Michael P. DiGiovanna

Relationship of Epidermal Growth Factor Receptor Expression to ErbB-2 Signaling Activity and Prognosis in Breast Cancer Patients

PURPOSE To examine the relationship of epidermal growth factor receptor (EGFR) expression to ErbB-2 signaling activity in breast cancer and the impact that this interaction has on the prognosis of patients with early-stage breast cancer. PATIENTS AND METHODS Paraffin tumor sections were collected retrospectively from 807 breast cancer patients diagnosed between 1976 and 1983. Immunohistochemical assays for ErbB-2, phosphorylated (activated) ErbB-2, and EGFR were performed, and the results were correlated with clinicopathologic variables and outcome. RESULTS EGFR expression was detectable in 15% of 807 invasive breast cancers, including 35% of the 306 ErbB-2-positive patients. Conversely, the majority (87%) of EGFR-positive tumors co-overexpressed ErbB-2. Ninety-seven percent of tumors with phosphorylated ErbB-2 co-overexpressed EGFR. Patients whose cancers demonstrated ErbB-2 phosphorylation or co-overexpression of ErbB-2 and EGFR had the shortest survival. In contrast, patients whose tumors were negative for all three markers and those tumors that expressed only EGFR or only nonphosphorylated ErbB-2 had a relatively favorable outcome. CONCLUSION These data provide the first clinical evidence that EGFR expression is linked to activation of ErbB-2 in human breast cancers. We have further shown that the adverse prognostic value of ErbB-2 overexpression is observed only when ErbB-2 is in the phosphorylated (activated) state or coexpressed with EGFR. These data suggest that ligand-dependent mechanisms of ErbB-2 activation are important in human breast cancer. These results also suggest that agents targeting EGFR may be useful in the treatment of tumors with activated ErbB-2.

Activation (Tyrosine Phosphorylation) of ErbB-2 (HER-2/neu): A Study of Incidence and Correlation With Outcome in Breast Cancer

PURPOSE We hypothesize that phosphorylated ErbB-2 (P-ErbB-2, identified by a novel antibody PN2A) may provide either more significant or additional prognostic marker data for breast cancer patients. This study was designed to compare the incidence and prognostic value of ErbB-2 (HER-2/neu) and P-ErbB-2 immunoexpression in archival breast cancer samples. MATERIALS AND METHODS Eight hundred sixteen invasive breast cancers with a median of 16.3 years of follow-up were immunostained for ErbB-2 (using antibody CB11) and P-ErbB-2 (using antibody PN2A). ErbB-2 and P-ErbB-2 data were compared with clinical, histologic, immunohistochemical, and outcome variables. RESULTS Of 816 primary breast cancers, 307 (38%) were positive for ErbB-2 and 37 (12% of ErbB-2 positive and 5% of the study population) expressed P-ErbB-2. P-ErbB-2 was not detected in ErbB-2-negative cases (n = 509). ErbB-2 immunohistochemical data were bimodal; patients with > or = 80% cellular expression had the shortest disease-free and disease-specific survival. P-ErbB-2 was associated with a higher percentage of ErbB-2-positive cells, a higher number of positive lymph nodes, and cellular proliferation. ErbB-2 and P-ErbB-2 were indicators of poor prognosis in node-positive patients in both univariate and multivariate analyses. We found that either P-ErbB-2 expression or high (> or = 80%) ErbB-2 expression provided the most significant prognostic value in node-positive cases by multivariate analyses. There were too few P-ErbB-2-positive cases and events in the node-negative patient group to allow statistical analysis of P-ErbB-2 in that subgroup. CONCLUSION PN2A immunostaining identified a subset (approximately 12% of ErbB-2-positive breast cancers) with activation (phosphorylation) of the receptor ErbB-2. P-ErbB-2 expression was strongly associated with higher levels of ErbB-2 expression (> or = 80%), although it was not a surrogate. Identification of cases with a high percentage of invasive breast cancer cells expressing ErbB-2 or determination of receptor activation via P-ErbB-2 may provide additional prognostic value in node-positive breast cancers.

Sequence of Radiotherapy With Tamoxifen in Conservatively Managed Breast Cancer Does Not Affect Local Relapse Rates

PURPOSE To evaluate whether the sequencing of tamoxifen (TAM) relative to radiation (RT) affects outcome in breast cancer patients treated with conservative surgery (CS) plus RT (lumpectomy with RT). METHODS Between 1976 and 1999, 1,649 patients with stage I or II breast cancer were treated with CS plus RT at Yale-New Haven Hospital (New Haven, CT). TAM was administered to 500 patients. The timing of TAM relative to RT was documented for each patient. Of the 500 patients, the timing of TAM was unclear in five patients, was administered concurrently with RT in 254 patients (CON-TAM), and was administered sequentially after completion of RT in 241 patients (SEQ-TAM). RESULTS There were no differences between the CON-TAM and SEQ-TAM group in T stage, estrogen and progesterone status, nodal status, histology, or margin status. The CON-TAM group was slightly older than the SEQ-TAM group (62 v 58 years) and received chemotherapy in addition to TAM less frequently (14% v 38%). As of September 2002, with a median follow-up of 10.0 years, there were no significant differences between the CON-TAM and SEQ-TAM groups in overall survival (84% v 82%; hazard ratio [HR], 1.234; 95% CI, 0.42 to 2.05; P = .45), distant-metastasis-free rate (82% v 78%; HR, 1.55; 95% CI, 0.89 to 2.68; P = .12), ipsilateral breast-relapse-free rate (90% v 86%; HR, 0.932; 95% CI, 0.42 to 2.05; P = .86), or contralateral breast-relapse-free rate (95% v 93%; HR, 0.892; 95% CI, 0.53 to 1.48; P = .66). CONCLUSION Although the concurrent use of TAM with RT may theoretically render cancer cells less responsive to RT, this retrospective study suggests that in practical application, concurrent administration of TAM with RT does not compromise local control.

Association of constitutively activated hepatocyte growth factor receptor (Met) with resistance to a dual EGFR/Her2 inhibitor in non-small-cell lung cancer cells

There is a pressing need to identify new drug targets and novel approaches for treatment of non-small-cell lung carcinoma (NSCLC). Members of the epidermal growth factor receptor (EGFR) and Met receptor families have been identified as important molecular targets for NSCLC. Two EGFR tyrosine kinase inhibitors (TKIs; erlotinib and gefitinib) are in current clinical use, but a majority of patients do not respond to these targeted therapies. We used receptor TK (RTK) capture arrays to identify receptors active in NSCLC cell lines. As Met and ErbBs were active, we explored the potential therapeutic advantage of combined targeting of Met with ErbB receptor family inhibitors for treatment of NSCLC. We found that Met physically interacts with both EGFR and Her2 in a NSCLC cell line with overexpression/overactivation of Met. Combined use of a dual EGFR/Her2 inhibitor with a Met inhibitor yields maximal growth inhibition compared with the use of EGFR and/or Met inhibitors. This suggests that simultaneous inhibition of multiple RTKs may be needed to effectively abrogate tumour cell growth. Phosphoproteomic analysis by RTK capture arrays may be a valuable tool for identifying the subset of tumours with functional receptor activation, regardless of mechanism.

Synergistic Interactions between Tamoxifen and Trastuzumab (Herceptin)

Purpose: HER-2/neu and estrogen receptor (ER) are critical in the biology of breast carcinoma, and both are validated therapeutic targets. Extensive interactions between the signaling pathways of these receptors have been demonstrated. This suggests that targeting both receptors simultaneously may have a dramatic effect on the biology of breast cancer. This hypothesis was tested in cell culture experiments. Experimental Design: ER-positive, HER-2/neu-overexpressing BT-474 human breast carcinoma cells were cultured in the presence of the anti-HER-2/neu therapeutic antibody trastuzumab (Herceptin), the antiestrogen tamoxifen, or both. The effects on cell growth, cell cycle distribution, clonogenicity, survival, and the level and activity of HER-2/neu were examined. Results: The combination of tamoxifen and Herceptin resulted in synergistic growth inhibition and enhancement of cell accumulation in the G0-G1 phase of the cell cycle, with a decrease in cells in S phase. Clonogenicity was inhibited in the presence of each drug and more so by the combination, although prior exposure to drugs did not affect subsequent clonogenicity in drug-free media, and neither drug nor the combination induced apoptosis. Herceptin, but not tamoxifen, inhibited signaling by HER-2/neu. Conclusions: The combination of tamoxifen and Herceptin is formally demonstrated to result in synergistic growth inhibition and enhancement of G0-G1 cell cycle accumulation. In vitro, the individual drugs or combination produces a cytostatic effect. These results suggest that combined inhibition of ER and HER-2/neu signaling may represent a powerful approach to the treatment of breast cancer.

Co-Targeting Insulin-Like Growth Factor I Receptor and HER2: Dramatic Effects of HER2 Inhibitors on Nonoverexpressing Breast Cancer

The insulin-like growth factor I receptor (IGFIR) and HER2 display important signaling interactions in breast cancer. We examined the effect of combinations of antagonists of these receptors using two human breast cancer cell lines: BT474 (HER2+, IGFIR low) and MCF7 (HER2 low, IGFIR high). In BT474 cells, growth was inhibited by HER2 antagonists but not by IGFIR antagonists; however, IGFIR antagonists enhanced the effect of HER2 inhibitors. In MCF7 cells, growth was inhibited by IGFIR antagonists but not by HER2 antagonists; however, HER2 antagonism enhanced the effect of IGFIR inhibitors. Synergistic inhibition of soft agar growth was also observed. Although HER2 and IGFIR antagonists individually only minimally affected cell cycle, their combination gave a small enhancement of their effects. No single receptor-targeting drug was capable of inducing apoptosis, but combining antagonists of both receptors induced a dramatic degree of apoptosis in both cell lines. Induction of apoptosis was most striking in MCF7 cells using a Herceptin/IGFIR antagonist combination despite these cells being HER2 nonoverexpressing. Toward understanding the mechanism of these effects, we detected coassociation IGFIR and HER2 in both cell lines. Specific inhibitors of one of these receptors could cross-inhibit the activity of the other. Targeting both receptors gave the maximal inhibition of their downstream extracellular signal-regulated kinase 1/2 and AKT signaling pathways. Hence, such drug combinations may be clinically useful and may be beneficial even in tumors in which single drugs are inactive, as exemplified by the effect of the HER2/IGFIR inhibitor combination in HER2 nonoverexpressing MCF7 cells.

Quantitative assessment Ki-67 score for prediction of response to neoadjuvant chemotherapy in breast cancer

Measurement of Ki-67, a marker of cell proliferation, has been associated with response to therapy, but methods of measurement are controversial. Here we use a quantitative objective measurement for Ki-67 to determine the best method for assessment of Ki-67 for prediction of response to neoadjuvant chemotherapy. Analysis was conducted on a cohort of 105 consecutive invasive breast cancer patients that received neoadjuvant therapy between 2002 and 2010, and on whom pre-surgical biopsies were obtainable. Ki-67 expression was measured using quantitative immunofluorescence automated quantitative analysis (AQUA) technology. Images for each specimen were collected for 5 to 115 fields of view (FOVs) and summary scores were obtained, corresponding to the average and maximum of all the FOVs. AQUA scoring (using both intensity and area) was comparable to automated calculation of percentage of positive nuclei for prediction of response to chemotherapy (OR: 2.832 vs 2.712). Both the average and maximum AQUA score showed Ki-67 expression was directly correlated to pathological complete response (pCR; average P=0.0002; maximum P=0.0011). Although examining the maximum FOV was more predictive of response to therapy (OR: 3.546 vs 2.832), averaging all fields provided more sensitivity and specificity (AUC 0.769 vs 0.732). Ki-67 average (P=0.0025) and maximum (P=0.0239) AQUA score were also significant predictors of pCR in a multivariable analysis, including tumor size, nuclear grade, nodal status, ER status, and HER2 status. Measurement of Ki-67 expression by objective quantitative methods shows increased Ki-67 levels are an independent predictor of response to neoadjuvant chemotherapy.

Co-targeting the insulin-like growth factor I receptor enhances growth-inhibitory and pro-apoptotic effects of anti-estrogens in human breast cancer cell lines.

The insulin-like growth factor I receptor (IGF1R) interacts with estrogen receptor-α (ERα) and HER2. We examined the effect of combinations of IGF1R antagonists (α-IR3, AG1024) and anti-estrogens (4-hydroxy tamoxifen, fulvestrant) in two human ER+ breast cancer cell lines: BT474 (HER2 overexpressing, IGF1R low) and MCF7 (HER2 non-overexpressing, IGF1R high). In BT474 cells, growth was inhibited by anti-estrogens, but not by IGF1R antagonists; however, adding IGF1R inhibitors to anti-estrogens enhanced growth inhibition. In MCF7 cells, growth was inhibited by IGF1R and ER antagonists and more so by their combination. In both cell lines, no single agents could induce apoptosis, but combining IGF1R inhibitors with anti-estrogens induced dramatic levels of apoptosis. IGF1R antagonists enhanced the ability of the anti-estrogens to inhibit ER transcriptional activity in BT474 cells, but not in MCF7 cells. The drug combination synergistically inhibited ER and IGF1R activity. Such combinations may be useful therapy for breast cancer.

Functional assay for HER-2/neu demonstrates active signalling in a minority of HER-2/neu-overexpressing invasive human breast tumours

Overexpression of HER-2/neu in human breast carcinomas correlates with poor prognosis, although its strength as a prognostic indicator varies widely in different reports. Variability may be due to active signalling by HER-2/neu in a subset of the tumours in which it is overexpressed. To study this hypothesis, we have developed an activation state-specific anti-HER-2/neu monoclonal antibody. In this report, we use this antibody to analyse the signalling status of HER-2/neu in a large series of invasive breast carcinomas. Overexpression of HER-2/neu was detected in 9% of 223 cases. Of the cases demonstrating overexpression, active signalling by HER-2/neu was detected in only 35%. The clinicopathological characteristics of these cases are described. This functional assay is predicted to improve the utility of HER-2/ neu as a prognostic indicator.

Phosphorylated/Activated HER2 as a Marker of Clinical Resistance to Single Agent Taxane Chemotherapy for Metastatic Breast Cancer

Purpose. To determine the association of phosphorylated/activated HER2 (P-HER2) and response to taxane chemotherapy in patients with metastatic breast cancer (MBC). Materials and Methods. Archived tumor specimens of patients with MBC treated on clinical trials with taxane monotherapy were analyzed by immunohistochemistry (IHC) for the presence of phosphorylated HER2 using the PN2A monoclonal antibody. Chi-squared analysis was performed to evaluate the association of P-HER2 status and efficacy of single agent taxane therapy. Results. One hundred twenty-six cases were identified as evaluable for both IHC and clinical outcome. Twelve cases (10 percent) were positive for P-HER2, of which 5 had evidence of clinical progression and 7 had evidence of clinical benefit with taxane therapy. Of the 114 cases that were negative for P-HER2, 20 demonstrated progression and 94 had clinical benefit. Chi-squared analysis revealed a significant correlation between the presence of P-HER2 and resistance to taxane therapy, χ2 = 3.9724 and p = 0.046. Conclusions. Phosphorylated/activated HER2 is associated with clinical resistance to single agent taxane therapy for MBC. The likelihood of direct progression of disease on taxane was greater in cases of PN2A-positive tumors (42 percent) as opposed to PN2A-negative ones (18 percent, p = 0.046). Functional assessment of HER2 status may provide unique predictive information not seen with conventional assessments.