation between p105, HER-2/neu tissue expression, and the other prognostic factors, a statistically significant correlation between high serum p105 levels and ER-negative status in breast cancer patients was found. Kaplan-Meier analysis confirmed that patients with positive HER-2/neu tissue expression had a significantly shorter survival than those with negative expression. Analysis with the Cox model demonstrated that tumor size was the only significant independent prognostic factor. CONCLUSIONS: This research failed to demonstrate a relationship between preoperative tissue overexpression and circulating HER-2/neu, suggesting that p105 does not represent a valid alternative to predict a worsened prognosis in breast cancer, but it could be a diagnostic marker to discriminate healthy subjects from breast cancer patients. 20 Souder C, Leitzel K, Ali S, et al. Serum epidermal growth factor receptor/HER-2 predicts poor survival in patients with metastatic breast cancer. Cancer. 2006 Nov 15;107(10):2337-45. ABSTRACT BACKGROUND: Epidermal growth factor receptor (EGFR, HER-1, and erbB1) is overexpressed in primary breast cancer and had been identified as a poor prognostic factor. METHODS: Pretreatment serum EGFR levels were quantified by using an enzyme-linked immunoadsorbent assay in a Phase III first-line trial of letrozole and tamoxifen and were correlated with patient outcomes. RESULTS: Serum EGFR levels in a control group of 117 healthy, postmenopausal women measured 64.1 +/- 13.3 ng/mL (mean +/- standard deviation). Using a cutoff EGFR level of 44.1 ng/mL from the control group (5% nonparametric method), 53 of 535 patients (10%) had decreased serum levels of EGFR. Patients with decreased serum EGFR had no significant difference in objective response rate (ORR), clinical benefit rate (CBR), time to progression (TTP), or time to treatment failure (TTF); however, they did have significantly reduced survival compared with patients who had normal serum EGFR levels (median survival, 23.3 months vs. 30.9 months; P = .007). A combined analysis of pretreatment serum EGFR and HER-2 yielded no additional predictive information for ORR, CBR, TTP, or TTF compared to serum HER-2 alone. However, in the current analysis, a subgroup of patients who had decreased serum EGFR and normal serum HER-2 was identified (n = 39 of 535 patients; 7.3%) that had significantly reduced survival compared with patients who had normal serum levels of both EGFR and HER-2 (median survival, 23.5 months vs. 37.1 months; P = .005). In multivariate analysis, a decreased serum EGFR level remained a significant independent prognostic factor for decreased survival (hazards ratio, 1.58; P = .007). CONCLUSIONS: In patients who had metastatic breast cancer, decreased serum EGFR/normal serum HER-2 predicted shorter survival compared with patients who had normal levels of serum EGFR/HER-2. This patient subgroup deserves further study to assess their response to and selection for anti-EGFR-directed therapies. 21 Witzel I, Thomssen C, Krenkel S, et al. Clinical utility of determination of HER-2/neu and EGFR fragments in serum of patients with metastatic breast cancer. Int J Biol Markers. 2006 Jul-Sep;21(3):131-40. ABSTRACT INTRODUCTION: The growth factor receptors EGFR and HER-2/neu are targets for new treatment strategies and are of potential use as prognostic and predictive factors. However, the optimal method of determination in order to obtain clinically relevant information remains a source of controversy. METHODS: HER-2/neu and EGFR expression was examined by immunohistochemistry in primary tumors of patients with breast cancer. In addition, serum was tested for the extracellular domains of HER-2/neu (HER-2/neu ECD) and EGFR (sEGFR) before initiation of therapy for metastatic disease (n=76). The course of disease from the time of metastasis with regard to these parameters was evaluated by univariate and multivariate analyses. RESULTS: HER-2/neu ECD levels at the time of metastatic disease were correlated with HER-2/neu expression determined by immunohistochemistry from primary tumors (p=0.001). No correlation was observed between expression of EGFR in primary tumors and sEGFR serum levels. HER-2/neu ECD and sEGFR levels at the onset of metastatic disease did not show a significant impact on overall survival. CONCLUSIONS: Determination of HER-2/neu ECD levels in the serum measured by ELISA at the onset of metastatic disease could offer an alternative to immunohistochemistry of the primary tumor since serum levels are correlated with protein expression in primary tumors. In contrast, no such correlation was observed for EGFR. 22 Carney WP. HER2 status is an important biomarker in guiding personalized HER2 therapy. Personalized Medicine, 2005. Vol. 2, No. 4, Pages 317-324 ABSTRACT: The human epidermal growth factor receptor (HER)2 oncoprotein has emerged as an important cellular target for the development of a variety of new cancer therapies. The method used to define the HER2 status is a major factor in determining who will receive these targeted therapies. The HER2 status can be determined by using either tissue tests to look at the primary tumor cells, or an enzyme-linked immunosorbent assay (ELISA) that measures the circulating levels of the extracellular portion of HER2 protein. Tissue test (immunohistochemistry and fluorescence in situ hybridization) results indicate that approximately 20–30% of patients with primary breast cancer have a HER2-positive tumor, whereas ELISA results demonstrate that an average of 45% (range: 23–80%) of metastatic breast cancer (MBC) patients can have an abnormally high (> 15 ng/ml) serum HER2 level, which is evidence that a HER2-positive tumor is present. Published studies show that the HER2 status of a breast cancer patient can differ both by the test method used and the time at which HER2 status is assessed. In this review, data will be shown that demonstrates that not all HER2 test results obtained from the primary breast cancer are correct, and that there is a population of patients categorized as HER2 negative by tissue tests that, in fact, have HER2-positive tumors. This observation has important therapeutic implications for breast cancer patients with HER2-positive tumors that are classified as HER2 negative, since they are not eligible for anti-HER2 therapy, such as trastuzumab. If a patient is found to have an elevated (> 15 ng/ml) serum HER2 level in MBC, then either the original tumor should be re-evaluated for HER2 status, or a metastatic lesion should be tested for HER2 positivity, to determine if the patient is eligible for anti-HER2 therapy. Studies have also shown that lack of adequate validation of a testing method can result in false conclusions concerning the HER2 status. If the goal of personalized medicine is to deliver the right treatment to the right patient at the right time then we need to ensure the validity of all test methods, regardless of whether they are for research purposes or are registered as in vitro diagnostics. In the case of establishing HER2 status, it takes more than one type of test to identify patients with HER2-positive tumors. It is highly likely that the introduction of additional targeted drugs to growth factor receptors or to angiogenesis targets will take a variety and combinations of tests to tailor the most appropriate therapy to the patient. 23 Esteva FJ, Cheli CD, Fritsche H, et al. Clinical Utility of Serum HER-2/neu in Monitoring and Prediction of Progression-free Survival in Metastatic Breast Cancer Patients Treated with Trastuzamab-based Therapies. Breast Cancer Res. 2005;7(4):R436-R443 ABSTRACT INTRODUCTION: The purpose of this retrospective study was to determine the clinical utility of serum HER2/neu in monitoring metastatic breast cancer patients undergoing trastuzumab-based therapy and to compare these results with those obtained using cancer antigen (CA) 15-3. We also sought to determine whether early changes in serum HER2/neu concentrations could be a predictor of progression-free survival. METHODS: Sera were obtained retrospectively from 103 women at four medical institutions. Patients eligible for participation were women with metastatic breast cancer who had HER2/neu tissue overexpression and were scheduled to be treated with trastuzumab with or without additional therapies as per the established practices of the treating physicians. A baseline serum sample for each patient was taken before trastuzumab-based therapy was started. Patients were subsequently monitored over 12 to 20 months and serum samples were taken at the time of clinical assessment and tested with Bayer's HER2/neu and CA15-3 assays. RESULTS: Concordance between clinical status in patients undergoing trastuzumab-based treatment and HER2/neu and CA15-3 used as single tests was 0.793 and 0.627, respectively, and increased to 0.829 when the tests were used in combination. Progression-free survival times did not differ significantly in patients with elevated baseline HER2/neu concentrations (¡Ý 15 ng/mL) and those with normal concentrations (<15 ng/mL). However, progression-free survival differed significantly (P = 0.043) according to whether the patient's HER2/neu concentration at 2 to 4 weeks after the start of therapy was >77% or ¡Ü 77% of her baseline concentration. The median progression-free survival times for these two groups were 217 and 587 days, respectively. A similar trend was observed for a subcohort of patients treated specifically with a combination of trastuzumab and taxane. CONCLUSION: These findings indicate that serum HER2/neu testing is clinically valuable in monitoring metastatic breast cancer patients undergoing trastuzumab-based treatment and provides additional value over the commonly used CA15-3 test. The percentage of baseline HER2/neu concentrations in the early weeks after the start of therapy may be an early predictor of progression-free-survival. 24 Fornier MN, Seidman AD, Schwartz MK, et al. Serum HER2 extracellular domain in metastatic breast cancer patients treated with weekly trastuzumab and paclitaxel: association with HER2 status by immunohistochemistry and fluorescence in situ hybridization and with response rate. Ann Oncol. 2005 Feb;16(2):234-9. ABSTRACT PURPOSE: We explored the relationship between circulating HER2 extracellular domain (ECD) and tissue HER2 status as determined by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). We also examined its predictive value in a cohort of metastatic breast cancer patients treated with weekly trastuzumab and paclitaxel. METHODS: Eligible patients had pre- and post-treatment stored serum specimens and were treated on a previously reported phase II trial. Retrospective analysis evaluated: the association between pretreatment serum HER2 ECD and tissue HER2 status by IHC and FISH; and the association between change in serum HER2 ECD after 12 weeks of therapy and response proportion. RESULTS: Stored serum samples were available for 55/95 (58%) patients. Statistically significant associations were found between HER2 status as assessed by IHC and FISH, and baseline serum HER2 ECD level. Patients whose ECD normalized after 12 weeks of therapy had a higher response proportion compared with patients with persistently high ECD levels (68% versus 15%, P=0.005). A relative decline of over 55% from baseline HER2 ECD predicted response to therapy. CONCLUSION: A statistically significant association was observed between pretreatment serum HER2 ECD and tissue HER2 status as assessed by IHC and FISH. A decrease in serum HER2 ECD level was a significant predictor of response to trastuzumab-based therapy. 25 Lipton A, Leitzel K, Ali SM, et al. Serum HER-2/neu conversion to positive at the time of disease progression in patients with breast carcinoma on hormone therapy. Cancer. 2005 Jul 15;104(2):257-63. ABSTRACT BACKGROUND: Prolonged exposure of breast carcinoma cells in vitro to tamoxifen results in tamoxifen resistance. Tamoxifen-resistant cells express increased HER-2/neu mRNA and protein. The objective of this study was to determine whether patients with metastatic or locally advanced breast carcinoma who have negative serum HER-2/neu status at the initiation of first-line hormone therapy with letrozole or tamoxifen convert to positive serum HER-2/neu status at the time of disease progression and to determine whether serum HER-2/neu conversion to positive status is associated with response to therapy and overall survival. METHODS: Serum samples were obtained at baseline and at the time of disease progression from 240 patients who initially had negative serum HER-2/neu status (< 15 ng/mL). A manual microtiter, enzyme-linked immunosorbent assay that was specific for the extracellular domain of the HER-2/neu (c-erbB-2) oncoprotein product was used to quantitate serum levels. RESULTS: Among 240 patients, 61 patients (26%) converted from serum HER-2/neu negative to positive (> 15 ng/mL) at the time of disease progression. Thirty-two of 129 patients (25%) who were treated with tamoxifen and 29 of 111 patients (26%) who were treated with letrozole became converted to positive serum HER-2/neu status at the time of disease progression. The response rate and the time to disease progression on first-line hormone therapy were not affected by serum HER-2/neu conversion. The survival of patients who converted to positive serum HER-2/neu status was significantly shorter compared with the survival of patients who remained negative for serum HER-2/neu. A multivariate analysis revealed that conversion to positive serum HER-2/neu status was an independent prognostic variable for survival. CONCLUSIONS: Conversion to positive serum HER-2/neu status occurred in approximately 25% of patients who received first-line hormone therapy. Conversion to serum HER-2/neu-positive status occurred with equal frequency in antiestrogen and aromatase-inhibitor therapy. The current results showed that serum conversion to HER-2/neu-positive status was an independent risk factor for decreased survival in patients with breast carcinoma. 26 Salvadori B, Pinzani P, Distante V, et al. Comparison of pre- and postsurgical concentrations of blood HER-2 mRNA and HER-2 extracellular domain reflects HER-2 status in early breast cancer. Clin Chem. 2005 Jan;51(1):254-6. NO ABSTRACT AVAILABLE 27 Stemmler HJ, Stieber P, Lassig D, et al. Re-evaluation of HER2 status in metastatic breast cancer and tumor-marker guided therapy with vinorelbine and trastuzumab. Onkologie. 2005 Feb;28(2):95-7. ABSTRACT BACKGROUND: HER2 is overexpressed in 20-30% of breast cancers. Compared to chemotherapy alone, chemotherapy with trastuzumab improves clinical outcome in patients with HER2-positive metastatic breast cancer (MBC). In general, HER2 status in a primary lesion predicts the status of metastases, so that biopsy of metastatic lesions appears unnecessary. CASE REPORT: A 39-year old woman was diagnosed with primary breast cancer in November 2000. Using the method and scoring system of the DAKO Hercep Test, the tumor has shown low HER2 expression (DAKO score 1+). After failure of several chemotherapy regimens for metastatic disease (liver, skeletal), the patient underwent CT-guided needle biopsy of the liver which showed HER2 positive adenocarcinoma (DAKO score 3+). In consequence, the patient was treated with vinorelbine (30 mg/m2 d1,8,15 q4w) and trastuzumab (4 mg/kg loading dose, 2 mg/kg weekly). During a treatment period of 4 months imaging results as well as tumor marker kinetics indicated an excellent response with sustained decrease of tumor markers. A retrospective analysis of the HER2 shed antigen in metastatic stage revealed excessively increased serum levels and supports HER2 overexpression observed in liver metastasis. The kinetics of the HER2 shed antigen during therapy for metastatic disease were found to be in phase with the kinetics of CEA and CA15-3. CONCLUSION: This case report demonstrates that re-evaluation of the HER2 status may be helpful in single patients not sufficiently responding to treatment of metastatic disease. Determination of HER2 overexpression may be facilitated by a determination of the HER2 shed antigen level in peripheral blood. 28 Bethune-Volters A, Labroquere M, Guepratte S, et al. Longitudinal changes in serum HER-2/neu oncoprotein levels in trastuzumab-treated metastatic breast cancer patients. Anticancer Res. 2004 Mar-Apr;24(2C):1083-9. ABSTRACT BACKGROUND: To evaluate longitudinal variations of serum HER-2/neu extracellular domain (sHER-2) in metastatic breast cancer patients receiving combined trastuzumab treatment. PATIENTS AND METHODS: Thirty-three patients were monitored by serial sHER-2 ELISA (Oncogene Science). Results were compared to time to progression (TTP) and survival from treatment initiation. Non parametric statistical tests were used. RESULTS: Median sHER-2 before first injection was 41.37 ng/ml (range 7.54-1597.00 ng/ml, n=32). Mean sHER-2 levels differed significantly between responders (n=20) and non responders (n=13) (p<0.0001). Median TTP (266 days, range 35-1000 days) was unrelated to clinico-biological variables at diagnosis or number and site of metastases before treatment. Patients with pre-treatment sHER-2 levels < or = 30 ng/ml (n=14) had a significantly longer TTP than the group with sHER-2 > 30 ng/ml (n=18) (p=0.0346) and sHER-2 levels were of prognostic value for overall survival from first injection (p=0.0150). CONCLUSION: Our results show that monitoring serum HER-2/neu levels during metastatic breast cancer can provide a real time assessment of a woman's HER-2/neu status and can provide important information for therapeutic decisions. 29 Carney WP, Neumann R, Lipton A, et al. Monitoring the circulating levels of the HER2/neu oncoprotein in breast cancer. Clin Breast Cancer. 2004 Jun;5(2):105-16. ABSTRACT: The HER2/neu oncoprotein is a major target for the development of new cancer therapies and is similar to the estrogen receptor, which guides hormone therapy. The HER2/neu status is used to guide therapy decisions in patients with HER2/neu-overexpressing breast cancer tumors. The HER2/neu oncogene, or c-erbB-2, encodes a transmembrane receptor protein that is expressed on normal epithelial cells and can be overexpressed in breast cancer cells. Studies have shown that the extracellular domain (ECD) of the HER2/neu oncoprotein is released from the cell and can be measured in the circulation of women with breast cancer. Enzyme-linked immunosorbent assay methods used to measure the circulating HER2/neu ECD have shown that the prevalence of elevated ECD levels is approximately 18.1% in women with primary breast cancer and approximately 45.6% in women with metastatic breast cancer (MBC). Many studies have monitored the circulating ECD levels after surgery and indicate that increasing ECD levels can indicate recurrence of breast cancer earlier than clinical diagnosis. Studies in women with MBC showed that serial changes in circulating HER2/neu ECD levels paralleled the clinical course of disease, regardless of the treatment regimen. Several studies identified a subgroup of patients with MBC who had HER2/neu-negative disease by tissue testing but developed elevated ECD levels with MBC. In contrast to tissue testing, which is a one-time event, monitoring the circulating levels of the HER2/neu ECD in patients with breast cancer provides a real-time assessment of the HER2/neu status and provides important information for managing the therapy of patients with MBC. 30 Colomer R, Llombart-Cussac A, Lluch A, et al. Biweekly paclitaxel plus gemcitabine in advanced breast cancer: phase II trial and predictive value of HER2 extracellular domain. Ann Oncol. 2004 Feb;15(2):201-6. ABSTRACT BACKGROUND: We wanted to assess the toxicity and efficacy of paclitaxel plus gemcitabine in advanced breast cancer and to confirm whether circulating HER2 extracellular domain (ECD) correlates with treatment response. PATIENTS AND METHODS: Forty-three patients received paclitaxel 150 mg/m2 followed by gemcitabine 2500 mg/m2, both on day 1 of 14-day cycles, with a maximum of eight cycles. Serum levels of HER2 ECD were assessed by ELISA. RESULTS: All patients were evaluable for toxicity and 42 for efficacy. Overall toxicity was low. Grade 3 neutropenia occurred in 12% of patients and grade 4 in 17%, and other grade 3 toxicities in <5%. One patient had an allergic infusion reaction. Overall response rate was 71% [95% confidence interval (CI) 62% to 81%], with 11 patients achieving a complete response (26%). With a median follow-up of 26 months, the median time to progression was 16.6 months. Response rate correlated significantly with HER2 ECD, with 42% of HER2 ECD-positive patients responding versus 83% of HER2 ECD-negative patients (P = 0.02). Furthermore, response duration was shorter in patients with positive HER2 ECD levels (7.9 versus 14.4 months; P = 0.04). CONCLUSIONS: Paclitaxel plus gem