p95HER2 Expression Research Articles

Hyperactivation of the PI3K-AKT Pathway Commonly Underlies Resistance to Trastuzumab in HER2 Amplified Breast Cancer.

Abstract BACKGROUND: Targeted therapy of HER2 amplified breast cancers with Trastuzumab has resulted in a survival benefit for patients with this disease. However, de novo and acquired resistance to Trastuzumab is commonly encountered. Many laboratory models of Trastuzumab resistant cancer have been developed and suggest that hyperactivation of the HER3-PI3K-AKT pathway downstream of HER2 may enable resistance. It has also been suggested that changes to HER2 itself, such as through loss of HER2 amplification or induction of p95-HER2 expression, may mediate resistance. A comprehensive in vivo assessment of multiple mechanisms of resistance has yet to be reported. Moreover, it is not known if these lesions are always present in pretreatment tumor samples or can be acquired over time. The aim of this study was to identify which mechanisms of resistance are present in human tumor samples after progression of disease on a Trastuzumab containing regimen. METHODS: Patients with HER2 amplified breast cancer with progression of disease by RECIST criteria on a Trastuzumab containing regimen were enrolled on an IRB approved protocol to obtain biopsies of progressive tumors. One tumor biopsy was flash frozen for nucleic acid studies and one biopsy preserved in formalin. For the pre-Trastuzumab comparison, archival formalin-fixed paraffin embedded (FFPE) blocks were obtained. Manual microdissection was performed on the frozen blocks to isolate tumor-containing sections for DNA isolation. PIK3CA hotspot mutations were identified using the Sequenom MassARRAY system. Following antibody optimization, IHC staining for HER3, PTEN and P-AKT (S473) was performed on freshly cut sections. Stained sections were scored by two, independent reviewing pathologists. HER3, PTEN and P-AKT expression were scored relative to staining in adjacent normal ductal epithelium. Staining and scoring for ER, PR, and HER2 was performed in CLIA-certified clinical laboratory. RESULTS: 19 patients have been enrolled and completed biopsy on protocol. Changes in HER2 overexpression status from the pretreatment biopsy to the post-progression biopsy were noted in 2/15 evaluated samples, with the remaining cases demonstrating stability. PTEN expression was diminished or absent in four cases. Activating mutations in hotspot regions of PIK3CA were identified in 3/10 cases. Enhanced expression of the HER3 receptor was identified in 4/11 cases with 2 of the cases showing an increase in expression compared to the pretreatment sample. CONCLUSIONS: HER2 overexpression status infrequently changed from the pre-Trastuzumab to post-Trastuzumab biopsy. Abnormalities in HER3, PI3K, and PTEN were not coexistent (e.g. PTEN loss and PI3K mutation were not seen in the same samples) in most cases. In some instances, the pretreatment tumor sample did not demonstrate lesions that were found on the post-progression sample, suggesting the possibility that molecular lesions may arise in response to therapy. Finally, in most cases, an abnormality in HER3, PI3K, or PTEN was identified implying a critical role for this pathway in mediating resistance to Trastuzumab. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 709.

Abstract B250: Clinical benefit of lapatinib-based therapy in patients with HER2-positive breast tumors expressing p95HER2

Abstract Purpose: A subgroup of HER2 overexpressing tumors also express p95HER2, a truncated fragment of the HER2 receptor that lacks the extracellular domain, retains kinase activity and correlates with intrinsic trastuzumab resistance. We tested the hypothesis that the small molecule lapatinib, an EGFR/HER2 dual tyrosine kinase inhibitor, would be active in these tumors. We studied the correlation between p95HER2 expression and response to lapatinib, both in preclinical models and in the clinic [lapatinib monotherapy (Study EGF20009) or lapatinib in combination with capecitabine (Study EGF100151)]. Patients and Methods: Expression of p95HER2 in primary tumors was evaluated by immunofluorescence and correlated with clinical benefit rate (complete response + partial response + stable disease ≥ 24 weeks) and progression-free survival using logistic regression and Cox-proportional hazard models. Results: Lapatinib was efficacious in inhibiting tumor growth and HER2 downstream signaling of p95HER2 expressing tumors in two different animal models. In human samples, expression of p95HER2 was evaluable in 76% (105/138) and 56% (223/399) of patients from EGF20009 and EGF100151 studies, respectively. The percentage of p95HER2 positive patients was 20.5% in the EGF20009 study and 28.5% in the EGF100151 study. In neither study there was a statistically significant difference in progression-free survival between p95HER2-positive and p95HER2-negative patients. Similarly, in both studies the clinical benefit rate was not significantly associated with p95HER2 expression. Conclusions: Lapatinib as a monotherapy or in combination with capecitabine is equally effective in patients with p95HER2-positive and p95HER2-negative HER2-positive breast tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B250.

Inhibitors of HSP90 block p95-HER2 signaling in Trastuzumab-resistant tumors and suppress their growth

The anti-HER2 antibody Trastuzumab (Herceptin) has been proven to be effective in the treatment of HER2 overexpressing breast cancer; resistance, however invariably emerges in metastatic tumors. The expression of p95-HER2, a form of HER2 with a truncated extracellular domain that lacks the Trastuzumab binding epitope, has been implicated as a mechanism of resistance to the antibody. We utilized an in vivo tumor model that overexpresses p95-HER2 and demonstrate it to be resistant to the signaling and antitumor effects of Trastuzumab. We find that both full length and p95-HER2 interact with the HSP90 chaperone protein and are degraded in tumor cells exposed to HSP90 inhibitors in tissue culture and in vivo. Loss of expression of p95-HER2 is accompanied by downregulation of the PI3K/AKT and ERK signaling pathways and inhibition of cell proliferation. Chronic administration of HSP90 inhibitors in vivo results in sustained loss of HER2 and p95-HER2 expression and inhibition of AKT activation together with induction of apoptosis and complete inhibition of tumor growth in Trastuzumab-resistant, p95-HER2-overexpressing models. Thus, p95-HER2 is an HSP90 client protein, the expression and function of which can be effectively suppressed in vivo by HSP90 inhibitors. HSP90 inhibition is therefore a potentially effective therapeutic strategy for p95-HER2-mediated Trastuzumab-resistant breast cancer.

Clinical benefit of lapatinib-based therapy in patients with HER2-positive breast tumors expressing p95HER2

1048 Background: Approximately 25% of HER2 overexpressing breast tumors express a truncated form of the receptor (p95HER2) that lacks the extracellular domain but retains kinase activity. p95HER2-positive tumors are associated with a worse prognosis and resistant to trastuzumab therapy. In preclinical models, lapatinib (L), a tyrosine kinase inhibitor of EGFR and HER2, is active in p95HER2 expressing tumors. The aim of this analysis was to test the hypothesis that benefit from L-based therapy is independent of p95HER2 expression in 2 clinical trials in patients (pts) with HER2-positive tumors treated with either L monotherapy (Study EGF20009 ) or L in combination with capecitabine (Study EGF100151). Methods: Pre-treatment tumor tissue from both trials (N=201) was analyzed for p95HER2 expression by immunofluorescence as previously described (Scaltriti M. et al, JNCI 2007). Expression of p95HER2 was correlated with clinical benefit rate (CBR: complete response [CR] + partial response [PR] + stable disease [SD] ≥ 24 weeks) and progression-free survival (PFS) using logistic regression and Cox-proportional hazard models. Results: 68/105 and an initial 72/96 tissue samples were evaluable for p95HER2 expression from studies EGF20009 and EGF100151, respectively. The Table summarizes the results from the L treated pts. The CBR and PFS in L treated pts were not significantly different between the p95HER2 subgroups. Expanded data for p95HER2 expression in additional EGF100151 tumor samples will be presented. Conclusions: L as a monotherapy or in combination with capecitabine has similar response activity in pts with p95HER2-positive and p95HER2-negative HER2-positive breast tumors. [Table: see text] [Table: see text]

Determination of HER2 phosphorylation at tyrosine 1221/1222 improves prediction of poor survival for breast cancer patients with hormone receptor-positive tumors

IntroductionHigh expression of total HER2 protein confers poor prognosis for breast cancer patients. HER2 is a member of the HER family consisting of four receptors, HER1 to HER4. HER receptor activity is regulated by a variety of mechanisms, and phosphorylation of the C-terminal part of the HER receptors is a marker for active signaling. The importance of phosphorylation and thereby activation of the HER1 to HER4 receptors, however, has not been investigated concomitantly in breast tumors. In the present study we examined the importance of active HER signaling in breast tumor biopsies and paired metastases, by evaluating the expression of phosphorylated HER1, HER2, HER3, Erk, Akt and the total level of HER4 and HER2.MethodsImmunohistochemical analysis was performed on 268 primary breast tumors and 30 paired metastatic lesions from postmenopausal women with hormone receptor-positive breast tumors, who had received adjuvant tamoxifen therapy. The observed protein expression levels were analyzed for co-expression, for correlation to clinicopathological parameters and for prognostic value in relation to disease-free survival and overall survival. Lastly, the difference between protein levels in primary tumors versus metastasis was evaluated.ResultsIn the primary tumors, 8%, 18%, 14% and 15% of cases were scored positive for total HER2, pHER1, pHER2 and pHER3 expression, respectively. HER4 was expressed with strong intensity in 68% and at moderate intensity in 29% of cases. The activated forms of Akt and Erk were quite uniformly expressed in the categories; negative, moderate or strong. In univariate analysis, expression of total HER2, pHER1, pHER2 and pHER3 was significantly associated with poor disease-free survival. Strong HER4 expression was associated with prolonged disease-free as well as with overall survival. Expression of pAkt and pErk was not correlated with survival. In multivariate analysis, pHER2 expression was clearly an independent marker for poor disease-free survival and overall survival when tested against tumor size, tumor grade, nodal status and HER2. Lastly, comparison of HER receptor expression in metastatic versus primary tumors showed a significant increase in expression of pHER1 and pHER3 in the metastases.ConclusionsIn hormone receptor-positive breast cancer, determination of pHER2 yields additional prognostic information about poor prognosis compared with the current clinical standard for measuring HER2.

Lapatinib is active in patients with HER2-amplified breast tumors expressing p95HER2.

Abstract Abstract #708 Background: A subgroup of HER2 overexpressing tumors also express p95HER2, a truncated fragment of the HER2 receptor that lacks the extracellular domain and retains kinase activity. Tumors expressing p95HER2 have a worse clinical outcome and are resistant to trastuzumab therapy (Scaltriti M. et al, JNCI 2007). The hypothesis that lapatinib, a small molecule inhibitor that targets the intracellular kinase domain of EGFR and HER2, would be active in these tumors was tested by correlating p95HER2 expression with clinical response to lapatinib in EGF20009, a lapatinib monotherapy study
 Methods: Archival tumor blocks were obtained from patients (pts) participating in study EGF20009. In this study, pts with untreated locally advanced or metastatic HER2 amplified breast tumors either received lapatinib 1500mg daily or 500mg BID (H.L. Gomez et al, J Clin Oncol 2008). Pre-treatment samples were analyzed for p95HER2 expression by immunofluorescence using 2 antibodies against HER2, binding to the intracellular and extracellular domain, respectively, plus a pan-cytokeratin antibody. Tumors were scored as p95HER2 positive (p95HER2+) [if any cytoplasmic staining was detected with the intracellular HER2 antibody and this staining co-localized with the pan-cytokeratin antibody], negative (p95HER2-) [no intracellular HER2 cytoplasmic staining) or non-evaluable (N/E) [no cytokeratin or HER2 staining by immunofluorescence]. The presence of p95HER2 was then correlated with response rate (RR), stable disease for over 100 days (SD) and progression-free survival (PFS) to lapatinib. A Cox-proportional hazards model was used to compare the groups. In order to correlate the presence of p95HER2 with HER2 extracellular domain in the serum, a quantitative determination of the 105 kD ECD level in serum (Oncogene Science HER2/neu Microtiter ELISA) has also been performed.
 Results: 68/105 tumors were evaluable for p95HER2 expression. Fourteen were p95HER2+ (20.5%) and 54 were p95HER2- (79.5%). The observed percentage of p95HER2+ tumors is concordant with published reports. Among the 14 pts with p95HER2+ tumors, 7 responded (RR: 50%) and 1 had SD for > 100 days for an overall clinical benefit rate of 57% (RR+SD). In the p95HER2- population (N=54), 24 pts responded (RR: 44.4%) and 16 pts had SD >100 days for an overall clinical benefit rate of 56% (RR+SD). PFS was similar in both groups, with a median PFS of 24 wks in pts with p95HER2+ and p95HER2- tumors (p=0.97, HR 1.1). These findings compare with the median PFS of 20.7 wks for the entire population in the trial (N=138pts). Further correlation of p95 status and HER2 ECD status of the tumors will be presented.
 Conclusion: These data demonstrate that lapatinib has similar activity in pts with p95HER2+ or p95HER2-, HER2 amplified breast tumors. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 708.

HER2 overexpression and activation, and tamoxifen efficacy in receptor-positive early breast cancer

Tamoxifen is a partial ER antagonist that is highly effective in the treatment of receptor positive breast cancer. It significantly reduces recurrence and improves survival in both pre- and postmenopausal women. Unfortunately, many ER+ positive tumors progress despite tamoxifen treatment and until now, no possibility exists to prospectively identify tamoxifen-resistant tumors. It has been suggested that that in HER2 over-expressing tumors, cross-talk via activated HER2 receptors is a key mechanisms by which tumors become tamoxifen-resistant. We have therefore used immunohistochemistry to analyze the expression of HER2 and activated ptyr-1248 HER2 in 408 women of ER+, early breast cancer who had received at least 2 years of adjuvant tamoxifen. We then analyzed possible associations between HER2 and pHER2 expression, and prognostic parameters, and evaluated the effect of HER2 expression and survival. With HER2 being positive in 12 of 208 (2.9%) of ER+ positive tumors, HER2 overexpression was found to be considerably less common in ER+ tumors than what has been thought previously. The majority of HER2 overexpressing tumors, however, also expressed the activated receptor form (r = 0.664; P < 0.0001). Both HER2 and pHER2 are moderately correlated with Grading (r = 0.138; P = 0.0052 and r = 0.118; P = 0.0241, respectively) and nodal involvement (r = 0.163; P = 0.0018 and r = 0.134; P = 0.016, respectively), but neither HER2 nor its activated form are significant predictors of RFS, DFS, or OS. Taken together, we have demonstrated that in ER+ breast cancer, the HER2 receptor is commonly activated, but its low prevalence in ER+ tumors does not render it a useful prognostic parameter in tamoxifen-treated patients.

Tyrosine1248-phosphorylated HER2 expression and HER2 gene amplification in female invasive ductal carcinomas

Phosphorylated HER2 (pHER2) may more accurately reflect the signaling and functional activity of the HER2 protein than detection of HER2 itself. The detection of HER2 gene amplification using fluorescence in situ hybridization (FISH) provides superior prognostic information for the diagnosis of breast cancer. However, the relationship between pHER2 expression in tissue samples and HER2 gene amplification remains unclear. A total of 210 cases were recruited. The expression of HER2 and tyrosine (Tyr)1248-pHER2 was investigated by immunohistochemistry, and HER2 gene amplification was analyzed by FISH. Spearman's rank correlation test was employed to confirm correlation between HER2 and Tyr1248-pHER2. Chi-square and Student's t test were used to determine a significant difference between the baseline characteristics of tumors and the FISH, HER2 and Tyr1248-pHER2 results. The phosphorylation rate of HER2 was calculated using a digital-analysis system. HER2 expression was significantly (P < 0.001) associated with Tyr1248-pHER2 expression. HER2 gene amplification could be detected in 55 (26.2%) of the 210 tumors; 40 were HER2 positive and 32 were Tyr1248-pHER2 positive. The sensitivity and specificity of HER2 and Tyr1248-pHER2 for HER2 gene amplification were 72.7 and 58.2%, and 91.6 and 95.5%, respectively. In cases with an HER2 score of 2, and a phosphorylation score of 2 or 3, gene amplification was observed in 4 (80.0%) out of 5 tumors. Tyr1248-pHER2 expression is highly specific for HER2 gene amplification. The phosphorylation status might provide an adjunct to the assessment of gene amplification in patients with an HER2 score of 2.

Synergistic effect of a novel antiandrogen, VN/124-1, and signal transduction inhibitors in prostate cancer progression to hormone independence in vitro

This study was carried out to determine the mechanisms associated with loss of androgen dependency and disease progression in prostate cancer. We investigated the role of the androgen receptor and its relationship to other signal transduction proteins. A hormone-refractory prostate cancer cell line [high-passage LNCaP (HP-LNCaP)] was established in vitro. Cells were treated with inhibitors of mammalian target of rapamycin and tyrosine kinase receptors. Expression of these proteins and the androgen receptor were measured by Western immunoblotting. Analysis of the model and various treatments was also assessed through proliferation assays, luciferase activation assays, binding assays, and ELISA. Our novel antiandrogen, VN/124-1, effectively inhibited proliferation of hormone-resistant prostate cancer cell lines (HP-LNCaP), which were no longer sensitive to bicalutamide and had increased expression of the androgen receptor. Treatment with everolimus or gefitinib resulted in an increase in protein expression and activation of the androgen receptor. Conversely, inhibition of the androgen receptor resulted in increased expression of IGFR1beta, pHER2, pmTOR, and pAkt. The addition of bicalutamide to everolimus or gefitinib inhibited cell proliferation in HP-LNCaP cells. However, the addition of VN/124-1 has proven to be superior to bicalutamide, and the combination was synergistic (P<0.05) compared with either agent alone. This study suggests that compensatory cross-talk between the androgen receptor and various signaling pathways may account for decreased sensitivity to androgen receptor antagonists and the progression to hormone-resistant prostate cancer. Furthermore, these findings suggest that inhibition of both pathways may provide effective control in hormone-resistant prostate cancer and restore sensitivity to androgen antagonists in hormone-refractory patients.

Expression of p95HER2, a Truncated Form of the HER2 Receptor, and Response to Anti-HER2 Therapies in Breast Cancer

Women with HER2-overexpressing breast cancers have poor prognosis, and many are resistant to the HER2 monoclonal antibody trastuzumab. A subgroup of HER2-overexpressing tumors also express p95HER2, an amino terminally truncated receptor that has kinase activity. Because p95HER2 cannot bind to trastuzumab but should be responsive to the HER2 tyrosine kinase inhibitor lapatinib, we compared the sensitivity of tumors expressing p95HER2 and tumors expressing the full-length HER2 receptor to these agents. MCF-7 and T47D breast cancer cells were stably transfected with either full-length HER2 or p95HER2. We studied the effects of trastuzumab and lapatinib on receptor signaling, cell proliferation, and the growth of xenograft tumors. A paraffin-based immunofluorescence assay was developed to study the association between p95HER2 expression and sensitivity to trastuzumab in patients with advanced breast cancer. All statistical tests were two-sided. Treatment of p95HER2-expressing cells with lapatinib inhibited p95HER2 phosphorylation, reduced downstream phosphorylation of Akt and mitogen-activated protein kinases, inhibited cell growth (MCF-7p95HER2 clones, lapatinib versus control, mean growth inhibition = 57.6% versus 22.6%, difference = 35%, 95% confidence interval [CI] = 22.5% to 47.3%; P<.001; T47Dp95HER2 clones, lapatinib versus control, mean growth inhibition = 36.8% versus 20%, difference = 16.8%, 95% CI = 11.3% to 22.3%, P<.001), and inhibited growth of MCF-7p95HER2 xenograft tumors (lapatinib versus control, mean = 288.8 versus 435 mm3, difference = 146.2 mm3, CI = 73.8 to 218.5 mm3, P = .002). By contrast, treatment with trastuzumab had no effect on any of these parameters. Of 46 patients with metastatic breast cancer who were treated with trastuzumab, only one of nine patients (11.1%) expressing p95HER2 responded to trastuzumab (with a partial response), whereas 19 of the 37 patients (51.4%) with tumors expressing full-length HER2 achieved either a complete (five patients) or a partial (14 patients) response (P = .029). Breast tumors that express p95HER2 are resistant to trastuzumab and may require alternative or additional anti-HER2-targeting strategies.

P95HER-2 Predicts Worse Outcome in Patients with HER-2-Positive Breast Cancer

The HER-2 receptor undergoes a proteolytic cleavage generating an NH(2)-terminally truncated fragment, p95HER-2, that is membrane-associated and tyrosine-phosphorylated. We have reported that p95HER-2, but not the full-length receptor, p185HER-2, correlated with the extent of lymph node involvement in patients with breast cancer and its expression was significantly enhanced in nodal metastatic tissue. These facts suggested an important role for p95HER-2 either as a marker or cause of metastasis and poor outcome in breast cancer. In this work, we have studied the prognostic value of p95HER-2 in breast cancer. Primary breast tumor tissues (n = 483) were from surgical resections conducted in hospitals in two different countries: the U.S. (n = 334) and Spain (n = 149). HER-2 protein forms, including p185HER-2 and p95HER-2, were examined in extracts of primary breast tumors by Western blot analysis. The levels of the two forms (high or low) were tested for association with other clinicopathologic factors and for correlation with disease-free survival. The median follow-up was 46 months. A high level of p95HER-2 in primary tumor tissue correlated with reduced 5-year disease-free survival (hazard ratio, 2.55; 95% confidence interval, 2.13-8.01; P < 0.0001). The median time for disease-free survival was 32 versus 139 months in patients with low levels of p95HER-2. In comparison, high levels of the full-length p185HER-2 did not significantly correlate with poor outcome (P > 0.1). Multivariate analysis revealed that high p95HER-2 was an independent predictor of disease-free survival (hazard ratio, 1.59; 95% confidence interval, 1.246-1.990; P = 0.0004). p95HER-2 expression is an independent prognostic factor in breast cancer and defines a group of patients with HER-2-positive breast cancer with significantly worse outcome.

Expression of heregulin, phosphorylated HER-2, HER-3 and HER-4 in HER-2 negative breast cancers

A significant number of HER-2 amplified breast cancers is effectively treated by trastuzumab and further shows receptor-enhanced chemosensitivity. Recent studies have postulated transactivation of HER-2 also in tumors expressing phosphorylated/activated HER-2 (pHER-2) and of the HER-3/HER-4 ligand heregulin (HRG), independent of HER-2 amplification. As a consequence, a subset of tumors without HER-2 overexpression would be sensitive to trastuzumab chemotherapy. To investigate the potential transactivation of HER-2, in 171 breast cancers from the GENICA study with negative/low expression of HER-2 we analyzed the expression of pHER-2, HRG, HER-3 and HER-4 by immunohistochemistry. None of the tumors examined displayed expression of pHER-2. Moderate or strong cytoplasmic staining of HRG, HER-3 and HER-4 was observed in 44 (26%), 67 (39%) and 33 (19%) cases, respectively. No association of HRG, HER-3 and HER-4 with the survival of patients or with known prognostic clinical factors was seen. In conclusion, our data obtained on a well-characterized cohort of breast cancers provide no evidence of HER-2-activation in the absence of HER-2 overexpression. The biological function and clinical implications of HRG, HER-3 and HER-4 in this group of tumors remain unclear. Our results cannot support the hypothesis of a transactivation of HER-2 and thus a possible therapeutic benefit of trastuzumab in HER-2 negative breast cancers.