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Keywords:

  • HER2/neu;
  • erbB2;
  • metastatic breast cancer;
  • trastuzumab;
  • predictive biomarker;
  • fluorescence in situ hybridization;
  • VeraTag;
  • HERmark;
  • immunohistochemistry

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES

BACKGROUND:

Only a portion of breast cancer patients currently selected for trastuzumab therapy respond.

METHODS:

Using a novel assay (HERmark) to quantify total human epidermal growth factor receptor 2 (HER2) expression, the authors examined outcomes in 102 trastuzumab-treated metastatic breast cancer patients previously assessed as immunohistochemistry (IHC) 3+ by local but not central IHC, or fluorescence in situ hybridization (FISH) positive, and then retested by central FISH.

RESULTS:

Of 102 MBC patients previously scored as IHC 3+ or 2+/FISH-positive and treated with trastuzumab-containing regimens, 98 had both central FISH and HER2 total expression values. Sixty-six of 76 central FISH-positive patients (87%) had high HER2 total expression levels (concordant positive), and 19 of 22 central FISH-negative patients (86%) were HER2 total expression low (concordant negative). Fourteen percent (3 of 22) of central FISH-negative patients were HER2 total expression high (discordant HER2 total expression high), and 13% (10 of 76) of central FISH-positive patients were HER2 total expression low (discordant HER2 total expression low). The concordant positive group had a significantly longer time to progression (TTP, median = 11.3 months) compared with the concordant negative group (median TTP, 4.5 months; hazard ratio [HR] = 0.42, P < .001), and also compared with the discordant HER2 total expression low group (median TTP, 3.7 months; HR = 0.43, P = .01). The discordant HER2 total expression low group behaved similarly compared with concordant negatives (HR = 1, P = .99). In analyses restricted to central FISH-positive patients only (n = 77), Cox proportional hazards multivariate regression identified HER2 total expression as an independent predictor of TTP (HR = 0.29, P = .0015) and overall survival (HR = 0.19, P < .001).

CONCLUSIONS:

A subset of patients with HER2 gene amplification by FISH express low levels of HER2 protein and have reduced response to trastuzumab-containing therapy, similar to FISH-negative patients. This cohort represents a training dataset, and the observed relationships and derived cutoffs require validation in an independent cohort of trastuzumab-treated metastatic breast cancer patients. Cancer 2010. © 2010 American Cancer Society.

The monoclonal antibody trastuzumab, targeting the human epidermal growth factor receptor 2 (HER2), has been shown to offer significant disease-free and overall survival (OS) advantages in both the metastatic and adjuvant settings in patients with HER2-overexpressing breast cancer.1-5 Normally, HER2 activates signaling pathways by dimerizing with ligand-bound epidermal growth factor receptor-family members such as HER1 and HER3. To date, no ligand has been identified for HER2 itself; thus, it is thought that the regulation of signaling pathway activity is under the control of the receptor requiring ligand activation.6 In situations where HER2 is pathologically overexpressed, the receptor may homodimerize and activate signaling cascades in the absence of the normal regulatory control imposed by the requirement for ligand binding of its heterodimerization partners.7-10

Current methodologies for the selection of HER2-positive patients include immunohistochemistry (IHC) to detect HER2 protein overexpression, and fluorescence in situ hybridization (FISH) to detect amplification of HER2. Using these methods, only a fraction of HER2-positive patients with metastatic breast cancer (MBC) respond to trastuzumab, and a significant proportion of the responders relapse within 1 year. Prior efforts to correlate quantitative measures of HER2 status (eg, HER2 gene copy number) with clinical outcome have been disappointing.11-16 Whereas IHC and FISH assays performed by experienced pathologists at central laboratories have been shown to be reliable and predictive of trastuzumab benefit,16 recent data from the adjuvant setting have called the accuracy of local IHC and FISH into question, suggesting that some patients who may benefit from trastuzumab are being missed using the current assays.17, 18 New approaches to the assessment of HER2 status may allow for more accurate identification of patients who are likely to benefit from trastuzumab.19

This study involved the use of formalin-fixed paraffin-embedded (FFPE) specimens from a cohort of patients with MBC who were all assessed as HER2 overexpressed (IHC 3+) and/or gene-amplified (FISH positive), and who received trastuzumab at a single institution. We used a novel assay to make precise quantitative measurements of HER2 protein expression, and correlated them with results obtained by local IHC and central FISH, as well as with clinical outcomes.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES

Study Population

The study population comprised patients who were prospectively observed during trastuzumab-based therapy at the Medical University of Vienna between 1999 and 2006 (only outpatients, Eastern Cooperative Oncology Group performance status 0-2, age >18 years, estimated life expectancy >12 weeks) with HER2-overexpressing (>10% of tumor cells IHC 3+ as determined by the HercepTest; Dako Diagnostics, Vienna, Austria) and/or HER2-amplified MBC (with positive FISH testing mandatory in all IHC 2+ cases). In addition, patients were required to be trastuzumab-naive and have bidimensionally measurable disease progressing within 4 weeks before initiation of treatment (excluding previously irradiated lesions). Pregnant or breast-feeding patients, patients with a history of congestive heart failure, ischemic heart disease, second malignancy (except in situ cervical cancer, or adequately treated basal cell or squamous cell carcinoma of the skin), severe hepatic or renal dysfunction, or altered mental status were not eligible. Patients' written informed consent was obtained for documentation of disease and treatment-related data as well as subsequent analysis of tumor tissue before initiating trastuzumab; however, not all consented patients had tissue available for testing. All treatment decisions were made by the treating physicians, not the investigators. All consented patients received planned therapy. Tissue for analysis was derived from the primary tumor. Response to treatment was documented by review of all imaging studies according to Southwest Oncology Group criteria as previously described,20 and data on HER2 and hormone receptor status were retrieved from pathology reports. Archived FFPE tumor specimens were retrieved from 131 (89%) of 147 patients. The research protocol was approved by the Penn State University/Hershey Medical Center and Medical University of Vienna institutional review boards. A description of the cohort characteristics (Table 1) and a REMARK (National Cancer Institute-European Organization for Research and Treatment of Cancer Reporting Recommendations for Tumor Marker Prognostic Studies)21 diagram is provided (Fig. 1).

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Figure 1. Patient selection flow (REMARK) diagram is shown. FFPE indicates formalin-fixed paraffin-embedded; DCIS, ductal carcinoma in situ; IHC, immunohistochemistry; FISH, fluorescence in situ hybridization; H2T, total HER2 expression.

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Table 1. Characteristics of the Patient Cohort
CharacteristicNo. (Range, %)
  1. H2T indicates total human epidermal growth factor receptor 2 (HER2); ER, estrogen receptor; +, positive; PR, progesterone receptor; −, negative; FISH, fluorescence in situ hybridization.

Total patients102
Mean follow-up, mo34 (11.8-77.9)
Mean age, y55.4 (27.6-85.4)
H2T (>cutoff)69 (68)
No. of metastatic sites 
 <360 (59)
 ≥342 (41)
Hormonal status 
 ER+/PR+17 (17)
 ER+/PR−19 (19)
 ER−/PR+3 (3)
 ER−/PR−61 (60)
 Unknown2 (2)
HER2 status 
 IHC 2+5
  FISH+5
 IHC 3+95
  FISH+70
  FISH−22
  FISH unknown3
 IHC Unknown2
  FISH+2
Treatment 
 Trastuzumab and chemotherapy90 (88)
 Trastuzumab only12 (12)
Line of chemotherapy 
 First line75 (74)
 Second line17 (17)
 Third line8 (8)
 Unknown2 (2)

Blinding and Analysis of Data

Anonymized clinical data and FFPE sections provided by investigators at the Medical University of Vienna, Austria were shipped to the coordinating center at Penn State University/Hershey Medical Center, Hershey, Pennsylvania. After securing the completeness of clinical information, 127 FFPE samples were shipped to Monogram Biosciences (South San Francisco, Calif). Twenty-five specimens were deemed inadequate for the generation of final results; 17 patients had insufficient tumor, 6 patients had ductal carcinoma in situ only on pathology review at Monogram Biosciences, and 2 patient sample assays failed for technical reasons. Final results were generated for 102 patients (see Fig. 1). All HERmark assays were performed in blinded fashion. The treating physicians were blinded to the results of central FISH and the HERmark results. Subsequent to testing, HER2 total expression results were transmitted to Penn State/Hershey before receipt of clinical data at Monogram Biosciences. The patients were exposed to a variety of different chemotherapy regimens, either before trastuzumab or during trastuzumab therapy (10 in all).

The HERmark Assay

The HERmark HER2 total expression assay is an application of the VeraTag technology platform designed specifically for breast cancer. VeraTag is a proximity-based method designed to accurately and reproducibly quantify protein expression and protein-protein complexes, including cell surface dimers, in FFPE specimens (Fig. 2).22 The assay has been technically validated according to the specifications prescribed by the Clinical Laboratory Improvement Amendment and is performed in a College of American Pathologists-certified clinical reference laboratory at Monogram Biosciences.

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Figure 2. The HERmark assay is shown. Activation of the molecular scissors by red light yields singlet O2, resulting in cleavage of the fluorescein reporter. HERmark's standard unit of measurement is (relative peak area × illumination buffer volume) / (invasive tumor area in mm2). Adapted from Shi Y, Huang W, Tan Y et al. 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. Diagn Mol Pathol. 2009;18:11-21.22

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Central FISH Testing

Detection of HER2 gene amplification in HERmark-tested specimens was performed in 1 batch by a single pathologist at the Medical University of Vienna using the Vysis assay (Abbott-Vysis, Downers Grove, Ill). The central pathologist was blinded to the local HER2 results and to the HERmark results. FISH assays were performed using contiguous sections from the same tissue block as the HERmark measurements. For each specimen, the HER2 and CEP17 signals of >50 nuclei of invasive tumor areas were recorded. Tumors were classified as FISH-positive if the HER2/CEP17 ratio exceeded 2.2.

Statistical Analysis

Statistical analyses were performed independently at Monogram Biosciences and Penn State and then compared. Determination of clinical cutoffs for HER2 total expression occurred before the performance of central FISH assays in Vienna, by positional scanning analysis and selection of the cutoff associated with the lowest P value for time to progression (TTP). Clinical cutoffs did not change during subsequent analyses comparing central FISH and HERmark results.

Correlation of HER2 total expression with objective response rate (complete response [CR] + partial response [PR]/all patients) and clinical benefit (CR + PR + stable disease [SD] ≥6 months/all patients) used the test for trend, Kaplan-Meier analyses used the log-rank test, and multivariate analyses used Cox proportional hazards regression in the R statistical package. Graphpad (La Jolla, Calif) Prizm was used to generate all graphs. All P values are 2-tailed. Measurements of HER2 total expression were tested for correlation with TTP and OS. TTP was defined as the time from the initiation of trastuzumab-containing treatment to progression (Southwest Oncology Group) or censoring, and OS was defined as the time from initiation of trastuzumab-containing treatment to death or censoring. Patients were classified as either high or low for HER2 total expression based on the clinical cutoff originally identified for TTP. High and low groups were compared for time-to-event endpoints using Kaplan-Meier analysis. Cox proportional hazards models were fitted to determine the most significant correlates with outcome, and the assumption of proportional hazards was tested for each variable. Potential prognostic factors included HER2 total expression ≥ versus < cutoff, continuous values of HER2 total expression, HER2 status by FISH (amplified vs not amplified, where amplified was defined as having ≥2.2 HER2 copies per chromosome 17 centromere), FISH total copy number, FISH copy number per chromosome 17 centromere number (FISH/CEP17), IHC score, age, treatment group (trastuzumab-only or trastuzumab plus chemotherapy), estrogen and progesterone receptor status, time from metastasis to treatment, and time from surgery to treatment. Total number of sites of metastasis was considered both as a continuous and categorical (<3 vs ≥3 sites) variable. A log transformation was applied to HER2 total expression to reduce the asymmetry of the distribution of this variable. All possible Cox models were tested, and those with the lowest P values were considered the best models.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES

Description of the Clinical Cohort

HERmark testing was performed on 102 patients who had previously been assessed as IHC 3+ (95 of 102), IHC 2+/ FISH positive (5 of 102), or IHC unknown/ FISH positive (2 of 102). Central FISH results were obtained for 99 of 102 patients (missing data because of <50 tumor cells [1 patient] and inadequate material [2 patients] ). Twenty-two of 99 patients were assessed as central FISH negative. All of these tumors were IHC 3+ on prior testing. Seventy-seven of 99 tumors were central FISH positive.

Correlation of HER2 Expression With IHC and Central FISH

The distribution of HER2 total expression was determined for the entire cohort and compared with IHC (Fig. 3Top) and central FISH (Fig. 3Bottom) assessments. The HER2 total expression values ranged over approximately 2 to 2.5 logs, and the distributions for specimens scored by IHC as 2+ or 3+ overlapped (Fig. 3Top). The medians of the HER2 total expression values in central FISH-positive versus central FISH-negative subpopulations were significantly different (Mann-Whitney P value <.0001); however, significant overlap in the distributions of values was observed (Fig. 3Bottom).

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Figure 3. HER2 total expression (H2T) correlated with (Top) immunohistochemistry (IHC) and (Bottom) central fluorescence in situ hybridization (FISH) calls.

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Correlation of HER2 Protein Expression With Clinical Outcomes After Treatment With Trastuzumab

Patients were sorted into 4 groups according to quartiles of measured HER2 total expression. Objective response rate was calculated for each subgroup of patients, and an identical analysis was performed using clinical benefit as the outcome measure (Fig. 4). In both analyses, a statistically significant trend favoring better responses at higher levels of HER2 total expression (test for trend P value = .007 for objective response rate and .034 for clinical benefit) was observed.

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Figure 4. Objective response and clinical benefit by Southwest Oncology Group (SWOG) are shown. Correlation of HER2 total expression (H2T) with objective response rate (ORR) (complete response [CR] + partial response [PR]) and clinical benefit (CR + PR + stable disease [SD] >6 months) are determined by SWOG responses. Quartile 1 = lowest 25% of the HER2 total expression distribution, quartile 4 = highest 25% of the HER2 total expression distribution. PD indicates progressive disease.

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Univariate Kaplan-Meier analyses examining TTP for the subgroups defined as central FISH negative and central FISH positive demonstrated, as expected, a statistically significant advantage for the FISH-positive subgroup (hazard ratio [HR] = 0.45, P <. 001) (Fig. 5Top). Median TTP was 4.4 months for the FISH-negative subgroup and 10.3 months for the FISH-positive subgroup.

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Figure 5. Univariate Kaplan-Meier plots of time to progression by (Top) central fluorescence in situ hybridization (FISH) status and (Bottom) HER2 total expression (H2T) are shown. The HER2 total expression cutoff was derived by positional scanning analysis with selection of the cutoff having most significant (lowest) P value. HR indicates hazard ratio.

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Identical Kaplan-Meier analyses were performed using HER2 total expression. Cutoffs determined by positional scanning (Fig. 6) were used to discriminate high from low HER2 total expression (≥ vs <log10 HER2 total expression = 1.25). Statistically significant relationships were observed with those patients exhibiting higher levels of HER2 total expression (HR = 0.40, P < .001) experiencing longer TTP than those patients with lower HER2 total expression (Fig. 5Bottom). Median TTP was 4.4 months for the subgroup with HER2 total expression values below the cutoff, and 11.3 months for those with HER2 total expression values above the cutoff. Univariate Kaplan-Meier analyses were also performed using OS as the clinical outcome endpoint. Neither FISH nor HER2 total expression were significantly associated with OS. The median OS for patients in the FISH-negative subgroup was 28.7 months compared with 31.8 months for those who were FISH-positive (P = .29). For HER2 total expression, those below versus those above the cutoffs had a median OS of 28.7 versus 37.4 months (P = .16). Cox proportional hazards multivariate analyses were performed including all patients in the cohort (N = 102) (Table 2) to identify variables that were significantly associated with clinical outcome on trastuzumab. Initial analyses excluding HER2 total expression demonstrated, as expected, that FISH status was a significant correlate of TTP (HR = 0.48, P = .006). When the categorical variable HER2 total expression (≥ vs <cutoff) was added to the analysis, FISH status dropped out of the model (HR = 1.0, P = .94) and was replaced by HER2 total expression as a significant correlate of TTP (HR = 0.44, P = .042). The presence of 3 or more metastatic sites (HR = 2.6-2.7, P < .001) was a significant correlate in both models.

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Figure 6. HER2 total expression positional scanning cutoff determination is shown. All possible cutoffs for HER2 total expression were tested using positional scanning, and the cutoff with the lowest associated P value was selected. For HER2 total expression, this cutoff was log10 (H2T) = 1.25.

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Table 2. Cox Proportional Hazards Multivariate Analysis
VariableEntire Cohort (N=102)
Without H2TWith H2T
HRPHRP
  1. H2T indicates total human epidermal growth factor receptor 2 (HER2); HR, hazard ratio; TTP, time to progression; FISH, fluorescence in situ hybridization; OS, overall survival; ER, estrogen receptor.

  2. Models were generated using the entire cohort (N=102), and examining the significance of central FISH status without and then with the inclusion of H2T. Subsequently, multivariate Cox analyses were performed on the subset of the cohort that was FISH-positive by central testing (n=77).

TTP    
 No. of metastatic sites (≥3 vs <3)2.6<.0012.7<.001
 FISH (positive vs negative)0.48.0061.94
 H2T (≥ vs <cutoff)0.44.042
OS    
 Age (per year)0.97.0480.97.021
 No. of metastatic sites (continuous)1.6<.0011.6<.001
 ER status (positive vs negative)0.49.030.48.03
 FISH status (positive vs negative)0.65.231.4.57
 H2T (≥ vs <cutoff)0.4.058
FISH-Positive Patients Only (n=77)
TTP    
 No. of metastatic sites (≥3 vs <3)3.3<.001  
 H2T (≥ vs <cutoff)0.29.0015  
 Line of therapy (more vs less)1.7.011  
 Time from sample to trastuzumab therapy (more vs less) (per year)0.86.045  
OS    
 No. of metastatic sites (continuous)1.8<.001  
 H2T (≥ vs <cutoff)0.19<.001  
 Age (per y)0.97.02  

Similar Cox multivariate analyses using OS as the outcome measure showed that FISH was not a significant correlate of OS in either model, but HER2 total expression trended toward statistical significance (HR = 0.4, P = .058) (Table 2). The presence of 3 or more metastatic sites (HR = 1.6, P < .001), estrogen receptor positivity (HR = 0.48-0.49, P = .03), and age (HR = 0.97, P = .021-.048) were also significant correlates in both models.

Cox proportional hazards multivariate analyses were also conducted on the subgroup of patients who were central FISH positive (n = 77) (Table 2) to examine the prognostic ability of HER2 total expression in a stringently selected population of patients with HER2 gene amplification. For these analyses, both HER2 total copy number and HER2/CEP17 ratio were included as continuous variables along with the categorical (≥ vs <cutoff) and continuous measures of HER2 total expression. In both the TTP (HR = 0.29, P = .0015) and OS (HR = 0.19, P < .001) analyses, the categorical variable HER2 total expression was identified as an independent correlate of outcome. Patients with HER2 total expression values above the cutoff exhibited longer TTP and longer OS than those who had HER2 total expression values below the cutoff. Other significant variables included number of metastatic sites, line of therapy, time from surgery to initiation of trastuzumab therapy, and age.

Checking the assumption of proportional hazards, 2 variables: showed evidence of time dependence 1) time from sample to trastuzumab therapy in the TTP model using the subset of 77 FISH-positive patients (P = .02) and 2) number of metastatic sites (continuous) in the OS model using the subset of 77 FISH-positive patients (P = .04). No other variables in any model demonstrated evidence of time dependence.

Clinical Outcomes of Tumors Exhibiting HER2 Gene Amplification But Expressing Low Amounts Of HER2 Protein

As previously noted in Figure 3Bottom, the distribution of HER2 total expression in subgroups of patients characterized as central FISH negative or central FISH positive overlap. Application of the cutoff for HER2 total expression to the distribution yields 4 groups: 1) patients who are central FISH negative and below the cutoff for HER2 total expression (concordant negative), 2) patients who are central FISH positive and above the cutoff for HER2 total expression (concordant positive), 3) patients who are FISH negative but above the cutoff for HER2 total expression (discordant high), and 4) patients who are central FISH positive but below the cutoff for HER2 total expression (discordant low) (Fig. 7). The number of patients in the discordant high group is too small to permit any further analysis. However, the discordant low group contains 10 patients. We performed univariate Kaplan-Meier analyses of TTP and OS to examine the behavior of the discordant low group compared with those patients who were either concordant negative or concordant positive.

The concordant negative group had a mean total HER2 gene copy number of 3.2 (range, 1.7-6.0) and a mean HER2/CEP17 copy number ratio of 1.4 (range, 1.1-1.9). The concordant positive group had a mean total HER2 copy number of 10 (range, 5-16) and a mean HER2/CEP17 copy number ratio of 4.5 (range, 2.3-8.4). The discordant low group had a mean total HER2 copy number of 8.8 (range, 5.4-12.7) and a mean HER2/CEP17 copy number ratio of 3.4 (range, 2.2-5).

The discordant low group generates a Kaplan-Meier curve that tracks with the concordant negative group rather than the concordant positive group (Fig. 7). Compared with the concordant negative group, the concordant positive group experiences a significantly longer TTP (HR = 0.42, P < .001). Compared with the discordant low group, the concordant positive group also experiences a significantly longer TTP (HR = 0.43, P = .01). However, compared with the concordant negative group, the discordant low group experiences a TTP that is indistinguishable (HR = 1.0, P = .99). Kaplan-Meier curves for OS are also shown (Fig. 7).

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Figure 7. Kaplan-Meier analyses examine outcomes (time to progression [TTP] and overall survival [OS]) associated with the 3 subpopulations defined by the fluorescence in situ hybridization (FISH) and HERmark cutoffs: 1) concordant negative (FISH negative, <HER2 total expression [H2T] cutoff), 2) concordant positive (FISH positive, ≥HER2 total expression cutoff), and 3) discordant low (FISH positive, <HER2 total expression cutoff).

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All of the aforementioned analyses were also performed using HER2 homodimer levels as the VeraTag variable, yielding nearly identical results as those using HER2 total expression. As HER2 homodimer levels offered no additional insight in these analyses, we have chosen to focus our attention on the simpler HER2 total expression measurement.

In summary, HERmark identified a subpopulation of patients who were FISH positive for HER2 gene amplification, but expressed low levels of total HER2 protein. These patients experienced a significantly shorter TTP on trastuzumab compared with FISH-positive patients with high total HER2 protein expression, and their outcomes were indistinguishable from those of FISH-negative patients.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES

Until now, the only method available for the direct measurement of the HER2 protein target has been IHC. Although high-quality IHC testing is possible and predictive of clinical outcome to trastuzumab, this is most uniform only in a well-controlled central IHC setting. Local IHC is often complicated by lack of sensitivity, poor specificity in FFPE specimens, and suboptimal reproducibility because of its subjective scoring system and widespread use across many laboratories, presenting challenges to standardization.14-16 The HERmark assay provides an accurate and reproducible measure of HER2 protein expression in breast tumors prepared as FFPE specimens.22 Assay validation data developed at Monogram Biosciences comparing HERmark measurements to centrally performed IHC in cell lines prepared as FFPE pellets, as well as to quantitative HER2 by enzyme-linked immunosorbent assay and quantitative HER2 expression by flow cytometry, demonstrate excellent concordance with all 3 measurements, but greater ability to accurately measure HER2 expression at low levels than IHC.23 As a result, HERmark is able to measure a distribution of HER2 expression extending over a large dynamic range corresponding to approximately 2500 to 2 million receptors/cell.22,23 Measurements of gene copy number, although predictive, capture the amplification of a segment of DNA that includes not only HER2, but other genes that reside in contiguous regions on chromosome 17. HERmark makes a direct measurement of HER2, and therefore can capture the impact of (post)transcriptional and/or (post)translational regulatory events that may influence HER2 protein expression specifically.

In this study, we have demonstrated, in a population of patients with MBC who were IHC 3+ or FISH positive, a continuum of HER2 expression that extends over a 2 to 2.5 log dynamic range. By using a clinical cutoff determined before generation of the central FISH results, we identified a subset of IHC 3+ patients with HER2 values below the cutoff who responded less well than patients above the cutoff. Approximately two-thirds of these patients were subsequently found to be central FISH negative (19 of 29). Examination of the discordant subgroup who were FISH positive but had HER2 total expression values below the cutoff demonstrated that those patients experienced clinical outcomes that were indistinguishable from the concordant negative subgroup and significantly different from the concordant positive subgroup. An important limitation of this analysis is that we did not have enough material to gather central IHC data for this discordant subgroup of patients. It is possible that these represent false-positive calls by local IHC that might have been discovered if central IHC had been performed on these tumors.

Multivariate Cox proportional hazards analyses involving the entire population as well as the central FISH-positive subgroup demonstrated that quantitative HER2 measurements outperformed FISH as a correlate of TTP and OS.

Although existing data tell us that patients who are gene-amplified for HER2 as measured by FISH experience better outcomes than patients who are not gene-amplified when considered as a group, our results suggest that not all patients who are gene-amplified for HER2 necessarily express high levels of the HER2 protein. Beyond gene dosage effects attributable to HER2 gene amplification, HER2 protein levels critically depend on multiple regulatory networks involving transcription factors, post-transcriptional and translational regulators (eg, microRNAs), and post-translational mediators of protein stability or degradation such as the chaperone HSP90 and the ubiquitin ligase CHIP, respectively. Thus, upstream measurements may be less reliable as indicators of response to drugs that target the protein than measurements made at the protein level. Indeed, there appear to be at least 20 predicted microRNA binding sites in the human HER2 mRNA (see http://microrna.sanger.ac.uk/cgibin/targets/v5/detail_view.pl?transcript_id=ENST00000269571), suggesting the possibility that HER2 gene expression may be regulated post-transcriptionally.

Identification of a subset of FISH-positive patients with low HER2 expression who experience outcomes that are similar to FISH-negative patients may provide at least a partial explanation for the heterogeneity of clinical outcomes that are observed for HER2-positive patients treated with trastuzumab. Other protein biomarkers (eg, HER3 and p9524) may also allow the identification of patients who experience suboptimal outcomes on trastuzumab, and studies examining the relationship between several of these and clinical outcomes after trastuzumab treatment in the MBC setting are currently in progress.

Limitations of this study include its small size, its retrospective nature, the lack of a trastuzumab-untreated control arm, the lack of central IHC measurements for all patients to confirm that some of the cases included were not HER2 IHC false positives, and the heterogeneous chemotherapy to which patients were exposed and that could potentially confound these analyses. All of these elements demand that we consider these results preliminary and hypothesis-generating until they can be confirmed in additional, larger, well-controlled studies involving patients with breast cancer. It is not unexpected that we should observe significant associations between HER2 total expression and TTP in this cohort, because the cutoffs used to define high and low HER2 total expression were derived from this cohort. As such, this cohort represents a training dataset, and the observed relationships and derived cutoffs require validation in an independent cohort of trastuzumab-treated MBC patients before we can say with confidence that these measurements are clinically useful. Confirmation of these results in larger clinical trials will be necessary to use HERmark to optimize the treatment of HER2-positive MBC patients. That said, this is the first dataset to describe such relationships using this new technology, and the results are consistent with the view that measurements made at the protein level, where the drug is targeted, may offer advantages over measurements made further upstream at the level of the gene.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES

We thank the investigators who contributed samples and data to this study; Yolanda Lie, Ahmed Chenna, Ellen Paxinos, and the staff of the oncology clinical reference laboratory at Monogram Biosciences; and the patients who participated in this clinical study.

CONFLICT OF INTEREST DISCLOSURES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES

Supported by grants from Komen for the Cure (BCTR0707722), Medical-Scientific Fund of the Mayor of Vienna (MWFB 47-07), and Monogram Biosciences. Dr. Köstler received research funding from Monogram Biosciences. Drs. Weidler, Sperinde, Jin, Tan, Winslow, Goodman, Larson, Whitcomb, Sherwood, Bates, and Paquet are employees of Monogram Biosciences. Dr. Lipton received a research grant from and is a member of the Advisory Board for Monogram Biosciences. Dr. Leitzel received a research grant and honoraria from Monogram Biosciences.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES
  • 1
    Cobleigh MA, Vogel CL, Tripathy D, et al. Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. J Clin Oncol. 1999; 17: 2639-2648.
  • 2
    Piccart-Gebhart MJ, Procter M, Leyland-Jones B, et al. Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med. 2005; 353: 1659-1672.
  • 3
    Romond EH, Perez EA, Bryant J, et al. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med. 2005; 353: 1673-1684.
  • 4
    Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med. 2001; 344: 783-792.
  • 5
    Vogel CL, Cobleigh MA, Tripathy D, et al. Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol. 2002; 20: 719-726.
  • 6
    Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol. 2001; 2: 127-137.
  • 7
    Slamon DJ. Studies of the HER-2/neu proto-oncogene in human breast cancer. Cancer Invest. 1990; 8: 253.
  • 8
    Slamon DJ, Clark GM. Amplification of c-erbB-2 and aggressive human breast tumors? Science. 1988; 240: 1795-1798.
  • 9
    Slamon DJ, Clark GM, Wong SG, et al. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science. 1987; 235: 177-182.
  • 10
    Slamon DJ, Godolphin W, Jones LA, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science. 1989; 244: 707-712.
  • 11
    Dressler LG, Berry DA, Broadwater G, et al. Comparison of HER2 status by fluorescence in situ hybridization and immunohistochemistry to predict benefit from dose escalation of adjuvant doxorubicin-based therapy in node-positive breast cancer patients. J Clin Oncol. 2005; 23: 4287-4297.
  • 12
    Pauletti G, Dandekar S, Rong H, et al. Assessment of methods for tissue-based detection of the HER-2/neu alteration in human breast cancer: a direct comparison of fluorescence in situ hybridization and immunohistochemistry. J Clin Oncol. 2000; 18: 3651-3664.
  • 13
    Perez EA, Suman VJ, Davidson NE, et al. HER2 testing by local, central, and reference laboratories in specimens from the North Central Cancer Treatment Group N9831 intergroup adjuvant trial. J Clin Oncol. 2006; 24: 3032-3038.
  • 14
    Press MF, Sauter G, Bernstein L, et al. Diagnostic evaluation of HER-2 as a molecular target: an assessment of accuracy and reproducibility of laboratory testing in large, prospective, randomized clinical trials. Clin Cancer Res. 2005; 11: 6598-6607.
  • 15
    Press MF, Slamon DJ, Flom KJ, et al. Evaluation of HER-2/neu gene amplification and overexpression: comparison of frequently used assay methods in a molecularly characterized cohort of breast cancer specimens. J Clin Oncol. 2002; 20: 3095-3105.
  • 16
    Wolff AC, Hammond ME, Schwartz JN, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol. 2007; 25: 118-145.
  • 17
    Paik S, Kim C, Wolmark N. HER2 status and benefit from adjuvant trastuzumab in breast cancer. N Engl J Med. 2008; 358: 1409-1411.
  • 18
    Tuma RS. Inconsistency of HER2 test raises questions. J Natl Cancer Inst. 2007; 99: 1064-1065.
  • 19
    Singer C, Kostler W, Hudelist G. Predicting the efficacy of trastuzumab-based therapy in breast cancer: current standards and future strategies. Biochim Biophys Acta. 2008; 1786: 105-113.
  • 20
    Kostler WJ, Schwab B, Singer CF, et al. Monitoring of serum Her-2/neu predicts response and progression-free survival to trastuzumab-based treatment in patients with metastatic breast cancer. Clin Cancer Res. 2004; 10: 1618-1624.
  • 21
    McShane LM, Altman DG, Sauerbrei W, et al. Reporting recommendations for tumor marker prognostic studies. J Natl Cancer Inst. 2005; 97: 1180-1184.
  • 22
    Shi Y, Huang W, Tan Y, et al. 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. Diagn Mol Pathol. 2009; 18: 11-21.
  • 23
    Larson J, Goodman L, Tan Y, et al. Analytical validation of a highly quantitative, sensitive, accurate, and reproducible assay (HERmark™) for the measurement of HER2 total protein and HER2 homodimers in FFPE breast cancer tumor specimens. Pathol Res Int.In press.
  • 24
    Sperinde J, Jin X, Banerjee J, et al. Quantitation of p95HER2 in paraffin sections using a p95-specific antibody and correlation with outcome in a cohort of trastuzumab-treated breast cancer patients. Clin Cancer Res. In press.