HER2 Homodimers Research Articles

Different Mechanisms for Acquired Resistance to Trastuzumab and Lapatinib in HER2 Positive Breast Cancers: Role of ER and HER2 Reactivation.

Abstract About 25% of human breast cancers are amplified for HER2 with half of these tumors also expressing estrogen receptor (ER). Therapies targeting HER2 are very effective in the metastatic and the adjuvant settings, especially, although de novo or acquired resistance are still major problems. Trastuzumab (T) and lapatinib (L) are approved drugs now used in the clinic for treatment of HER2+ tumors. Data suggest that T works primarily by blocking signals generated by HER2 homodimers, while L is a small molecule tyrosine kinase inhibitor that more completely blocks the pathway by inhibiting HER1 in addition to HER2. In the clinic, these drugs demonstrate incomplete cross-resistance since L is active in some patients with T-resistant tumors. However, the mechanisms for this resistance have not been clarified.To investigate the mechanisms for acquired resistance, we developed a panel of HER2+ cell lines resistant to T, L, and L+T by long-term exposure to increasing drug concentration in vitro. Two of these lines, BT474 and UACC812, are amplified for HER2 and also express ER, and they, together with subclones resistant to L, T, and L+T, were used to better understand potential resistance mechanisms. Western blot analysis of the parental BT474 and its resistant subclones showed that subclones resistant to T had reactivated the HER2 signaling pathway, while subclones resistant to L or L+T in which the HER receptor layer was more completely inhibited showed continued complete blockade of the HER2 pathway at the receptor layer but high levels of ER activity and phosphorylated-AKT. L, but not L+T, subclones after more prolonged time in culture did reactivate the HER pathway. UACC812 resistant cells were similar to BT474: T-resistant clones showed evidence of reactivation of HER signaling while L and L+T resistant clones showed enhanced ER activity. These cells showed no reactivation of HER signaling even after prolonged exposure in vitro. Consistent with these data, both BT474 and UACC812 T-resistant clones were still sensitive to and cell proliferation was inhibited by L. L-resistant clones, however, were also resistant to T. The potent anti-estrogen fulvestrant (F) was used to evaluate the role of ER in these resistant clones. T-resistant clones from both parental lines were resistant to F, indicating that ER had no role in resistance. In contrast, L and L+T-resistant clones, but not parental cells, were extremely sensitive to F with significant inhibition of cell proliferation in vitro.These data demonstrate that only partial inhibition of the HER2 pathway in breast cancer cells by T can be overcome by activating other components of the HER pathway. Resistance to more complete HER2 blockade with L or L+T requires reactivation of a redundant cell survival pathway, in this case ER, which is upregulated by HER2 blockade. Optimal therapy in those tumors may require both ER and HER2-targeted therapy. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 708.

Correlation of quantitative total HER2 expression and HER2 homodimers with histopathologic characteristics of breast cancers in the FinHer study

11061 Background: We recently reported that the HERmark assay (Monogram Biosciences) accurately measures continua of total HER2 expression (H2T) and HER2 homodimers (H2D) over a wide (∼3 logs) dynamic range, and that a higher concordance was observed between H2T and HER2 status with more stringent central tests as compared with IHC tests performed locally (Joensuu et al, 2008 SABCS,abstract 2071). H2D/H2T ratio was reported as a marker of activated HER2 and a prognosticator of disease progression in HER2+ patients not treated with trastuzumab in the adjuvant setting (Bates et al, 2008 SABCS,abstract 1074). In this follow-up analysis, H2T, H2D, and H2D/H2T ratio were correlated with histopathologic characteristics of breast cancers in the FinHer study. Methods: The HERmark assay was used to measure H2T and H2D in 899 formalin-fixed, paraffin-embedded FinHer specimens. The results were correlated with histopathologic characteristics of breast cancers in the FinHer study (Joensuu et al, N Engl J Med2006;354), including estrogen receptor/progesterone receptor (ER/PR), tumor grade, tumor size, lymph node metastasis, and stage. Results: Higher H2T and H2D levels correlated with ER/PR negativity and high tumor grade (P<0.0001). 42% (102/244) of ER- and 37% (137/374) of PR- cases were HERmark Positive; while 17% (110/655) of ER+ and 14% (75/524) of PR+ cases were HERmark Positive. 10% (13/136) of grade 1, 18% (65/353) of grade 2, and 35% (131/375) of grade 3 tumors were HERmark Positive. No significant association was found between H2T or H2D and tumor size, lymph node metastasis or stage. ER/PR negative and poorly differentiated cancers had higher H2D/H2T ratios (P=0.013), and H2D/H2T ratios >0.6 were associated with smaller primary tumor diameters at the time of cancer detection (P=0.009). Conclusions: The quantitative H2T measurement confirms the known correlations between HER2 expression and histopathologic characteristics of breast cancer. The novel H2D measurement and H2D/H2T ratio may provide further insights into HER2 activation and better diagnostic tests for targeted HER2 therapy. [Table: see text]

A Novel Proximity Assay for the Detection of Proteins and Protein Complexes: Quantitation of HER1 and HER2 Total Protein Expression and Homodimerization in Formalin-fixed, Paraffin-Embedded Cell Lines and Breast Cancer Tissue

The availability of drugs targeting the EGFR/HER/erbB signaling pathway has created a need for diagnostics that accurately predict treatment responses. We have developed and characterized a novel assay to provide sensitive and quantitative measures of HER proteins and homodimers in formalin-fixed, paraffin-embedded (FFPE) cell lines and breast tumor tissues, to test these variables. In the VeraTag assay, HER proteins and homodimers are detected through the release of fluorescent tags conjugated to specific HER antibodies, requiring proximity to a second HER antibody. HER2 protein quantification was normalized to tumor area, and compared to receptor numbers in 12 human tumor cell lines determined by fluorescence-activated cell sorting (FACS), and with HER immunohistochemistry (IHC) test categories and histoscores in cell lines and 170 breast tumors. HER1 and HER2 expression levels determined by the VeraTag assay are proportional to receptor number over more than a 2 log10 range, and HER homodimer levels are consistent with crosslinking and immunoprecipitation results. VeraTag HER2 measurements of breast tumor tissue and cell lines correlate with standard IHC test categories (P<0.001). VeraTag HER2 levels also agree with IHC histoscores at lower HER2 protein levels, but are continuous and overlapping between IHC test categories, extending the dynamic range 5-fold to 10-fold at higher HER2 levels. The VeraTag assay specifically and reproducibly measures HER1 and HER2 protein and homodimers in FFPE tissues. The continuous measure of HER2 protein levels over a broad dynamic range, and the novel HER2 homodimer measure, are presently being assessed as predictive markers for responses to targeted HER2 therapy.

HER2 protein expression predicts response to trastuzumab in FISH-positive patients.

Abstract Abstract #32 BACKGROUND: Fluorescent in Situ Hybridization (FISH) is currently employed to select patients for trastuzumab or lapatinib therapy. However, only a portion of patients will respond to these HER2-directed therapies. Using a novel assay to quantitate total HER2 (H2T) and HER2 homodimer (H2D) levels, we examined the relationship between these measurements and clinical outcomes in a cohort of trastuzumab-treated metastatic breast cancer patients previously assessed as FISH positive by central testing.&amp;#x2028; METHODS: The VeraTag assay was used to measure H2T and H2D. A cutoff previously defined by positional scanning was used to classify patients as H2T high or low (Leitzel et al. ASCO abstract # 1002, 2008). FISH status was determined using the Vysis assay and was performed by a single pathologist under standardized conditions (central testing). FISH + was defined as HER2/CEP17 ratio greater than 2.2. Kaplan-Meier and Cox proportional hazards analyses (TTP and OS) were conducted.&amp;#x2028; RESULTS: 99 MBC patients previously scored as IHC 3+ or 2+, FISH+ and treated with first line trastuzumab-containing regimens were re-tested by central FISH. 22 patients were found to be FISH- and 77 were FISH+. 67 FISH+ patients had high H2T levels, and 19 FISH- patients were H2T low. 14% (3/22) of FISH- patients were H2T high, and 13% (10/77) of FISH+ patients were H2T low (overall discordance rate = 13%). In Kaplan Meier analyses, patients assessed as FISH+, H2T high had a significantly longer TTP (median TTP=11.3 mos.) compared to those who were FISH-, H2T low (median TTP=4.5 mos. HR=0.42, p=0.0006), and also compared to those who were FISH+, H2T low (median TTP=3.7 mos. HR=0.43, p=0.01). Those patients who had discordant results (FISH+, but H2T low) behaved similarly compared with FISH-, H2T low (HR=1, p=0.99). Similar results were observed using H2D. In analyses restricted to central FISH+ patients only (N=77), Cox proportional hazards multivariate regression identified H2T as an independent predictor of TTP (HR=0.29, p=0.0015) and OS (HR=0.37, p=0.02).&amp;#x2028; CONCLUSION: In metastatic breast cancer patients previously assessed as FISH-positive by central testing, we observed an approximate 55-fold distribution of HER2 protein expression. Patients with HER2 gene amplification by FISH but low HER2 total protein expression or homodimer levels as measured by the VeraTag assay respond poorly to trastuzumab-containing therapy. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 32.

Quantitative measurements of HER2 expression and HER2 homodimer using a novel proximity based assay: comparison with HER2 status by immunohistochemistry and chromogenic in situ hybridization in the FinHer study.

Abstract Abstract #2071 Background: The accuracy and reliability of current methods, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), to assess HER2 status has recently been a subject of debate. The best method to assess HER2 status remains controversial. We developed a novel assay (HERmark, Monogram Biosciences) that provides precise quantification of HER2 expression (H2T) and HER2 homodimer (H2D) in formalin-fixed paraffin-embedded (FFPE) tissues. We compared H2T and H2D to local IHC, central chromogenic in situ hybridization (CISH) and central IHC retesting of breast cancers from the FinHer study.&amp;#x2028; Methods: H2T and H2D were detected through light-dependent release of fluorescent tags (VeraTag reporters) conjugated to a HER2 antibody, requiring proximity to a second HER2 “scissors” antibody. The VeraTag signal was quantified by capillary electrophoresis and normalized to tumor area. Assay comparisons correlated H2T and H2D with HER2 testing by local IHC and central CISH from FinHer (Joensuu et al, N Engl J Med 2006;354), as well as central HER2 status reassessment by combination of externally performed central IHC retesting (PhenoPath labs, Seattle, WA) and central CISH (FinHer) according to ASCO/CAP guideline for HER2 testing in breast cancer (Wolff et al, Arch Pathol Lab Med 2007;131).&amp;#x2028; Results: H2T and H2D in 899 evaluable FinHer samples described a continuum over a wide dynamic range (∼ 3 logs), in contrast with conventional IHC categories (0-3+). The correlation between H2T and IHC categories was significant (P &amp;lt; .0001). Overlap of H2T among the IHC categories was observed. H2D showed a similar correlation with IHC and a general correlation with H2T (P &amp;lt; .0001). A H2T cutoff value, based on its ability to distinguish high and low responders in a cohort of metastatic breast cancer patients treated with trastuzumab-based regimens (log10 H2T= 1.14, Leitzel et al, 2008 ASCO, abstract 1002), was used to define HERmark negative (-) and positive (+), which were then compared with IHC and CISH results. The concordances between HERmark (-) and local IHC (-), central CISH (-), and central HER2 reassessment (-) were 89%, 84%, and 91%, respectively. The concordances between HERmark (+) and local IHC (+), central CISH (+), and central HER2 reassessment (+) were 71%, 89%, and 92%, respectively. The HERmark test showed greater overall concordance with central HER2 reassessment (91%) than with local IHC (84%) and central CISH (87%)&amp;#x2028; Conclusions: HERmark reliably measures H2T and H2D in FFPE tissues. H2T showed excellent concordance with central HER2 status according to ASCO/CAP guideline for HER2 testing. The precise quantification of H2T and H2D may provide novel, quantifiable, predictive tests for targeted HER2 therapy. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2071.

Quantitative HER2 homodimer levels correlate with time to first recurrence in HER2-positive breast cancer patients who did not receive trastuzumab in the adjuvant setting.

Abstract Abstract #1074 BACKGROUND: HER2-positive breast cancer patients are currently treated with trastuzumab in the adjuvant setting, but prior to 2005 these patients were not routinely offered trastuzumab, creating an opportunity to examine the relationship between HER2 expression and disease progression. The VeraTag technology is a proximity-based assay system that permits quantitative measurements of total HER2 protein expression (H2T) as well as HER2 homodimers (H2D) in formalin-fixed paraffin-embedded (FFPE) tissues. We measured H2T and H2D in a cohort of patients who were HER2-positive but did not receive trastuzumab in the adjuvant setting, and correlated those measurements with time to first recurrence of disease.&amp;#x2028; METHODS: Patients were selected for study because they were treated with a trastuzumab-containing regimen for HER2+ metastatic breast cancer. HER2-positivity was defined as IHC (Herceptest) 3+ or 2+, FISH+ (Vysis). 96 HER2+ patients who had been treated (but not with trastuzumab) in the adjuvant setting were identified. The VeraTag assay was used to quantitate H2T and H2D. Kaplan-Meier and Cox proportional hazards analyses were employed.&amp;#x2028; RESULTS: The distribution of H2T ranged over approximately 135-fold, and H2D varied over approximately 100-fold. In univariate Cox proportional hazards regression analysis, H2T trended toward significance (HR=1.44, p= 0.088) while both H2D (HR=1.39, p=0.022) and the ratio H2D/H2T (HR=2.01, p=0.03) were significantly correlated with time to first recurrence. Kaplan-Meier analysis of the H2T distribution divided into tertiles showed no significant difference in time to first recurrence among the tertiles (HR=0.7, p=0.16 for low vs. high comparison) and no trend was observed (Log Rank test for trend p=0.2). However, the same analyses performed using H2D (HR=0.59, p=0.03) or H2D/H2T (HR=0.56, p=0.01) demonstrated significantly longer time to first recurrence for the lowest tertile compared with the highest tertile respectively. A trend was observed in both cases (H2D: Log Rank p=0.048; H2D/H2T: Log Rank p=0.026)&amp;#x2028; CONCLUSION: In a population of HER2+ patients who did not receive trastuzumab in the adjuvant setting, and who subsequently developed metastatic disease, higher levels of HER2 homodimers and the ratio of homodimers to HER2 total expression correlated with time to first recurrence while total HER2 expression levels did not. These data suggest that measures of the activated forms of HER2 (dimers) may be better predictors of disease progression than simple quantitation of HER2 alone. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1074.

The use of proximity ligation assay to identify HER2:HER3 heterodimers in early breast cancers.

Abstract Abstract #2068 Background: The HER receptor family includes four members: HER1, HER2, HER3 and HER4. Expression of these proteins is linked to breast cancer development and progression. HER2 gene amplification is found in 20-30% of breast cancers. Overexpression of HER2 has been linked with high grade tumours and poor prognosis. Clinical studies have demonstrated a strong relationship between HER2 and HER3 with 70% of HER3-positive tumours also being positive for HER2. Co-expression of HER2/HER3 has been suggested to drive endocrine and chemotherapy resistance. However such resistance is driven not by co-expression but by signalling via HER2/HER3 dimers which have previously been impossible to detect in situ. Proximity ligation assays (PLAs) can detect homo- and heterodimers in situ at a single-molecule level and allow study of signalling pathways. Paired antibodies are used to detect protein complexes in tissues.&amp;#x2028; Methods: Using tissue microarrays containing 100 breast cancer samples, we analysed HER2 homodimers and HER2:HER3 heterodimers by PLA. HER2 amplification was detected using FISH analysis.&amp;#x2028; Results: PLA was used to detect HER2 homo and heterodimers in vivo and in vitro. HER2 homodimers were detected in amplified and non-amplified cases. A significant association (p&amp;lt;0.00001) was identified between HER2 homodimerisation and amplification. HER2 homodimerisation was not associated with any clinicopathological data.&amp;#x2028; HER2:HER3 heterodimers were detected in vivo and in vitro through PLA and western blot analysis. The median number of HER2:HER3 heterodimers (7.8 signals per cell) was greater than HER2:HER2 homodimers (2.0 signals per cell) in this series indicating HER2:HER3 may be the preferred dimer. HER2:HER3 was significantly associated with nodal status (p=0.003) and grade (p=0.02).&amp;#x2028; Conclusions: Using an in situ PLA we demonstrated the in vivo expression of HER2 homodimers in human breast cancer and increased HER2 homodimerisation linked to HER2 gene amplification. We have demonstrated HER2:HER3 heterodimers to be linked to nodal status and grade. Our observations suggest that PLA is an invaluable method to detect homo and heterodimers in situ and could potentially be used a diagnostic tool in the future. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2068.

Comparison of 3D and 2D tumor models reveals enhanced HER2 activation in 3D associated with an increased response to trastuzumab

Three-dimensional (3D) cell culture techniques are frequently used to model alterations in tissue architecture critically important for tumor development. Here, we report on a detailed comparison of a spheroid model of human epidermal growth factor receptor (HER2) overexpressing cancer cells with the traditional monolayer culture. In 2D culture, HER2 and HER3 form heterodimers, whereas in multicellular spheroids HER2 homodimers are formed. These homodimers localize in membrane rafts, resulting in enhanced inhibition of the proliferation of cancer cells with trastuzumab (Herceptin), a monoclonal antibody specifically targeting HER2. Within the tumor spheroids, HER2 homodimerization leads to enhanced activation of HER2 and results in a switch in signaling pathways from phosphoinositide 3-kinase (PI3K) to mitogen-activated protein kinase (MAPK). Diminished PI3K signaling is accompanied by the activation of the integrin beta4/Rac1/PAK 2 signaling cascade. We propose that the described 3D culture system may better reflect some in vivo aspects of HER signaling and can be used to further improve the understanding of the molecular mechanisms of trastuzumab action. Furthermore, the described human multicellular tumor spheroids may allow identification of new targets for the treatment of HER2-positive breast cancer patients who currently benefit suboptimally from trastuzumab treatment.

Use of total HER2 and HER2 homodimer levels to predict response to trastuzumab

1002 Background: Current methods used to select HER2 positive patients with metastatic breast cancer for treatment with trastuzumab are semi-quantitative and yield response rates of less than fifty percent. Using a novel assay to quantitate total HER2 and HER2 homodimer levels, we examined the relationship between these measurements and clinical outcomes in a cohort of trastuzumab-treated metastatic breast cancer patients, previously assessed as IHC 3+ or FISH+ for HER2 overexpression or amplification. Methods: The VeraTag assay was used to measure total HER2 expression (H2T) and HER2 homodimers (H2D) in 106 formalin-fixed, paraffin-embedded primary tumor specimens. Using test for trend, multivariate Cox proportional hazards, and Kaplan-Meier analyses, the results were correlated with objective response, time-to-progression (TTP), and overall survival following trastuzumab-containing treatment. Results: Higher levels of H2T (p= 0.008) or H2D (p= 0.001) correlated with higher probability of objective response to trastuzumab therapy in a quartile analysis. Using the median value of the H2T distribution as a cutoff, higher H2T correlated with longer TTP (median TTP 11.6 mo vs. 5.4 mo, p=0.055), as did higher H2D (median TTP 11.6 mo. vs. 5.8 mo., p=0.055). Using incremental scanning to identify an optimal cutoff, higher H2T correlated with longer TTP (median TTP 11.3 mo. vs. 4.2 mo., p=0.003), as did higher H2D (median TTP 11.1 mo. vs. 4.2 mo., p=0.016). Using the optimal cutoff, higher H2T showed a non-significant trend toward longer overall survival (median survival 37.4 mo. vs. 28.7 mo., p=0.2). Cox multivariate models identified H2T above vs. below the optimal cutoff as a statistically significant correlate of both TTP (HR=0.41, p=0.0002) and overall survival (HR=0.45, p=0.009). Also in multivariate analyses, H2D above vs. below the optimal cutoff was a statistically significant correlate of both TTP (HR=0.64, p= 0.03) and overall survival (HR=0.6, p =0.02). Conclusions: In metastatic breast cancer patients previously selected by IHC or FISH, higher total HER2 and HER2 homodimer levels as measured by the VeraTag assay predict those patients more likely to respond to trastuzumab-containing therapy. Grant support: Supported by a grant from Susan G. Komen for the Cure Foundation. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Monogram Biosciences Monogram Biosciences Monogram Bioscience Monogram Biosciences

Biomarkers as potential predictors of pathologic complete response (pCR) in the NOAH trial of neoadjuvant trastuzumab in patients (pts) with HER2-positive locally advanced breast cancer (LABC)

504 Background: The NOAH trial evaluated the addition of trastuzumab (H) to chemotherapy (CT) for pts with HER2-positive LABC. A significant improvement in pCR has been reported (Gianni. ASCO 2007; abs 532). Methods: 228 pts were randomized to receive 3 cycles of doxorubicin (60 mg/m2) and paclitaxel (150 mg/m2) q3w, 4 cycles of paclitaxel (175 mg/m2 q3w) and 3 cycles of CMF (C 600 mg/m2, M 40 mg/m2, F 600 mg/m2 d 1+8 q4w) with or without concomitant H (8 mg/kg loading dose then 6 mg/kg q3w for 1 year) before surgery. 171 (75%) core biopsies obtained before treatment were available for assays of c-Myc (by FISH), PTEN (by IHC) and IGF1R (by IHC) in addition to estrogen receptor (ER) and progesterone receptor (PgR). Results: The 171 pts with available biopsies were representative of the overall NOAH population, although with a lower incidence of ER+ tumors. Negative PgR status was strongly associated with pCR in the H group (51% vs 16% in PgR-positive tumors; p=0.008). Overall, 32 pts had c-Myc amplification (cut-off ratio >2). Of these, 11/21 (52%) H pts but only 1/11 (9%) CT pts had pCR. PTEN expression was not predictive of pCR after H but was associated with a higher pCR rate after CT alone (p=0.05). IGF1R membrane staining was significantly lower in H-treated pts with pCR than those with residual disease (24% vs 48%; p=0.05). In logistical regression analysis, PgR status and H treatment were significant predictors of pCR. In the H-treated group, PgR status (p=0.012) was the most significant predictor of pCR; ER and IGF1R membrane staining showed borderline significance (p=0.089 and p=0.075, respectively). Conclusions: PgR-negative status and c- Myc amplification are associated with higher pCR rates after addition of H compared to CT alone. Overexpression of membranous IGF1R is associated with a higher likelihood of residual disease after H-based CT. These results will be combined with those of ongoing studies of additional biomarkers (HER3, p95HER2 and HER2 homodimers) to define a multivariate residual disease predictor. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Genentech™ BioOncology, Roche MGRM Genentech™ BioOncology, Roche Roche

Quantitative measurement of HER2 expression and HER2 homo-dimerization in formalin-fixed paraffin-embedded (FFPE) breast cancer tissues using a novel proximity-based assay

21023 Background: The best method to assess HER2 status remains controversial. Here we describe a novel assay that provides reliable quantitation of total HER2 (H2T) and HER2 homodimer (H22D) in FFPE tissues. The assay was cross-validated with conventional HER2 tests - immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Characteristics of the new assay and its potential application as predictive test were explored. Methods: Electrophoretic Tag (eTag) is a proprietary proximity-based technology that measures gene and protein expression. We have developed a novel application of the technology to quantify H2T and H22D in FFPE tissues. The validity of the assay was evaluated by analyzing its performance in cell lines and FFPE breast cancers. IHC (clone CB-11, Ventana) was performed in 174 breast cancers, scored according to standard IHC criteria and Histoscore (H-score), and HER2 gene amplification was evaluated by FISH (PathVysion, Vysis) in 19 breast cancers. eTag assays were performed on the same specimens and the results were compared with those of IHC and FISH. Results: H2T and H22D were proportional to the known expression levels in cell lines. H2T demonstrated a continuous measurement over a wide dynamic range (&gt;2 logs), in contrast with conventional IHC (0, 1+, 2+, 3+). The correlation between H2T and IHC categories was significant (correlation=0.84, p&lt;0.0001 by the Jonckheere-Terpstra test for trend), but overlap of H2T values in adjacent IHC categories was observed. H-score showed good correlation within the lower range of H2T, but plateaued at higher range of H2T. HER2 gene amplification (HER2/CEP17 ratio and gene copy #) by FISH was generally correlated with IHC categories and H2T values. There was a good correlation between H22D and H2T (r2= 0.7, P &lt; 0.001). Conclusions: eTag reliably measures H2T and H22D in FFPE tissues. The continuous measure of H2T over a wide dynamic range and the novel H22D measure provide data superior to conventional HER2 tests by IHC and FISH, but clinical utility of the eTag assay remains to be investigated. eTag has potential as a predictive test for targeted HER2 therapy in breast cancer. No significant financial relationships to disclose.

HER2 expression and HER2:HER2 dimerization identifies subpopulations of metastatic breast cancer patients with different probabilities of long-term survival following trastuzumab treatment and with different requirements for concomitant chemotherapy

10557 Background: Selection of patients for treatment with trastuzumab is currently based on the semi-quantitative measurement of HER2 protein expression (IHC) or gene amplification (FISH). We made quantitative measurements of HER2 expression and homodimerization using a novel assay, eTag, and correlated them with overall survival (OS) and time-to-progression (TTP) following trastuzumab treatment in a cohort of patients with metastatic breast cancer (MBC). We also examined the benefit of concomitant chemotherapy (CT) depending on quantitative HER2 expression. Methods: The Jules Bordet cohort (Brussels, Belgium, N=71) had MBC and prior treatment with at least two CT regimens. Patients received trastuzumab alone or combined with predominantly paclitaxel. Independent medical data review and central confirmation of HER2 overexpression by FISH (N=64) or IHC (N=7) were mandatory. The eTag assay was used to quantitate HER2 protein expression and HER2:HER2 dimers in FFPE specimens. eTag measurements were correlated with OS and TTP using Kaplan-Meier and Cox Proportional Hazards regression analyses. Results: Cox analyses identified three independent correlates of OS and TTP: number of metastatic sites (HROS = 2.4/site, 95%CI 1.5–3.9, p = 0.00019), treatment with trastuzumab only (HROS = 2.8, 95%CI 1.1–7.3, p = 0.036), and HER2 expression level (HROS = 0.24/log10(HER2), 95%CI 0.09–0.7, p = 0.0058). Patients with high HER2 (above median expression) experienced better OS than those with low HER2 (HR 0.24, p = 0.0014). Patients with high HER2 expression did not benefit from added CT (HR = 0.95, 95%CI 0.24–3.8, p = 0.94), while patients with low HER2 expression did (trastuzumab alone HR 4.4, 95% CI 1.3–18, p = 0.018). Similar results were observed for HER2:HER2 dimerization. Conclusions: Within a population of patients selected by FISH or IHC to receive trastuzumab, higher HER2 expression or HER2:HER2 dimerization correlated with better outcomes. Patients with high HER2 expression derived no benefit from concomitant CT, while patients with low HER2 expression benefited significantly from the addition of CT to trastuzumab. No significant financial relationships to disclose.

Are HER1/EGFR interactions with ligand free HER2 related to the effects of HER1-targeted drugs?

This paper is aimed to describe consequences of possible dimerization modes between ligand binding HER1 and ligand free HER2 receptors on cellular membrane. Cells without HER2/neu need high exposure to HER1 ligands for growth stimulation since formation of HER1-ligand:HER1-ligand homodimers depends on the number of both HER1 and ligand molecules and an "all or none" threshold under which cell growth is not stimulated can be expected. Cells with HER2/neu molecules on their surface can react to moderate or even low HER1 ligand exposure through formation of HER2:HER1-ligand dimers, making them more sensitive to growth stimulation by EGF or other ligands without the "all or none" threshold in cell growth stimulation. Formation of some HER2:HER2 homodimers can provide the basal cell growth stimulation despite available ligands to HER1. In tumors, high expression of HER2 can lead to many HER2:HER2 homodimers and increased cell growth that contributes to a poor prognosis. Here presented concept is that some 75 millions of years ago, introduction of HER2/neu with increased sensitivity to low EGFR ligand exposure, might be the cause of increased variability of HER1 expression on normal cells and of the basal EGF secretion from the uninjured tissue. Spontaneous formation of HER2:HER2 homodimers in cells with HER2/neu expression might have substituted the low ligand exposure from uninjured tissue and thus slowly reduce importance of basal secretions of EGFR ligands. Reported variability in HER1 tumor expression and response to HER1-targeted agents, a wide range of EGF concentration in healthy women breasts fluid and the skin rash/tumor response relation to HER1-targeted drugs are discussed as possible examples of individual differences in tissue dependency on HER1 interactions with ligands in normal and cancer tissue.

Morphogenetic and proliferative responses to heregulin of mammary epithelial cells in vitro are dependent on HER2 and HER3 and differ from the responses to HER2 homodimerisation or hepatocyte growth factor.

The effects of heregulin on the cell line HB2, derived from immortalised human luminal mammary epithelial cells, have been examined. HB2 cells, which normally form smooth spherical colonies in collagen gels, exhibited a striking heregulin-induced morphological change to colonies projecting a large number of spiky branches. A mitogenic effect of heregulin on HB2 cells was also seen, which was more pronounced on collagen than on plastic, whereas cell motility was unaffected. HB2 cells were found to express the heregulin receptor subunits HER2 and HER3, but not HER4. Treatment of HB2 cells with heregulin also induced tyrosine phosphorylation of a band shown by immunoprecipitation to contain HER3. Using specific receptor-blocking antibodies, it was found that both the morphogenetic and proliferative responses of heregulin in HB2 cells were mediated by HER2 and HER3. To compare the effects of HER2 in heregulin signaling to heregulin-independent HER2 homodimerisation (thought to be a carcinoma-associated event), HB2 cells were transfected with the trk-neu hybrid receptor which could be induced to form homodimers by NGF. Although activated HER2 homodimers induced proliferation in the HB2 transfectants in collagen, a morphological response in collagen was not seen, suggesting that HER3 signaling is important for morphogenesis in this cell type.