HER2 ECD Levels Research Articles

Prognostic Value of the Serum HER2 Extracellular Domain Level in Breast Cancer: A Systematic Review and Meta-Analysis.

Simple SummaryWe performed a systematic review and meta-analysis evaluating the predictive value of the serum HER2 extracellular domain (sHER2 ECD) for breast cancer prognosis. Our review investigated 40 studies (12,229 patients) that assessed the impact of the sHER2 ECD levels on breast cancer prognosis and explored the clinical significance of sHER2 ECD levels in different treatment modalities. Our findings indicated that an elevated sHER2 was an unfavorable prognostic factor in breast cancer. More interestingly, sHER2 ECD was found to be a promising biomarker for predicting adverse clinical outcomes of trastuzumab-based treatment but did not affect the efficacy of tyrosine kinase inhibitors. In addition, the baseline cutoff value of sHER2 ECD in different treatment stages of breast cancer remains to be further explored. Overall, our study suggested that sHER2 ECD levels have important prognostic value in breast cancer and may be helpful for clinicians to select the appropriate anti-HER2 therapy for HER2-positive breast cancer, providing more evidence for guiding clinical practice.An elevated serum HER2 extracellular domain is associated with poor prognosis in breast cancer, but the relationship between sHER2 and the efficacy of different modalities remains controversial. Herein, we aimed to evaluate the prognostic value of serum HER2 extracellular domain (sHER2 ECD) in breast cancer and to identify its correlation with the efficacy of different treatment regimens. A systematic search of the PubMed, Embase, Cochrane Library, Web of Science, and Scopus databases was conducted to identify studies exploring the association between HER2 ECD level and clinical outcomes among patients with breast cancer. Using the random effects models, pooled hazard ratios (HRs), and odds ratios (ORs) with 95% confidence intervals (CI), were calculated for progression-free survival (PFS), overall survival (OS), disease-free survival (DFS), and the objective response rate (ORR). Heterogeneity was further evaluated by subgroup and sensitivity analysis. Overall, 40 studies comprising 12,229 patients were included in this systematic review and meta-analysis. Elevated HER2 ECD levels were associated with worse PFS (HR 1.74, 95% CI 1.40–2.17; p < 0.001), and this effect was observed in patients treated with chemotherapy (HR 1.81, 95% CI 1.37–2.39; p < 0.001), endocrine therapy (HR 1.91, 95% CI 1.57–2.32; p < 0.001), and trastuzumab (HR 1.74, 95% CI 1.31–2.30; p < 0.001). However, this association was not present in patients treated with tyrosine kinase inhibitors (TKIs) (HR 1.44, 95% CI 0.85–2.43, p = 0.17). The HRs/ORs for an elevated HER2 ECD level for DFS, OS, and ORR were 2.73 (95% CI 2.17–3.42; p < 0.001), 2.13 (95% CI 1.77–2.57; p < 0.001), and 0.80 (95% CI 0.49–1.31; p = 0.381), respectively. An elevated sHER2 ECD was an unfavorable prognostic factor in breast cancer but did not affect the efficacy of tyrosine kinase inhibitors such as lapatinib. Detection of sHER2 ECD may be helpful for clinicians selecting the appropriate anti-HER2 therapy for patients with HER2-positive breast cancer.

A high-performance electrochemical aptasensor based on graphene-decorated rhodium nanoparticles to detect HER2-ECD oncomarker in liquid biopsy

Evaluation of extracellular domain of human epidermal growth factor receptor-2 (HER2-ECD) oncomarker status is an impressive factor in screening, diagnosing and monitoring early-stage breast cancer (BC). Electrochemical aptamer-based nanobiosensor with high sensitivity and selectivity for quantitative and qualitative measurement of HER2-ECD oncomarker was developed. In this study, the nanocomposite made by distinct materials included reduced graphene oxide nano-sheets (rGONs) and rhodium nanoparticles (Rh-NPs) on the graphite electrode (GE) surface. This structure resulted in amplified electrochemical activity, high surface area, stability, and bio-compatibility. Each of the steps of preparing nanomaterials and setting up biosensor were carefully examined by analytical and electrochemical techniques. Various modified electrodes were constructed and analyzed in terms of electrochemical performance, morphology, size, and shape of nanomaterials. The GE-based aptasensor had a noteworthy and conducive results against HER2-ECD with a wide dynamic range of 10.0–500.0 ng/mL, a low limit of detection (LOD) of 0.667 ng/mL (significantly less than the clinical cut-off), and a low limit of quantification (LOQ) of 2.01 ng/mL. The benefits provided by this aptasensor such as broad dynamic range, high sensitivity, selectivity, stability, reproducibility, and low cost suggest tremendous potential for non-invasive detection and monitoring of the HER2-ECD levels of BC care and clinical diagnosis.

Utility of Serum HER-2/ neu in Prediction of Tissue HER-2 /neu Status of Primary Breast Cancer

Background: The use of biomarkers has become increasingly important in the diagnosis and treatment of several malignancies, including breast cancer. Among the established biomarkers, receptor tyrosine-protein kinase erbB-2 (HER-2/ neu) has been found to be of a predictive and prognostic role in breast cancer patients. Method: 94 patients with primary breast cancer were enrolled in our prospective study between 2016 and 2017. 5 cc clot blood samples were taken for serum HER-2/neu testing at the time of diagnosis of breast cancer. The patients’ demographic data, tumor characteristics, including tumor size, tumor grade, presence of lymph node involvement, and stage, hormone receptor, and tissue HER-2/neu status of the subjects were recorded following tumor removal. Results: All of them were female with the age range of 27 to 80 years (the mean of age was 49.66. 36 patients (38.3%) had positive tissue HER-2 results and 58 patients (61.7%) had negative results. In those with high level serum HER-2 ECD, 28(77.7%) had positive tissue HER-2 and 8(22.2%) had negative results. Moreover, in the patients with low level serum HER-2 ECD, 8(13.7%) had positive tissue HER-2 and 50(86.2%) had negative results with the sensitivity of 77.7% and specificity of 86.2%. HER2 ECD levels were highly concordant with tissue HER2 status, with a P value of less than 0.001, which was considered to be statistically significant. Among different clinic –pathologic variables, serum and tissue HER-2/nu were significantly correlated with only tumor grade. Conclusion: A significantly increased release of HER2 ECD levels may accurately predict tumor HER2 status as detected with IHC and/or in situ hybridizations studies.

Analysis of plasma HER2 copy number in cell-free DNA of breast cancer patients: a comparison with HER2 extracellular domain protein level in serum

HER2 (human epidermal growth factor receptor 2) status has been evaluated in breast cancer (BC) tissues by immunohistochemistry or in situ hybridization. We evaluated HER2 copy number (CN) assay in plasma cell-free DNA (cfDNA) from blood samples and compared it with protein measurements of HER2 extracellular domain (ECD) in serum. Serum HER2-ECD levels were measured by chemi-luminescence immunoassay using anti-HER2 monoclonal antibodies. Analyses were performed on 120 cases of primary BC, 30 cases of metastatic BC and 34 cases treated by neoadjuvant chemotherapy (NAC). This study was approved by Medical Research Review Advancement No. 1857 for Kumamoto University. There was a positive correlation between HER2-CN ratios and HER2-ECD levels, in primary (n = 54) and metastatic (n = 30) HER2-positive BC (P = 0.003 and P < 0.001, respectively). HER2-ECD levels were significantly higher in patients with a larger number of metastatic sites (P = 0.02). The usefulness of HER2 levels in discriminating primary and metastatic HER2-positive BC evaluated by ROC curve analysis was better in the HER2-ECD assay than in the HER2-CN assay. In 34 patients who received NAC, there was a small decrease in HER2-CN ratios between before and after NAC (P = 0.10), while there was an obvious decrease in HER2-ECD levels between before and after NAC (P < 0.001). Compared to HER2-ECD levels, the clinical usefulness of HER2-CN ratio was somewhat inferior. Improved measurement methods and further examination of the association with long-term prognosis and the response to anti-HER2 treatment analyzed by HER2-CN and HER2-ECD assay are required.

Remarkable response with pembrolizumab plus albumin-bound paclitaxel in 2 cases of HER2-positive metastatic breast cancer who have failed to multi-anti-HER2 targeted therapy

ABSTRACTIn clinical practice, one subgroup patients of breast cancer might have developed resistance to multi-anti-HER2 targeted drugs(trastuzumab, lapatinib and/or T-DM1) and can not benefit from the anti-HER2 targeted therapy continuously. We attempt to change the next therapic way for these patients. Two patients with metastatic breast cancer who have failed to multi-anti-HER2 targeted therapy were treated with pembrolizumab (2 mg/Kg, day1) plus albumin-bound paclitaxel (125 mg/m2, day1,8) every 3 weeks. CT evaluation and HER2 ECD test were performed every 2 cycles. Both of the two patients achieved remarkable response with Partial Remission (PR), meanwhile serum HER2 ECD levels (the upper normal limit is 15 ng/ml) showed a remarkable decreases(compared to the base line decreases 75% and 60% respectively). The results indicate that regimen of pembrolizumab combination with albumin-bound paclitaxel might produce response in patients with HER2-positive metastatic breast cancer who have failed to multi-anti-HER2 targeted therapy.

Value of serum human epithelial growth factor receptor 2 extracellular domain and circulating tumor cells in evaluating therapeutic response in advanced gastric cancer

To examine the correlation between serum human epithelial growth factor receptor 2 extracellular domain (HER2 ECD) and circulating tumor cells (CTC), as well as the dynamic variation of HER2 ECD and its correlation to the therapeutic efficacy. Fifty-three advanced gastric cancer (AGC) patients who treated in Peking University Cancer Hospital and ever enrolled into CTC study (ClinicalTrial gov. ID: NCT01625702) were retrospectively included in this study. the patients were histologically confirmed as locally advanced or recurrent and/or metastatic adencarcinoma; they received two or more cycles of fluorouracil-based chemotherapy or combination targeted therapy; serum CTC was counted before and after therapy; the clinical response was evaluated every 2 cycles of treatment by the presence of at least one measurable lesion according to RECIST version 1.1 criteria. This study was approved by Ethics Committee of Peking University Cancer Hospital, and informed consents were signed by patients. The sera before and after two cycles of treatment were collected for CTC enumeration and HER2 ECD detection, in which the levels of HER2 ECD were measured by chemiluminescence immunoassays method. The positive threshold value of HER2 ECD and CTC number were ≥15 μg/L and ≥3 CTCs/7.5 ml respectively. The progression-free survival (PFS) and overall survival (OS) were compared among different groups using Log-rank tests. In 53 enrolled patients, 39 were histologically identified as negative HER2, 9 as positive HER2 and another 5 cases were unknown. All the patients received fluorouracil-based chemotherapy, and 9 positive HER2 patients received combined anti-HER2 targeted therapy. Before therapy, the median HER2 ECD concentration of 53 cases was 10.45 (8.0 to 83.2) μg/L. Seven patients exhibited positive HER2 ECD levels, in whom 4 were histologically HER2 positive, but 3 were histologically HER2 negative. The median CTC number of 53 cases was 2 (0 to 668) CTCs/7.5 ml, and the positive rate of CTC was 47.2%(25/53). Following 2 cycles of therapy, a total of 10 histologically HER2 negative patients exhibited positive HER2 ECD levels, in whom 2 also possessed positive HER2 ECD levels, 83.3 μg/L and 46.9 μg/L before therapy, and 22.4 μg/L and 20.4 μg/L after therapy respectively, whereas another 8 patients (10.3 to 14.5 μg/L before therapy) acquired the elevated expression of HER2 ECD following therapy (15.1 to 19.5 μg/L). It seems that the increased level of HER2 ECD after therapy was, though not statistically significant, correlated to low number of CTCs. In histologically HER2 negative patients, pretherapeutic HER2 ECD level (positive vs. negative) was not significantly correlated to PFS (7.6 months vs. 4.4 months, P=0.328) and OS (13.6 months vs. 10.9 months, P=0.679). However, in histologically HER2 positive patients, patients with positive HER2 ECD level before therapy exhibited longer PFS (10.7 months vs. 4.2 months, P=0.025) and OS (16.5 months vs. 8.9 months, P=0.015) compared to those with negative HER2 ECD level. Additionally, CTC number was significantly correlated to prognosis in histologically HER2 negative patients. Patients with positive pretherapeutic CTC number showed longer PFS (5.3 months vs. 3.3 months, P=0.049) and OS (14.3 months vs. 7.6 months, P=0.001) as well. While in histologically HER2 positive patients, CTC number was not obviously correlated to the PFS and OS. In above 8 negative HER2 patients acquiring elevated expression of HER2 ECD following therapy, the increased HER2 ECD level was not correlated to PFS and OS (all P>0.05). In 9 histologically HER2 positive patients, 4 patients who exhibited decreased HER2 ECD level and reduced or constant CTC number had longer PFS (7.5 to 15.3 months) and OS (11.0 to 26.3 months) compared with those 2 patients who suffered from acquired HER2 ECD level following therapy (PFS 3.0 to 4.8 months and OS 7.3 to 8.6 months). In histologically HER2 positive patients, increased pretherapeutic HER2 ECD level predicts better prognosis. The acquired elevated HER2 ECD level following therapy is correlated to inefficient therapeutic response. The acquirement of elevated HER2 ECD level can also be found in histologically HER2 negative patients, which may be correlated to the corresponding variation of CTC number.

P1-5-61 - Characterization of serum HER2 extracellular domain (ECD) level in patients with metastatic colorectal cancer

Background: Overexpression of human epidermal growth factor receptor (HER) 2 occurs in approximately 20% of breast or gastric, and 5% of colorectal cancers. Moreover,activation of the HER2 pathway has been reported to be one of the mechanisms of primary or acquired resistace to anti-EGFR antibodies in RAS wild-type metastatic colorectal cancer (mCRC). The serum level of the HER2 extracellular domain (ECD) is used as a tumor marker or surrogate marker of the HER2 status in gastric and breast cancers. However, little is known about HER2-ECD levels and its characterization in mCRC patients (pts).Methods: We prospectively analyzed consecutive mCRC pts receiving palliative chemotherapy in our hospital. HER2-ECD level was measured in blood serum, and its positivity was defined as a serum HER2 level> 15.2 ng/ml, based on the manufacturers' recommended cutoff value.Results: A total of 145 pts were enrolled in this study. Pt characteristics were as follows: males/females (55%/45%), median age (range); 64 (24- 93) years, primary site; colon/rectum (52%/48%), RAS status; wild/RAS mutant/BRAF mutant/ unknown (57%/31%/5%/8%). The positive rate of HER2 ECD, CEA, and CA19-9 were 27.6, 80.0 and 51.7%, respectively. HER2 ECD showed a linear relationship with CEA level (correlation coefficient of 0.677). Regarding the association with the RAS status, serum levels of HER2 ECD in RAS wild type pts were higher than those in RAS mutant pts (positive rate of 33 vs. 20%, respectively). Moreover, in RAS wild type pts, pts resistant to anti-EGFR antibody within 1 year showed a higher positive rate of HER2 ECD than the others (55 vs. 28%,respectively; p = 0.0311).Conclusions: A Non negligible number of mCRC pts were positive for serum levels of HER2 ECD. HER2 ECD may be associated with tumor burden and acquired resistance to anti-EGFR therapy, but further studies are necessary to determine clinical utility of HER2 ECD. Background: Overexpression of human epidermal growth factor receptor (HER) 2 occurs in approximately 20% of breast or gastric, and 5% of colorectal cancers. Moreover,activation of the HER2 pathway has been reported to be one of the mechanisms of primary or acquired resistace to anti-EGFR antibodies in RAS wild-type metastatic colorectal cancer (mCRC). The serum level of the HER2 extracellular domain (ECD) is used as a tumor marker or surrogate marker of the HER2 status in gastric and breast cancers. However, little is known about HER2-ECD levels and its characterization in mCRC patients (pts). Methods: We prospectively analyzed consecutive mCRC pts receiving palliative chemotherapy in our hospital. HER2-ECD level was measured in blood serum, and its positivity was defined as a serum HER2 level> 15.2 ng/ml, based on the manufacturers' recommended cutoff value. Results: A total of 145 pts were enrolled in this study. Pt characteristics were as follows: males/females (55%/45%), median age (range); 64 (24- 93) years, primary site; colon/rectum (52%/48%), RAS status; wild/RAS mutant/BRAF mutant/ unknown (57%/31%/5%/8%). The positive rate of HER2 ECD, CEA, and CA19-9 were 27.6, 80.0 and 51.7%, respectively. HER2 ECD showed a linear relationship with CEA level (correlation coefficient of 0.677). Regarding the association with the RAS status, serum levels of HER2 ECD in RAS wild type pts were higher than those in RAS mutant pts (positive rate of 33 vs. 20%, respectively). Moreover, in RAS wild type pts, pts resistant to anti-EGFR antibody within 1 year showed a higher positive rate of HER2 ECD than the others (55 vs. 28%,respectively; p = 0.0311). Conclusions: A Non negligible number of mCRC pts were positive for serum levels of HER2 ECD. HER2 ECD may be associated with tumor burden and acquired resistance to anti-EGFR therapy, but further studies are necessary to determine clinical utility of HER2 ECD.

P-232 Characterization of serum HER2 ECD level in patients with metastatic colorectal cancer

P-232 Characterization of serum HER2 ECD level in patients with metastatic colorectal cancer

Is serum HER2-ECD testing significant for resectable gastric cancer?

48 Background: Accurate assessment of human epidermal growth factor receptor (HER) 2 is essential for patients with gastric cancer (GC) who may benefit from therapies targeting this surface receptor such as trastuzumab. The extracellular domain (ECD) of the HER2 protein may be shed into the serum and is detectable using an enzyme-linked immunosorbent assay. Correlations have been reported between raised baseline ECD levels, suggesting that serum ECD levels may be useful in making treatment decisions in patients with HER2 positive GC. We investigated this relationship in patients with resectable GC. Methods: This retrospective study was conducted in 71 patients with resectable GC between Apr. 2009 and May 2012. We assessed HER2 protein overexpression and gene amplification using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). HER2 positive was defined as IHC 3+ or IHC 2+/ positive FISH. The serum HER2-ECD level was measured using the blood serum before the surgical treatment. We examined the relationship between serum HER2-ECD level, HER2 expression in resected specimens, depth of invasion of cancer, lymph node metastasis, and clinical stage. Results: Baseline values were available in 71 patients, and of these, 2 (2.8%) had raised levels (&gt; 15 ng/mL). Median serum ECD levels was 11.7 (7.5-17.9) ng/mL. Two patients showing high HER2-ECD without HER2 over-expression. No clear relationship was found between baseline ECD levels and HER2 over-expression. Serum HER2-ECD level was not associated with invasion depth and lymph node metastasis, and clinical stage. Conclusions: There is no significance of serum HER2-ECD testing in resectable gastric cancer.

Serum HER2 in combination with CA 15-3 as a parameter for prognosis in patients with early breast cancer

AimHER2 in breast cancer tissue is a marker of high prognostic and predictive relevance. Soluble HER2, the extracellular domain of the HER2/neu receptor (HER2 ECD), which is shed into the blood, has been suggested to be a helpful tumor marker. We investigated the relationship between the concentrations of HER2 ECD, CEA and CA 15-3, the association of these markers with clinicopathological features and the impact of HER2 ECD alone and in combination with known prognostic factors on disease free survival (DFS) and cancer specific survival (CSS) in untreated early breast cancer patients. Patients and methodsHER2 ECD (ADVIA, Bayer), CEA (AxSYM, Abbott) and CA 15-3 (Elecsys, Roche) were measured at time of primary diagnosis in the pre-therapeutic (pre-operative) sera of 241 breast cancer patients and were correlated with clinicopathological parameters and outcome. ResultsHigher HER2 ECD levels were significantly correlated with postmenopausal status (p=0.016) and tissue HER2-overexpression (p<0.0001). Higher serum levels of CA 15-3 were associated with larger tumor size (p=0.019), positive lymph nodes (p=0.019), UICC stage III (p<0.01), positive tissue HER2-overexpression (p<0.05) and negative hormone receptor status (p=0.016). In multivariate analysis, serum HER2 ECD levels, CA 15-3 levels, large tumor size and negative hormonal status were independent prognostic factors in DFS. Patients with both high levels of HER2 ECD (>15ng/mL) and high serum levels of CA 15-3 (>24U/mL) had the poorest prognosis with a DFS after 3years of 50.0%. Patients without elevated serum levels had a better outcome with a DFS of 91.2%. ConclusionsIn our retrospective analysis, HER2 ECD and CA 15-3 were independent and better prognostic tools than HER2 in tissue. Prospective validation is necessary to confirm their usefulness in clinical practice.

Association of HER2 ECD and t-PSA serum levels for possible breast cancer diagnosis

Because of the high heterogeneity of breast cancer (BC), BC subtype-specific biomarker panel might be useful for diagnostic BC or becomes an adjunct assay to improve the accuracy of current screening methods. In this study, we measured the protein levels of human epidermal receptor-2 extracellular domain (HER2 ECD) and total prostate-specific antigen (t-PSA) in the serum from 14 BC patients and 8 healthy controls by utilizing our previously developed high-throughput metal-enhanced fluorescence biosensor. The experimental results showed that the summation of HER2 ECD and t-PSA serum levels is proposed in this study. We found that AUC >0.9 of better ability to distinguish HER2+ group from HER2− and healthy groups. In the mean time, if taking the ratio of t-PSA and HER2 ECD serum levels, it is very useful able to differentiate HER2− group from HER2+ and healthy groups at value of AUC >0.8. To our best knowledge, this is for the first time to integrate serum levels of HER2 ECD and t-PSA levels potentially suggested in the screening BC patients for further improving the specificity by using our developed high-throughput metal-enhanced fluorescence biosensor.

Erratum to: Evaluation of serum HER2-ECD levels in patients with gastric cancer

Erratum to: Evaluation of serum HER2-ECD levels in patients with gastric cancer

Serum HER2 ECD level and its clinical significance in advanced breast cancer patients with different molecular subtypes

To evaluate the serum HER2 ECD level and its significance in advanced breast cancer patients with different molecular subtypes. A total of 322 advanced breast cancer patients were enrolled. The serum HER2 ECD concentrations were quantitatively detected by enzyme-linked immunosorbent assay(ELISA). Its relationship with clinicopathological characteristics and clinical significance were analyzed. It was found that 55.9% (19/34)Luminal A, 42.7% (44/103) Luminal B-HER2(-), 70.6% (60/85) Luminal B-HER2(+), 73.8% (45/61)HER2-enriched and 23.1% (9/39) triple-negative patients had serum concentrations of HER2 ECD at least 15 ng/ml respectively. The prevalence of elevated ECD level in patients of different molecular subtypes differed significantly (P < 0.001). Tissue HER2 status, number of metastatic sites, visceral metastasis, CA15-3 and carcinoembryonic antigen(CEA) levels exhibited statistically significant correlations with the prevalence of elevated serum HER2 ECD level. The serum concentrations of HER2 ECD decreased after effective targeted therapy in tissue HER2-positive patients. The prevalence of elevated serum levels of HER2 ECD differed significantly in advanced breast cancer patients with different molecular subtypes. The serum HER2 ECD level may reflect both histological HER2 status and tumor load. And the dynamic changes of ECD concentrations are somewhat correlated with the efficacies of targeted treatment.

Evaluation of serum HER2-ECD levels in patients with gastric cancer

Determination of the human epidermal growth factor receptor 2 (HER2) status of gastric cancer patients is indispensable in clinical practice. However, the clinical value of serum HER2-extracellular domain (ECD) in gastric cancer has not yet been defined. The serum level of HER2-ECD was measured using the chemiluminescence immunoassay method, and its relationship with tissue HER2 status and clinicopathologic features was examined. Transition of serum HER2-ECD level was examined in patients during chemotherapy to clarify the correlation between changes in the level of HER2-ECD in serum and response to chemotherapy. A total of 150 gastric cancer patients were enrolled in this study; changes in HER2-ECD level were examined in 36 of these patients during chemotherapy. Serum levels of HER2-ECD ranged from 4.8 to 180.0ng/ml (median 9.2ng/ml) and were positive (cutoff value 15.2ng/ml) in ten patients (6.7%). There was a significant correlation between serum HER2-ECD level and tissue HER2 status (P<0.001); however, no correlation with TNM stage was found. Change in serum HER2-ECD level during chemotherapy was significantly correlated with response to chemotherapy in patients with HER2-positive tumor tissue. Serum HER2-ECD is a potential biomarker of gastric cancer and could be used as a diagnostic marker with regard to tissue HER2 status, and also as a monitoring marker in relation to response to chemotherapy.

Interference-Free HER2 ECD as a Serum Biomarker in Breast Cancer.

Over-expression of the HER2/neu receptor occurs in 20 to 30 percent of breast tumors and is linked to poorer prognosis. The HER2/neu expression status determines whether or not patient will receive trastuzumab-based treatment. In clinical practice, over-expression of HER2/neu is routinely identified using Immunohistochemistry (IHC) or Fluorescence in Situ Hybridization (FISH), both of which are invasive approaches requiring tissue samples. Serum assays for the Extra Cellular Domain of HER2/neu receptor (HER2 ECD) have been reported but the use is very limited due to serum interference factors (e.g. human anti-animal immunoglobulin antibodies) that lead to false test results and inconsistency with tissue Her2 status. We have developed an ELISA based approach using an MBB buffer to eliminate false results and to obtain more accurate assessment of HER2 ECD levels. Using this refined assay we retroactively measured HER2/neu levels from breast cancer patients and controls. Abnormal HER2 ECD levels were detected in about 32% of invasive breast cancer patients but not in controls or patients with benign diseases. In addition, we also showed that patients with elevated serum HER2 levels appeared to have worse survival regardless of treatments. In a small group of 12 Ductal Carcinoma in situ (DCIS) patients who received HER2/neu peptide vaccination and surgery, only one patient showed constantly rising HER2 levels after treatment and this patient had recurrence of HER2 positive tumor within 5 years. Our studies indicate that once the serum interference issue is resolved, serum HER2 ECD can have potential clinical utility to supplement the tissue based tests.

S1-4: Retrospective Analysis of Study EGF30008 by Mass-Spectrometry Based Serum Assay (VeriStrat®).

Abstract Background: In EGF30008 the addition of lapatinib (La) to first-line treatment with letrozole (Le) significantly improved progression-free survival (PFS) in patients (pts) with hormone receptor positive (HR+), HER2 positive (HER2+) metastatic breast cancer (hazard ratio (HR) = 0.71; p=0.019), but not in HR+, HER2 negative (HER2−) pts. This study assessed the ability of VeriStrat (VS), a mass-spectrometry blood-based pretreatment assay correlating with clinical outcome from EGFR-TKI therapy, to stratify pt outcome in EGF30008. The VS assay assigns VS Good (Good), VS Poor (Poor), or Indeterminate labels to a serum sample based on a specific 8-peak signature in the mass-spectra (Taguchi F et al., JNCI 2007). Methods: Blinded to clinical data, pretreatment serum was analyzed using standard VS procedurei. Statistical analyses were performed using Mantel-Haenszel and Cox proportional hazards methods; correlations were evaluated using Fisher's exact and χ2 tests. Results: Of the 1286 pts randomized (HER2+=219; HER2−=952; HER2 unknown=115), 1163 pts had serum available; a VS label was assigned to 1046 pts (961 Good; 80 Poor; 5 Indeterminate); 117 were not evaluable (hemolyzed). In the overall population there was no significant difference in PFS between Good and Poor groups within the Le+La arm (p=0.53). In contrast, PFS of the Good group was longer than that of the Poor group within the Le only arm (HR=0.36, p&amp;lt;0.001) and comparable with that of Good pts within the Le+La arm (median PFS 10.8 mo vs 11.0 mo). An interaction test showed significantly different HRs for PFS in Good vs Poor between treatment arms (p=0.002). In the HER2+ population both Good (n= 169) and Poor (n= 13) groups received significant PFS benefit from addition of La to Le: in Good HR=0.71, p=0.046, medians 3.0 mo (Le) vs 8.0 mo (Le+La); in Poor HR=0.17, p=0.02, medians 2.3 mo (Le) vs 8.6 mo (Le+La). In the HER2− population median PFS for the Poor group (n=58) increased from 3.1 to 11.0 mo (HR=0.57, p=0.068) with addition of La to Le, whereas the Good group (n=702) received no significant benefit, median 13.6 mo on Le and 13.8 mo on Le+La (PFS HR= 0.85, p=0.09). In multivariate analysis VS remained independently significant and predictive of differential treatment effect in the overall population. No significant correlation of VS classification with HER2 status (p=0.35) or prior adjuvant hormonal therapy (p=0.33) was found. VeriStrat was also not significantly correlated with baseline HER2 ECD levels (low &amp;lt;15 ng/ml vs. high ≥15 ng/ml), p = 0.23, or ER expression (H-score:lower quartile:&amp;lt;160 vs. rest), p=0.18. Conclusions: In the overall population VeriStrat predicted PFS in Le alone but not in the Le+La combination, with a significant differential treatment effect. VeriStrat identified a subset of pts with inferior PFS on Le therapy. Adding La to Le improved outcome in HER2+ pts, and interestingly suggested an improvement in the VS Poor subset of HER2− pts. HER2− VS Good pts do not gain benefit with the addition of La. No correlation between VS and investigated predictors of benefit from the addition of La to Le in HER2− pts was observed. If these results can be validated in a prospective study, the addition of La to Le may be a potential treatment option for HER2− HR+ VS poor pts. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S1-4.

Use of biomarkers and imaging to evaluate the treatment effect of AUY922, an HSP90 inhibitor, in patients with HER2+ or ER+ metastatic breast cancer.

e11024 Background: Thorough biomarker assessments are increasingly considered vital for compounds in early clinical development to demonstrate proof of drug target modulation. HSP90 chaperones key client proteins that drive breast cancer (BC) including HER2, and estrogen receptor (ER). Inhibition of HSP90 leads to the degradation of these client proteins and induction of co-chaperones, such as HSP70. AUY922 is a highly potent, non-geldanamycin HSP90 inhibitor, and data from a Phase I study in patients with advanced solid tumors resulted in a recommended AUY922 Phase II dose of 70mg/m2. Patients with HER2+ or ER+ metastatic BC (mBC), who are refractory to standard treatment, have been enrolled into the ongoing Phase II part of the study, which includes a comprehensive biomarker evaluation to demonstrate the effect of AUY922 on key HSP90 client proteins. Methods: Single-agent AUY922 was administered by IV infusion 70 mg/m2 once a week in 28 day cycles to pts with HER2+ or ER+ BC. HSP70 and soluble HER2 (HER2-ECD) levels were quantitated by ELISA assays in peripheral blood mononuclear cells (PBMCs) and serum samples, respectively. Molecular imaging with FDG-PET and radiolabeled trastuzumab- or bevacizumab-PET (for HER2+ or ER+ mBC, respectively) was performed prior to, and during, treatment. Results: 10 pts have been treated to date (8 HER2+ and 2 ER+), and biomarker and imaging data are partially reported due to various technical and logistical challenges with each type of assessment. The maximum increase of HSP70 fold change ranged 1.9–10.4, with a median of 6.4 and mean of 5.9 fold, which is similar to previous observations. Soluble HER2 levels decreased ~45% and ~80% in 2 HER2+ patients who remained on study >5 cycles and both had partial metabolic responses by FDG-PET, and 1 had a confirmed partial response by RECIST as per investigator’s assessment. In 3 pts, follow-up trastuzumab-PETs were assessable, indicating heterogeneous expression of HER2 in various lesions, and heterogeneous response in these lesions to therapy. Conclusions: AUY922 has shown preliminary activity in HER2+ or ER+ mBC and has demonstrated target modulation in keeping with its mechanism of action.

Serum HER2 Level Measured by Dot Blot: A Valid and Inexpensive Assay for Monitoring Breast Cancer Progression

Human epidermal growth factor receptor 2 (HER2) is one of the most important prognostic and predictive factors for breast cancer patients. Recently, serum HER2 ECD level of patients detected by enzyme-linked immunoabsorbent assay (ELISA) has been shown to predict tumor HER2 status and reveal its association with tumor progression, recurrence and poor prognosis. In this study, we established a new method, dot blot assay, to measure the serum HER2 level in breast cancer patients and further to evaluate the clinical value for monitoring breast cancer progression. We found that the serum HER2 level measured by dot blot assay was significantly correlated with tissue HER2 status in breast cancer patients (P = 0.001), and also significantly correlated with HER2 level measured by ELISA (P = 1.06×10−11). Compared with ELISA method, the specificity and sensitivity of dot blot assay were 95.3% and 65.0%, respectively. The serum HER2 levels of patients with grade III or ER-negative were higher than those with grade I–II (P = 0.004) and ER-positive (P = 0.033), respectively. Therefore, the novel dot blot method to detect serum HER2 level is a valid and inexpensive assay with potential application in monitoring breast cancer progression in clinical situations.

Fatty acid synthase activity regulates HER2 extracellular domain shedding into the circulation of HER2-positive metastatic breast cancer patients

Clinicopathological assessment of the functional relationship between the HER2 oncogene and tumor-associated fatty acid synthase (FASN) is largely precluded because immunohistochemical and/or mRNA studies should be performed in biopsies from breast cancer patients. We here sought to determine whether serum FASN (sFASN) could associate with circulating HER2 extracellular domain (HER2 ECD) in the blood of metastatic breast cancer (MBC) patients. Concentrations of serum FASN and HER2 ECD were measured with ELISA in sera retrospectively obtained from 201 patients with metastatic breast cancer (MBC) and 31 healthy subjects. Mechanistical in vitro studies were performed using pharmacological inhibitors of HER2 and FASN as well as cultured cancer cells engineered to overexpress HER2 and FASN human genes. When the upper limit of normal sFASN was defined as the mean + 2SD of the control group, sFASN was elevated above this cut-off (12 ng/ml) in 70 MBC patients (35%). Eighty-nine MBC patients (44%) had elevated levels of HER2 ECD (HER2 ECD cut-off = 15 ng/ml). HER2 ECD-positive MBC patients slightly increased their sFASN levels compared with HER2 ECD-negative MBC patients. sFASN-positive MBC patients had significantly increased levels of HER2 ECD when compared with sFASN-negative MBC patients (mean HER2 ECD=34 ng/ml, 95% CI 26-41 ng/ml and 18 ng/ml -95% CI 15-21 ng/ml, respectively; p=0.002). Sixty percent of sFASN-positive patients concurrently exhibited high levels of HER2 ECD whereas 64% of sFASN-negative patients were negative for circulating HER2 ECD. In vitro studies revealed that BC cells bearing HER2 gene-amplification released higher levels of extracellular FASN than HER2-negative BC cells. Trastuzumab-induced blockade of HER2 ECD shedding failed to prevent FASN release and retrovirally-induced HER2 overexpression in MCF-7 cells did not increase extracellular FASN. Of note, pharmacological inhibition of FASN activity significantly decreased HER2 ECD levels in the supernatant of HER2-overexpressing BC cells while transient overexpression of FASN gene in HBL100 cells promoted FASN protein release and concomitantly increased HER2 ECD shedding into the extracellular milieu. Subsequent studies should explore if quantitative determination of FASN molecules in blood could become a rapid and accurate non-invasive test to monitor disease progression and survival in HER2-overexpressing MBC undergoing HER2-targeted therapies.

Quantitative assessment of HER2 status and correlation with efficacy for patients (pts) with metastatic breast cancer (MBC) in a phase II study of trastuzumab-DM1 (T-DM1)

1003 Background: The antibody-drug conjugate T-DM1 combines the biological activity of trastuzumab with targeted delivery of an anti-microtubule agent (DM1) to HER-2-expressing cancer cells. This analysis examines correlation of response to T-DM1 with HER-2 status, as assessed by fluorescent in situ hybridization (FISH), immunohistochemistry (IHC), mRNA quantitative real-time polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA) (HER-2 extracellular domain [ECD]), for pts enrolled in TDM4258g, a phase II study of T-DM1 in pts with MBC. Methods: TDM4258g is an open-label, single-arm study of T-DM1 administered at 3.6 mg/kg IV q3w. Pts had progressed on HER-2-directed therapy and received chemotherapy in the metastatic setting and were HER-2 + based on local testing. Archival tissue (paraffin block or &gt;7 unstained tumor slides) was collected for retrospective central laboratory testing. HER-2 DNA amplification was determined by FISH, and protein levels by IHC. qRT-PCR for HER-2 was performed on extracted RNA; baseline HER-2 ECD ELISA was performed on pt sera. HER-2 data for each pt were compared with pt's best response. Results: As of August 29, 2008, 112 pts had enrolled; 107 were efficacy-evaluable pts with median 4.4 mos follow-up. There were 42/107 (39.3%) partial responses (PR) (investigator assessment). Of 86 pts centrally tested, 64 (74.4%) were confirmed HER-2+ (FISH+ and/or IHC 3+), with 32/64 (50%) PR. Of 76 pts tested by both FISH and IHC, 15/76 (19.7%) were confirmed HER-2- (FISH- and IHC 2+/1/0), with 2/15 (13.3%) PR. In HER-2+ pts, response rates did not correlate with high versus low FISH+ counts, nor with HER-2 ECD levels. Among 39 HER-2+ (FISH+ and/or IHC3+) efficacy-evaluable pts with qRT-PCR data, there were 13/19 (68.4%) PR for pts with qRT-PCR above median levels, and 7/20 (35.0%) PR for pts with qRT-PCR below median. Conclusions: HER-2+ pts (by central retesting) had better responses to T-DM1 than HER-2- pts, although a small number of PR were observed in HER-2- pts. Assessment of HER-2 expression by qRT-PCR may identify pts more likely to respond to T-DM1 therapy. Updated data, including additional diagnostic markers, will be presented at the meeting. [Table: see text]