Comparison of HER2 Status by Fluorescence in Situ Hybridization and Immunohistochemistry to Predict Benefit From Dose Escalation of Adjuvant
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《临床肿瘤学》
the Lineberger Comprehensive Cancer Center
Department of Medicine
Department of Pathology, University of North Carolina at Chapel Hill, Chapel Hill, NC
The University of Texas M.D. Anderson Cancer Center, Houston, TX
Cancer and Leukemia Group B (CALGB) Statistical Center, Duke University School of Medicine, Durham, NC
University of Kansas Medical Center, Kansas City, KS
Department of Pathology, Roswell Park Cancer Institute, Buffalo, NY
University of California at San Francisco, San Francisco, CA
Genome Institute of Singapore, Singapore
Department of Pathology, University of Oklahoma, Oklahoma City, OK
North Shore Long Island Jewish Health System, Manhasset, NY
University of Vermont, Burlington, VT
Memorial Sloan-Kettering Cancer Center, New York, NY
University of Michigan, Ann Arbor, MI
ABSTRACT
PURPOSE: HER2 is a clinically important tumor marker in breast cancer; however, there is controversy regarding which method reliably measures HER2 status. We compared three HER2 laboratory methods: immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR), to predict disease-free survival (DFS) and overall survival (OS) after adjuvant doxorubicin-based therapy in node-positive breast cancer patients.
METHODS: This is a Cancer and Leukemia Group B (CALGB) study, using 524 tumor blocks collected from breast cancer patients registered to clinical trial CALGB 8541. IHC employed CB11 and AO-11-854 monoclonal antibodies; FISH used PathVysion HER2 DNA Probe kit; PCR utilized differential PCR (D-PCR) methodology.
RESULTS: Cases HER2 positive by IHC, FISH and D-PCR were 24%, 17%, and 18%, respectively. FISH and IHC were clearly related ( = 64.8%). All three methods demonstrated a similar relationship for DFS and OS. By any method, for patients with HER2-negative tumors, there was little or no effect of dose of adjuvant doxorubicin-based therapy. For patients with HER2-positive tumors, all three methods predicted a benefit from dose-intense (high-dose) compared with low- or moderate-dose adjuvant doxorubicin-based therapy.
CONCLUSION: FISH is a reliable method to predict clinical outcome following adjuvant doxorubicin-based therapy for stage II breast cancer patients. There is a moderate level of concordance among the three methods (IHC, FISH, PCR). None of the methods is clearly superior. Although IHC-positive/FISH-positive tumors yielded the greatest interaction with dose of therapy in predicting outcome, no combination of assays tested was statistically superior.
INTRODUCTION
For more than 20 years, the only tumor markers used routinely in making treatment decisions in breast cancer have been the estrogen-receptor (ER) and progesterone-receptor (PR) status to predict response to hormone therapy. In recent years numerous markers have been evaluated for their ability to predict outcome following chemotherapy; however, none have been accepted for routine clinical use. HER2 is the first tumor marker to be significantly associated with outcome after adjuvant doxorubicin chemotherapy in node-positive breast cancer. Results from three large randomized clinical trials suggest that abnormal HER2, (amplification of the HER2 gene or overexpression of the HER2 protein), is associated with benefit from doxorubicin-based adjuvant chemotherapy.1-6
In addition to its putative role as a predictive factor following doxorubicin-based adjuvant therapy, HER2 status is clinically important for determining eligibility for HER2-directed immunotherapy (trastuzumab7,8). Currently, there is controversy regarding the best laboratory method to determine HER2 status in breast cancer.9-14 It is not certain whether different methods differ in their ability to predict outcome or response to systemic therapies associated with HER2.15 In the current study, we have evaluated the ability of an amplification method, fluorescence in situ hybridization (FISH), to determine HER2 status and its interaction with dose and schedule of doxorubicin-based therapy in a Cancer and Leukemia Group B (CALGB) breast cancer clinical trial. We have compared results of HER2 amplification by FISH with amplification by polymerase chain reaction (PCR) and overexpression by immunohistochemistry (IHC).
In 1985, the CALGB began a clinical trial in node-positive breast cancer patients (CALGB 8541) with three treatment arms evaluating different dose and scheduling of cyclophosphamide, doxorubicin, and fluorouracil (CAF): arm 1 (low dose), 300/30/300 mg/m2, respectively, Q 28 x 4 cycles; arm 2 (moderate dose), 400/40/400 mg/m2, respectively, Q 28 x 6 cycles; and arm 3, 600/60/600 mg/m2, respectively, Q 28 x 4 cycles, which was considered high dose at the time of the study and represented dose intensification.1,16,17 Although arm 3 is currently considered the standard dose for CAF, for the analysis in this manuscript we will refer to the three arms as they were intended at the time of the study, (ie, low, moderate, and high, respectively). The clinical trial accrued 1,549 patients between January 1985 and May 1991. Results from the clinical trial demonstrated that the high-dose arm compared favorably with the low-dose arm for both disease-free survival (DFS) and overall survival (OS) and although the overall results for high-dose were better than for moderate dose, this advantage was not statistically significant: "At a median follow-up of 9 years, DFS and OS for patients on the moderate- and high-dose arms are superior to the corresponding survival measures for patients on the low-dose arm (two-sided P < .0001 and two-sided P = .004, respectively), with no difference in DFS or OS between the moderate- and the high-dose arms".17
A laboratory companion study to the clinical trial was begun in 1988 to evaluate the prognostic value of a variety of tumor markers that could be measured in paraffin blocks of the primary tumors. These markers included DNA ploidy and S-phase fraction by flow cytometry, p53 and HER2 protein overexpression by IHC and, HER2 gene amplification by PCR. The initial report describing HER2 status and outcome included the initial 397 patients registered to the clinical trial1 and was the first report to indicate that node-positive breast cancer patients whose tumors have overexpressed or amplified HER2 benefit from higher doses of CAF while those with normal HER2 do not benefit from increasing dose.1 Following this observation, the laboratory companion study was expanded and tumor specimens from all patients who were registered to the complete clinical trial were solicited, resulting in 1,074 blocks. Analysis of HER2 status by IHC in 992 patients (the total number of blocks containing sufficient invasive breast cancer), showed a statistically significant interaction between HER2 and dose of CAF.2 A subset of 894 patients for whom HER2 amplification was assessed by differential PCR (D-PCR) also demonstrated the same significant interaction.2
In the current study we have evaluated amplification of the HER2 gene by FISH in a subset of the 992 cases previously studied by Thor et al.2 FISH methodology bridges the two techniques, IHC and D-PCR, allowing quantification of gene amplification while preserving tissue morphology in a slide-based tissue-assay. To our knowledge, this is the first report comparing these methods to predict clinical outcome to doxorubicin therapy in the setting of a randomized clinical trial.
METHODS
Patient Population and Tissue Specimens
The patient population consisted of node-positive breast cancer patients who were registered to the CALGB 8541 clinical trial and the laboratory companion study CALGB 8869. As part of the companion study, which began in 1988, representative formalin-fixed paraffin-embedded tissue blocks from the primary tumor were collected retrospectively from CALGB institutions that had registered patients to the original clinical trial. The companion study received institutional review board (IRB) approval at all institutions that submitted blocks (Appendix). Blocks were obtained from 992 patients and housed at the CALGB Pathology Coordinating Office (PCO) tissue bank. Sections from these blocks were previously assessed for a variety of markers including HER2 overexpression by IHC and amplification by D-PCR, p53 expression by IHC, and DNA ploidy and S-phase fraction by flow cytometry.1,2,18 For the current study, we randomly selected 750 cases from the total group of 992. On inspection of the block, 47 cases had minimal tissue remaining in the block and because CALGB tissue bank policy prohibits exhausting tissue, these blocks were not sectioned. Therefore, 703 blocks were freshly sectioned for this study. The current study, performed at the University of North Carolina (UNC) Linberger Comprehensive Cancer Center and part of a U01 grant, was approved by the UNC IRB.
Tissue Quality Control
All tissue sections used for this study were from formalin-fixed paraffin-embedded tumor blocks and showed adequate fixation. All sections used for FISH assay were obtained from the same block of tumor previously used for HER2, IHC, and PCR assays. Only those sections that had sufficient and representative invasive cancer were eligible for the current FISH study. Five to 10 4-μm serial sections were prepared fresh from the banked paraffin block at the CALGB PCO, placed on Fisher superfrost charged slides (Fisher Scientific; Pittsburgh, PA) and stored at 4°C. The first and last sections were stained by hematoxylin and eosin and reviewed by the study pathologist (D.N.) to determine whether sufficient invasive cancer nuclei were present on all newly cut sections. Five hundred sixty-nine cases met this criterion. Reasons for exclusion by pathology review included the following: only ductal carcinoma-in-situ remained on section (n = 10); insufficient quantity of invasive cancer present for reliable assay (n = 53); positive node, not primary breast cancer (n = 37); and no cancer remaining on section (n = 34). All FISH assays were performed within 4 weeks of sectioning.
FISH Assay
FISH was performed using the PathVysion HER-2 DNA FISH Kit, (Vysis Inc, Downers Grove, IL) according to the manufacturer's instructions. The kit incorporates two directly labeled fluorescence DNA probes: an orange probe directed against the HER2 gene (HER2, 17q11.2-q12) and a green probe directed against the pericentromeric region of chromosome 17, (CEP 17, 17p11.1-q11.1), the chromosome in which the HER2 gene resides. To breakdown formaldehyde cross-links, acid pretreatment and protease digestion were performed (Vysis paraffin pretreatment kit; Vysis Inc), followed by standard saline citrate (SSC) and formamide denaturation (72°C, 5 minutes). After dehydration, the HER2/CEP 17 probe cocktail was added, and coverslips were applied and sealed with rubber cement. Slides were incubated in a humidity chamber overnight for 18 hours at 37°C. On the following day, slides were washed in a stringency buffer (SSC, NP40), air-dried in the dark and incubated with 4,6 diamidino-2-phenylindole (DAPI) for nuclear identification. Slides were stored in the dark, at –20°C. Nonamplified and amplified control slides (MDA-MB-231 and Hs578T cell lines, respectively, fixed and embedded in paraffin; provided in the PathVysion kit; Abbott Labs, Vysis Inc) were analyzed with each assay. All assays were performed in one laboratory (UNC). Slides were scored within 1 week of FISH assay (most within 48 hours) with a Zeiss Axioplan Epifluorescence microscope (Carl Zeiss Inc, Thornwood, NY), equipped with a 100-watt mercury arc lamp, using a multifilter cube supplied by Vysis (DAPI single color bp; DAPI/9-orange [NB] dual bp; DAPI/green dual bp; DAPI/green/orange triple bp). To identify malignant cells for scoring, the underside of the slide was previously marked by the study pathologist with an indelible pen that can be distinguished under fluorescence microscopy. For each case, 60 nonoverlapping invasive cancer nuclei were scored for both CEP17 (green) and HER2 (orange) signals. In pilot studies, using non-CALGB breast cancer specimens, no difference in the average HER2:CEP17 ratio was observed when ratios were compared after counting 200, 120, or 60 nuclei per case. Therefore, 60 nuclei per case was selected for this study. Only cancer nuclei showing at least one orange and one green signal were counted. The 60 invasive cancer nuclei were selected from two-to-three different areas of the section to ensure representation of counts. When nuclei showed more than 10 signals, it was sometimes difficult to distinguish individual signals and therefore the raw count was categorized as 11 to 15, 16 to 20, or > 20. Raw counts (number of orange and green signals per nuclei; 60 nuclei per case) were submitted to the CALGB statistical center. The ratio of orange (HER2 gene) to green (chromosome 17 centromere) signal was calculated to normalize any increase in HER2 copy number due to polyploidy of chromosome 17. A case was considered amplified if the HER2:CEP17 ratio was 2.00. To allow scoring to be performed within 1 week of assay, cases were scored by two sites: UNC (scored 80% of cases) and University of Kansas (scored 20% of cases). Each technician followed the same procedure for scoring and used the same scoring criteria. All technicians and investigators were blinded to treatment assignment and clinical outcome. Before initiation of the FISH study, the two sites received identical training in scoring. Following training, a pilot study was performed in which both sites scored 20 non-CALGB breast cancer specimens. The specimens had a known range of amplification and had been previously assessed by D-PCR at the UNC Molecular Core lab. Results of the pilot study demonstrated perfect agreement in assigning a case as amplified or not amplified, with similar values for HER2:CEP17 ratios. To further address issues of inter-laboratory scoring variability, a blinded comparison was performed, in which 38 CALGB cases were scored by both labs. Similar to the pilot, there was perfect agreement in HER2 amplification status. To address assay reproducibility in the current study, an additional 22 CALGB cases were assayed twice on separate days. The agreement in HER2:CEP17 ratio was high (cc = +0.93). Independent studies have also reported on the reproducibility and portability of this FISH assay.19,20
IHC and D-PCR Assays
Results of other assays previously performed on the same cases were available in the CALGB database and were used for comparative analyses with FISH assays. Details of methods for measuring protein overexpression by IHC and amplification by D-PCR have been previously reported.1,2,21,22 In brief, IHC evaluations used two antibodies (CB11 and A0-11-854), directed against the internal domain of the HER2 protein. Prior studies have demonstrated excellent correlation between the two antibodies.2 The comparisons performed in the current study involved 346 cases stained with CB11 and 177 cases stained with A0-11-854. Cases were scored as overexpressing the HER2 protein if any membranous staining was found in malignant cells at a magnification of 100x. As described in the following statistical section, a cut point of 50% was used for some analyses. For D-PCR, DNA was extracted from 10-μm sections cut from the same block used for IHC and FISH. Stringent criteria was used to define a case as amplified: unequivocal amplification of two reference genes (as an assay control) and unequivocal HER2 gene amplification relative to a normal spleen control and an amplified standard (SKBR3 cell line).22
Statistical Analyses
Power calculations determined that we needed approximately 500 cases with assessable FISH assays. To arrive at that number, we estimated that approximately 30% of cases selected would not yield informative assays, mostly due to concerns that blocks on file would have insufficient tissue remaining for FISH assays (since these blocks had previously been sectioned for several assays) and to a lesser degree to account for assay failures. Therefore 750 cases were originally selected randomly from the total group of 992. Characteristics of patients included in the current FISH study were compared with those of patients not included in the study: age, menopausal status, tumor size, number of positive lymph nodes, ER status, PR status, proportion of patients who received tamoxifen, S-phase fraction, proportion of cells staining positive for p53 and HER2 by IHC, and assessment of HER2 gene amplification by D-PCR. 2 tests were carried out for dichotomous variables and t-tests were used to compare continuous variables. We also compared these same characteristics across the three dose schedules for patients in the present study, using 2 tests for dichotomous variables and F tests for continuous variables.
Correlations were calculated between assessment of HER2 by FISH and various clinical and tumor characteristics, including assessment of HER2 using other methods (IHC and D-PCR). 2 tests were carried out for dichotomous variables and t-tests were used to compare continuous variables. For tests of dichotomous variables the cutpoints for FISH and IHC were as follows: amplification by FISH used HER2:CEP17 ratio, where ratios 2 were considered positive; and overexpression by IHC, where cases with 50% invasive cells staining were considered positive.1,2 All analyses presented for D-PCR were dichotomized as amplified (positive [+]) or not amplified (negative [–]) based on the definition in the Methods section.
Comparisons were made based on overall agreement among the three measures of HER2. statistics (with their SEs) were used to quantify level of agreement.23 The Spearman rank correlation was applied to comparisons of continuous data between IHC and FISH methods.
The interaction between HER2 and dose was evaluated in predicting DFS and OS. This interaction relates to the therapeutic importance of HER2. The principal analysis of the interaction between dose and HER2 was multivariate proportional hazards modeling with 2 tests, as previously described.1,2,24 As in our previous reports1,2 the covariates considered were square root of the number of positive axillary lymph nodes (SQRT_NPN), tumor size (TSIZE = 2 for > 2 cm and TSIZE = 1 otherwise), menopausal status (PREMENO = 1 for premenopausal and PREMENO = 0 otherwise), dose of CAF (high v low and moderate v low), HER2 status (positive v negative), and interaction of HER2 with dose of CAF. For comparison, the three different methods for assessing HER2 (IHC, FISH, and D-PCR) were used, one set of models for each technique. The actual value of HER2:CEP17 ratio for FISH and the percentage of cells staining positive by IHC were used, whereas gene amplification by D-PCR was treated as a dichotomous variable, (amplified v not amplified). The most important question in these proportional hazards models was the existence of the "CAF by HER2" interaction. The strength of this interaction may suggest that some patients should be treated differently from other patients. Multivariate analyses were illustrated with Kaplan-Meier DFS and OS curves for the various CAF/HER2 groups.1,2,25
We also performed hazard ratio analysis for DFS for patients receiving high-dose CAF versus patients receiving moderate or low dose for each paired combination of methods (eg, IHC+/FISH+, IHC+/FISH–, IHC+/PCR–, and so on).
RESULTS
FISH Assessable Cases
Of the 569 cases assayed by FISH, 524 (92%) had assessable results. Technical reasons for unassessable cases included the following: 41 cases (7%) did not show appropriate hybridization signals even after repeat assay (ie, no orange [HER2] or green [CEP 17] signal detected or only one signal [orange or green] detected); in two cases (0.3%), tissue fell off the slide; and in three cases (0.5%), fewer than 60 nuclei showed both hybridization signals.
HER2 Status by Method
Assessable data were obtained for FISH and IHC assays from 524 and 523 cases, respectively. D-PCR results were obtained from 491 of these cases. HER2 protein, evaluated by IHC, was overexpressed in 24% of cases (127 of 523). HER2 gene copy, determined by FISH, was amplified in 17% of cases (90 of 524); and amplification by D-PCR was observed in 18% of cases (91 of 491).
Tables 1 through 3 show the 2 x 2 contingency tables comparing FISH, IHC, and PCR. The statistic, applied to dichotomous data, demonstrated a moderate level of agreement among the methods: 74% agreement between the amplification methods FISH and PCR; 65% agreement between IHC and FISH; and 53% agreement between IHC and PCR. We observed a high concordance rate, 92.3%, between the two measures of amplification (Table 1, FISH compared with PCR; n = 491). Thirty-eight cases (7.7%) were discordant in the FISH/PCR comparison. The concordance rate for FISH compared with IHC was 88.3% and for IHC compared with PCR was 83.9%. In the IHC/PCR pairing (n = 491) discordance was observed in 79 (16.1%) cases.
the statistic FISH and IHC are clearly related; however, we observed discordance in 61 cases (11.6%; Table 2). All 12 FISH+ cases were also positive by D-PCR. For this analysis, IHC+ cases were defined as those having 50% invasive cancer cells staining. Compared with other studies in the literature, many of which use 10% as a cut point for IHC positivity, most of the FISH+/IHC– cases might not be considered discordant (eg, only three cases showed < 10% cells staining in this group). Of the 49 FISH–/IHC+ cases, 42 cases were also PCR–, supporting a true lack of amplication. The small number of discordant cases overall precluded any definitive conclusions regarding the superiority of any one method to predict outcome.
We also evaluated the relationship between level of amplification by FISH and level of overexpression by IHC and found that tumors with a high percentage of HER2-positive (HER2+) cells by IHC were more likely to have a high FISH amplification ratio (r = 0.46; P < .001; data not shown). Nearly every case with more than 80% cells positive by IHC was amplified by FISH.
Clinical Correlations
Table 4 compares patient characteristics of cases included in the present study (n = 524) versus the remaining patients in CALGB 8541 (n = 469). Patient age; menopausal status; hormone-receptor status; HER2 status by IHC; tumor size; number of positive nodes; and percentage of patients receiving low (n = 178), moderate (n = 167), or dose-escalated (n = 179) CAF were similar between the current study of 524 patients and the remaining 469 patients not eligible or not assayed by FISH. However, we did observe differences in two parameters in the current study population compared with those patients not included. In the current study, a higher proportion of patients received tamoxifen (38.2% v 30.5%; P = .012). This was the result of a change in management of breast cancer patients in 1988 when the National Cancer Institute (NCI) recommended that postmenopausal ER-positive (ER+) breast cancer patients be considered for tamoxifen therapy. (The majority of patients in the current study entered the trial after 1988). In addition, the median S-phase fraction was higher in the current study (14% v 10%; P = .0001). Median S-phase values from the current study and those not included, however, are similarly categorized as representing a high proliferative capacity.
Comparison of FISH, IHC, and PCR to Predict Outcome After Dose-Intensive CAF Therapy
We considered proportional hazards models for DFS and OS using the three different HER2 methods (FISH, IHC, and PCR). For these models, we considered continuous measurement of FISH and IHC, whereas gene amplification by D-PCR was entered as a dichotomous variable (amplified v nonamplified). The covariates used are the same as those in our previous publications.1,2 Results of the multivariate analyses are summarized in Table 5 for DFS. Similar results were obtained for OS (data not shown). Of particular interest is the therapeutic implication of HER2 status: the interaction between HER2 status and dose schedule of CAF by likelihood ratio test was statistically significant for all three methods of HER2 assessment (FISH, P = .033; IHC, P = .0003; PCR, P = .043).
Hazard ratios for the multivariate Cox proportional hazards model for prediction of DFS are presented in Table 6. The hazard ratios for high-dose CAF versus moderate or low dose are 0.822, 0.834, and 0.732 for FISH, IHC, and PCR, respectively. This indicates that the high-dose arm has better DFS than the moderate and low-dose arms combined. The numerical values of the hazard ratios for the interaction of high-dose CAF compared to low and moderate dose with HER2 status are 0.919, 0.418, and 0.585, for FISH, IHC, and PCR, respectively. Each of the three methods for measuring HER2 indicates that patients receiving high-dose CAF tend to have better disease-free survival in the subset of HER2-positive cases, but no differences are apparent in HER2-negative cases. Hazard ratios of interaction terms are difficult to interpret. The inferential strength is provided by the overall interaction P values in Table 5. In addition, quantifications of the interactions are shown graphically using Kaplan-Meier survival curves in Figure 1.
The hazard ratio for DFS for those patients receiving high-dose CAF compared with patients receiving moderate or low-dose for the various pairs of methods are reported in Table 7. Although the categories of IHC+/FISH+ and IHC+/PCR+ show higher hazards ratios compared to the other combinations, as judged by the 95% CI, no combination of methods tested was found to be statistically superior.
Kaplan-Meier curves for DFS by HER2 status and CAF dose are shown for each of the three methods in Figure 1. By any method of assessment, for patients whose tumors are HER2 negative (HER2–), there was little or no effect of dose of CAF. Five-year DFS probabilities for patients with FISH-negative (FISH–) tumors are 60%, 64%, and 68% for low, moderate, and high (dose-escalated) doses, respectively. Similarly, patients with IHC– tumors had comparable DFS probabilities: 59%, 65%, and 64%, for low, moderate, and high doses, respectively. For patients whose tumors were HER2+, all three methods showed a benefit from dose-intense CAF compared with low or moderate doses. In the HER2+ subset, there is a significant difference between high versus low and high versus moderate CAF doses, whereas there is no difference between low and moderate doses. Patients with FISH+ tumors had a 5-year DFS of 43%, 42%, and 77% in the low, moderate, and high-dose groups, respectively. The corresponding 5-year DFS estimates for patients with IHC+ tumors were 53%, 47%, and 84%.
Kaplan-Meier plots for overall survival by HER2 status by each method are provided in Figure 2. There was no apparent difference between the three CAF doses for patients with HER2– tumors. However, similar to DFS curves, all three methods indicate a benefit with high-dose (dose-intense) compared with low- or moderate- dose doxorubicin-based therapy for patients with HER2+ tumors.
DISCUSSION
HER2 is a clinically important tumor marker with the potential to influence treatment decisions in the breast cancer patient, a role among tumor markers that previously has been achieved only with hormone receptors. Abnormal HER2 (gene amplification or protein overexpression) is also important in selecting metastatic breast cancer patients who are candidates for anti-HER2 therapy with trastuzumab.7,8,26,27 In the adjuvant setting,1-3,5,28 and recently in the neoadjuvant setting,29 several studies have suggested that HER2 status may be useful in determining a patient's benefit from doxorubicin-based chemotherapy. In prior CALGB reports,1,2 we observed a significant interaction between HER2 status and CAF dose in predicting outcome. The relative benefit of high-dose CAF was greater in patients with HER2+ (amplified by PCR or overexpressed by CB11 IHC) compared with HER2– (not amplified or overexpressed) tumors. This benefit however, was revealed only when patients were stratified by HER2 status. Looking at 9-year median follow-up results from the clinical trial, which evaluated all patients but ignored HER2 status, showed no difference in DFS or OS between the moderate- and high-dose arms.17
The National Surgical Adjuvant Breast and Bowel Project (NSABP), assessed HER2 status by IHC using a cocktail of two antibodies, mab-1 and pab-1, in 637 node-positive, ER-negative (ER–) breast cancer patients who were randomly assigned to melphalan and fluorouracil (FU) versus melphalan, FU, and doxorubicin.3 Patients with HER2+ tumors benefited from doxorubicin while those with HER2– tumors did not. In a Southwest Oncology Group study (SWOG 8814), HER2 status was determined by a third IHC system (antibody TAB250) in 595 postmenopausal, ER+, axillary node-positive patients who were randomly assigned to tamoxifen alone or tamoxifen plus CAF.5 Preliminary analyses of this study suggest that CAF offered a substantial advantage for HER2+ patients but little, if any, advantage for HER2– patients.5
Today, although HER2 is considered a clinically useful marker, there is no consensus regarding which method(s) should be used for clinical decision making. Optimally, the method should be reproducible and reliable, and validated for its clinical and biologic accuracy.9,30 Few methods meet these criteria. In the present study we have used clinical outcome to compare three different methods to measure HER2 status: HER2 gene amplification was measured by D-PCR and FISH using the Pathvysion FISH kit; protein overexpresssion was measured by IHC using CB-11 and AO-11-854 antibodies. We evaluated the relationship of each of these method systems to predict DFS and OS in breast cancer patients following treatment with CAF. We found that the three methods used in this study are not identical in assignment of HER2 status, nor are they identical in predicting outcome following CAF therapy. However, there is concordance among PCR, FISH, and IHC in determining HER2 status, and all three methods predict outcome following dose-intense CAF. Though it is tempting, from the multivariate analysis, to assign a greater predictive value for one method versus another in relation to interaction with dose, none of the methods is more clearly predictive than the others.
When making individual patient care decisions, one test method does not always provide all the necessary information. For example, knowing IHC, does FISH give us any further information Does the answer depend on whether IHC was positive or negative From the present study we may be misclassifying up to 12% of patients depending on the method selected (eg, IHC by CB11 v Vysis FISH). From our hazard ratio analysis for DFS of the different paired combination of methods (eg, IHC+/FISH– or FISH+/IHC–), many of the subgroups were small, and we could not identify a paired combination that was clearly superior in predicting outcome.
Much attention has focused recently on the use of FISH as an adjunct to the IHC assay to confirm ambiguous IHC results or as a substitute for IHC assays.10-12,14,31-34 The present study has demonstrated that FISH is a reliable method to predict clinical outcome; however, similar to IHC methods, not all FISH methods are alike. For example, among the two United States Food and Drug Administration–approved HER2 FISH kits, the criteria for defining a case as amplified differs. The Vysis Pathvysion kit used in this study incorporates two probes, the HER2 gene probe and a chromosome 17 centromeric probe to exclude polyploidy of chromosome 17 as a source of increased copy number of HER2. For a case to be considered amplified by this kit, the ratio of the HER2 copy number to chromosome 17 copies must be greater than or equal to 2.00, similar to the definition of amplification used in classical cytogenetics. The Ventana/Oncor FISH kit (Ventana; Tucson, AZ) uses a single probe system, where copy number of the HER2 gene alone is evaluated. For a case to be amplified, an average of 4 or more copies of HER2 must be observed in cancer nuclei. Clinically, it is uncertain whether an increase in HER2 gene copy number alone, or an increase of HER2 gene copies relative to copies of chromosome 17 is more useful in predicting outcome. Therefore, it was important, in the current study, to consider the influence of measuring chromosome 17 in the FISH assay. Chromosome 17 polyploidy, defined in this study as an average of three or more CEP17 signals in cancer nuclei, was observed in 8.5% or 45 of 523 cases. Due to the low rate of chromosome 17 polyploidy in this study, the use of the chromosome 17 probe to correct for HER2 amplification accuracy was not a major determinant of HER2 status or predicting outcome following treatment in this population. Kaplan-Meier curves were similar (data not shown) regardless of whether or not amplification was defined relative to chromosome 17. Use of the chromosome 17 probe in the clinical laboratory is still warranted, however, as an internal assay control for hybridization and to correct for differences in signals resulting from sectioning through nuclei. In other patient populations (eg, patients with metastatic breast cancer), which may have a higher rate overall of chromosome polyploidy, the use of CEP17 probe may prove to be more clinically useful.
In summary, the three HER2 methods tested, IHC by CB11, FISH by PathVysion, and PCR by D-PCR are similar but not identical in assigning a tumor the same HER2 status. All three methods showed a significant interaction between HER2 status and dose of CAF. All three methods result in similar but not identical Kaplan-Meier plots for disease-free survival and overall survival. Current data from this study do not support that one method or a paired combination of methods is statistically superior to the other in predicting clinical outcome following adjuvant doxorubicin therapy.
Appendix
The following institutions participated in the study: CALGB Statistical Office, Durham, NC: Stephen George, PhD, supported by CA33601; Christiana Care Health Services, Inc CCOP, Wilmington, DE: Irving M. Berkowitz, DO, supported by CA45418; Community Hospital-Syracuse CCOP, Syracuse, NY: Jeffrey Kirshner, MD, supported by CA45389; Dana-Farber Cancer Institute, Boston, MA: George P. Canellos, MD, supported by CA32291; Dartmouth Medical School—Norris Cotton Cancer Center, Lebanon, NH: Marc S. Ernstoff, MD, supported by CA04326; Duke University Medical Center, Durham, NC: Jeffrey Crawford, MD, supported by CA47577; Eastern Maine Medical Center CCOP, Bangor, ME: Philip L. Brooks, MD, supported by CA35406; Kaiser Permanente CCOP, San Diego, CA: Jonathan A. Polikoff, MD, supported by CA45374; Massachusetts General Hospital, Boston, MA: Michael L. Grossbard, MD, supported by CA12449; McGill Department of Oncology, Montreal, Quebec, Canada: Brian Leyland-Jones, MD, supported by CA31809; Memorial Sloan-Kettering Cancer Center, New York, NY: George Bosl, MD, supported by CA77651; Mount Sinai Medical Center, Miami, FL: Enrique Davila, MD, supported by CA45564; Mount Sinai School of Medicine, New York, NY: Lewis R. Silverman, MD, supported by CA04457; North Shore—Long Island Jewish Health System, Manhasset, NY: Daniel Budman, MD, supported by CA35279; Rhode Island Hospital, Providence, RI: Louis A. Leone, MD, supported by CA08025; Roswell Park Cancer Institute, Buffalo, NY: Ellis Levine, MD, supported by CA02599; South New Jersey CCOP, Camden, NJ: Jack Goldberg, MD, supported by CA54697; Southeast Cancer Control Consortium Inc CCOP, Goldsboro, NC: James N. Atkins, MD, supported by CA45808; Southern Maine Medical Center, Scarborough, ME: Thomas J. Ervin, MD, supported by CA37447; Southern Nevada Cancer Research Foundation CCOP, Las Vegas, NV: John Ellerton, MD, supported by CA35421; SUNY Maimonides Medical Center, Brooklyn, NY: Samuel Kopel, MD, supported by CA25119; SUNY Upstate Medical University, Syracuse, NY: Stephen L. Graziano, MD, supported by CA21060; University of Alabama, Birmingham, AL: Robert Diasio, MD, supported by CA47545; University of California at San Diego, San Diego, CA: Stephen L. Seagren, MD, supported by CA11789; University of Chicago Medical Center, Chicago, IL: Gini Fleming, MD, supported by CA41287; University of Iowa Hospitals, Iowa City, IA: Gerald Clamon, MD, supported by CA47642; University of Maryland Cancer Center, Baltimore, MD: David Van Echo, MD, supported by CA31983; University of Massachusetts Medical Center, Worcester, MA: F. Marc Stewart, MD, supported by CA37135; University of Missouri/Ellis Fischel Cancer Center, Columbia, MO: Michael C. Perry, MD, supported by CA12046; University of North Carolina at Chapel Hill, Chapel Hill, NC: Thomas C. Shea, MD, supported by CA47559; University of Tennessee Memphis, Memphis, TN: Harvey B. Niell, MD, supported by CA47555; Wake Forest University School of Medicine, Winston-Salem, NC: David D. Hurd, MD, supported by CA03927; Walter Reed Army Medical Center, Washington, DC: John C. Byrd, MD, supported by CA26806; Washington University School of Medicine, St Louis, MO: Nancy Bartlett, MD, supported by CA77440; Weill Medical College of Cornell University, New York, NY: Michael Schuster, MD, supported by CA07968.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
Acknowledgment
We thank the participating CALGB institutions, pathologists, and patients for their help, support, and contribution to this study.
NOTES
Supported in part by grants from the National Cancer Institute (National Institutes of Health, Bethesda, MD; CA31946) to the Cancer and Leukemia Group B (Richard L. Schilsky, MD, Chairman) and by NCI-U01-CA64061-05, the T.J. Martell Foundation for Leukemia, Cancer and AIDS Research, and Vysis Inc, Downers Grove, IL.
The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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Department of Medicine
Department of Pathology, University of North Carolina at Chapel Hill, Chapel Hill, NC
The University of Texas M.D. Anderson Cancer Center, Houston, TX
Cancer and Leukemia Group B (CALGB) Statistical Center, Duke University School of Medicine, Durham, NC
University of Kansas Medical Center, Kansas City, KS
Department of Pathology, Roswell Park Cancer Institute, Buffalo, NY
University of California at San Francisco, San Francisco, CA
Genome Institute of Singapore, Singapore
Department of Pathology, University of Oklahoma, Oklahoma City, OK
North Shore Long Island Jewish Health System, Manhasset, NY
University of Vermont, Burlington, VT
Memorial Sloan-Kettering Cancer Center, New York, NY
University of Michigan, Ann Arbor, MI
ABSTRACT
PURPOSE: HER2 is a clinically important tumor marker in breast cancer; however, there is controversy regarding which method reliably measures HER2 status. We compared three HER2 laboratory methods: immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR), to predict disease-free survival (DFS) and overall survival (OS) after adjuvant doxorubicin-based therapy in node-positive breast cancer patients.
METHODS: This is a Cancer and Leukemia Group B (CALGB) study, using 524 tumor blocks collected from breast cancer patients registered to clinical trial CALGB 8541. IHC employed CB11 and AO-11-854 monoclonal antibodies; FISH used PathVysion HER2 DNA Probe kit; PCR utilized differential PCR (D-PCR) methodology.
RESULTS: Cases HER2 positive by IHC, FISH and D-PCR were 24%, 17%, and 18%, respectively. FISH and IHC were clearly related ( = 64.8%). All three methods demonstrated a similar relationship for DFS and OS. By any method, for patients with HER2-negative tumors, there was little or no effect of dose of adjuvant doxorubicin-based therapy. For patients with HER2-positive tumors, all three methods predicted a benefit from dose-intense (high-dose) compared with low- or moderate-dose adjuvant doxorubicin-based therapy.
CONCLUSION: FISH is a reliable method to predict clinical outcome following adjuvant doxorubicin-based therapy for stage II breast cancer patients. There is a moderate level of concordance among the three methods (IHC, FISH, PCR). None of the methods is clearly superior. Although IHC-positive/FISH-positive tumors yielded the greatest interaction with dose of therapy in predicting outcome, no combination of assays tested was statistically superior.
INTRODUCTION
For more than 20 years, the only tumor markers used routinely in making treatment decisions in breast cancer have been the estrogen-receptor (ER) and progesterone-receptor (PR) status to predict response to hormone therapy. In recent years numerous markers have been evaluated for their ability to predict outcome following chemotherapy; however, none have been accepted for routine clinical use. HER2 is the first tumor marker to be significantly associated with outcome after adjuvant doxorubicin chemotherapy in node-positive breast cancer. Results from three large randomized clinical trials suggest that abnormal HER2, (amplification of the HER2 gene or overexpression of the HER2 protein), is associated with benefit from doxorubicin-based adjuvant chemotherapy.1-6
In addition to its putative role as a predictive factor following doxorubicin-based adjuvant therapy, HER2 status is clinically important for determining eligibility for HER2-directed immunotherapy (trastuzumab7,8). Currently, there is controversy regarding the best laboratory method to determine HER2 status in breast cancer.9-14 It is not certain whether different methods differ in their ability to predict outcome or response to systemic therapies associated with HER2.15 In the current study, we have evaluated the ability of an amplification method, fluorescence in situ hybridization (FISH), to determine HER2 status and its interaction with dose and schedule of doxorubicin-based therapy in a Cancer and Leukemia Group B (CALGB) breast cancer clinical trial. We have compared results of HER2 amplification by FISH with amplification by polymerase chain reaction (PCR) and overexpression by immunohistochemistry (IHC).
In 1985, the CALGB began a clinical trial in node-positive breast cancer patients (CALGB 8541) with three treatment arms evaluating different dose and scheduling of cyclophosphamide, doxorubicin, and fluorouracil (CAF): arm 1 (low dose), 300/30/300 mg/m2, respectively, Q 28 x 4 cycles; arm 2 (moderate dose), 400/40/400 mg/m2, respectively, Q 28 x 6 cycles; and arm 3, 600/60/600 mg/m2, respectively, Q 28 x 4 cycles, which was considered high dose at the time of the study and represented dose intensification.1,16,17 Although arm 3 is currently considered the standard dose for CAF, for the analysis in this manuscript we will refer to the three arms as they were intended at the time of the study, (ie, low, moderate, and high, respectively). The clinical trial accrued 1,549 patients between January 1985 and May 1991. Results from the clinical trial demonstrated that the high-dose arm compared favorably with the low-dose arm for both disease-free survival (DFS) and overall survival (OS) and although the overall results for high-dose were better than for moderate dose, this advantage was not statistically significant: "At a median follow-up of 9 years, DFS and OS for patients on the moderate- and high-dose arms are superior to the corresponding survival measures for patients on the low-dose arm (two-sided P < .0001 and two-sided P = .004, respectively), with no difference in DFS or OS between the moderate- and the high-dose arms".17
A laboratory companion study to the clinical trial was begun in 1988 to evaluate the prognostic value of a variety of tumor markers that could be measured in paraffin blocks of the primary tumors. These markers included DNA ploidy and S-phase fraction by flow cytometry, p53 and HER2 protein overexpression by IHC and, HER2 gene amplification by PCR. The initial report describing HER2 status and outcome included the initial 397 patients registered to the clinical trial1 and was the first report to indicate that node-positive breast cancer patients whose tumors have overexpressed or amplified HER2 benefit from higher doses of CAF while those with normal HER2 do not benefit from increasing dose.1 Following this observation, the laboratory companion study was expanded and tumor specimens from all patients who were registered to the complete clinical trial were solicited, resulting in 1,074 blocks. Analysis of HER2 status by IHC in 992 patients (the total number of blocks containing sufficient invasive breast cancer), showed a statistically significant interaction between HER2 and dose of CAF.2 A subset of 894 patients for whom HER2 amplification was assessed by differential PCR (D-PCR) also demonstrated the same significant interaction.2
In the current study we have evaluated amplification of the HER2 gene by FISH in a subset of the 992 cases previously studied by Thor et al.2 FISH methodology bridges the two techniques, IHC and D-PCR, allowing quantification of gene amplification while preserving tissue morphology in a slide-based tissue-assay. To our knowledge, this is the first report comparing these methods to predict clinical outcome to doxorubicin therapy in the setting of a randomized clinical trial.
METHODS
Patient Population and Tissue Specimens
The patient population consisted of node-positive breast cancer patients who were registered to the CALGB 8541 clinical trial and the laboratory companion study CALGB 8869. As part of the companion study, which began in 1988, representative formalin-fixed paraffin-embedded tissue blocks from the primary tumor were collected retrospectively from CALGB institutions that had registered patients to the original clinical trial. The companion study received institutional review board (IRB) approval at all institutions that submitted blocks (Appendix). Blocks were obtained from 992 patients and housed at the CALGB Pathology Coordinating Office (PCO) tissue bank. Sections from these blocks were previously assessed for a variety of markers including HER2 overexpression by IHC and amplification by D-PCR, p53 expression by IHC, and DNA ploidy and S-phase fraction by flow cytometry.1,2,18 For the current study, we randomly selected 750 cases from the total group of 992. On inspection of the block, 47 cases had minimal tissue remaining in the block and because CALGB tissue bank policy prohibits exhausting tissue, these blocks were not sectioned. Therefore, 703 blocks were freshly sectioned for this study. The current study, performed at the University of North Carolina (UNC) Linberger Comprehensive Cancer Center and part of a U01 grant, was approved by the UNC IRB.
Tissue Quality Control
All tissue sections used for this study were from formalin-fixed paraffin-embedded tumor blocks and showed adequate fixation. All sections used for FISH assay were obtained from the same block of tumor previously used for HER2, IHC, and PCR assays. Only those sections that had sufficient and representative invasive cancer were eligible for the current FISH study. Five to 10 4-μm serial sections were prepared fresh from the banked paraffin block at the CALGB PCO, placed on Fisher superfrost charged slides (Fisher Scientific; Pittsburgh, PA) and stored at 4°C. The first and last sections were stained by hematoxylin and eosin and reviewed by the study pathologist (D.N.) to determine whether sufficient invasive cancer nuclei were present on all newly cut sections. Five hundred sixty-nine cases met this criterion. Reasons for exclusion by pathology review included the following: only ductal carcinoma-in-situ remained on section (n = 10); insufficient quantity of invasive cancer present for reliable assay (n = 53); positive node, not primary breast cancer (n = 37); and no cancer remaining on section (n = 34). All FISH assays were performed within 4 weeks of sectioning.
FISH Assay
FISH was performed using the PathVysion HER-2 DNA FISH Kit, (Vysis Inc, Downers Grove, IL) according to the manufacturer's instructions. The kit incorporates two directly labeled fluorescence DNA probes: an orange probe directed against the HER2 gene (HER2, 17q11.2-q12) and a green probe directed against the pericentromeric region of chromosome 17, (CEP 17, 17p11.1-q11.1), the chromosome in which the HER2 gene resides. To breakdown formaldehyde cross-links, acid pretreatment and protease digestion were performed (Vysis paraffin pretreatment kit; Vysis Inc), followed by standard saline citrate (SSC) and formamide denaturation (72°C, 5 minutes). After dehydration, the HER2/CEP 17 probe cocktail was added, and coverslips were applied and sealed with rubber cement. Slides were incubated in a humidity chamber overnight for 18 hours at 37°C. On the following day, slides were washed in a stringency buffer (SSC, NP40), air-dried in the dark and incubated with 4,6 diamidino-2-phenylindole (DAPI) for nuclear identification. Slides were stored in the dark, at –20°C. Nonamplified and amplified control slides (MDA-MB-231 and Hs578T cell lines, respectively, fixed and embedded in paraffin; provided in the PathVysion kit; Abbott Labs, Vysis Inc) were analyzed with each assay. All assays were performed in one laboratory (UNC). Slides were scored within 1 week of FISH assay (most within 48 hours) with a Zeiss Axioplan Epifluorescence microscope (Carl Zeiss Inc, Thornwood, NY), equipped with a 100-watt mercury arc lamp, using a multifilter cube supplied by Vysis (DAPI single color bp; DAPI/9-orange [NB] dual bp; DAPI/green dual bp; DAPI/green/orange triple bp). To identify malignant cells for scoring, the underside of the slide was previously marked by the study pathologist with an indelible pen that can be distinguished under fluorescence microscopy. For each case, 60 nonoverlapping invasive cancer nuclei were scored for both CEP17 (green) and HER2 (orange) signals. In pilot studies, using non-CALGB breast cancer specimens, no difference in the average HER2:CEP17 ratio was observed when ratios were compared after counting 200, 120, or 60 nuclei per case. Therefore, 60 nuclei per case was selected for this study. Only cancer nuclei showing at least one orange and one green signal were counted. The 60 invasive cancer nuclei were selected from two-to-three different areas of the section to ensure representation of counts. When nuclei showed more than 10 signals, it was sometimes difficult to distinguish individual signals and therefore the raw count was categorized as 11 to 15, 16 to 20, or > 20. Raw counts (number of orange and green signals per nuclei; 60 nuclei per case) were submitted to the CALGB statistical center. The ratio of orange (HER2 gene) to green (chromosome 17 centromere) signal was calculated to normalize any increase in HER2 copy number due to polyploidy of chromosome 17. A case was considered amplified if the HER2:CEP17 ratio was 2.00. To allow scoring to be performed within 1 week of assay, cases were scored by two sites: UNC (scored 80% of cases) and University of Kansas (scored 20% of cases). Each technician followed the same procedure for scoring and used the same scoring criteria. All technicians and investigators were blinded to treatment assignment and clinical outcome. Before initiation of the FISH study, the two sites received identical training in scoring. Following training, a pilot study was performed in which both sites scored 20 non-CALGB breast cancer specimens. The specimens had a known range of amplification and had been previously assessed by D-PCR at the UNC Molecular Core lab. Results of the pilot study demonstrated perfect agreement in assigning a case as amplified or not amplified, with similar values for HER2:CEP17 ratios. To further address issues of inter-laboratory scoring variability, a blinded comparison was performed, in which 38 CALGB cases were scored by both labs. Similar to the pilot, there was perfect agreement in HER2 amplification status. To address assay reproducibility in the current study, an additional 22 CALGB cases were assayed twice on separate days. The agreement in HER2:CEP17 ratio was high (cc = +0.93). Independent studies have also reported on the reproducibility and portability of this FISH assay.19,20
IHC and D-PCR Assays
Results of other assays previously performed on the same cases were available in the CALGB database and were used for comparative analyses with FISH assays. Details of methods for measuring protein overexpression by IHC and amplification by D-PCR have been previously reported.1,2,21,22 In brief, IHC evaluations used two antibodies (CB11 and A0-11-854), directed against the internal domain of the HER2 protein. Prior studies have demonstrated excellent correlation between the two antibodies.2 The comparisons performed in the current study involved 346 cases stained with CB11 and 177 cases stained with A0-11-854. Cases were scored as overexpressing the HER2 protein if any membranous staining was found in malignant cells at a magnification of 100x. As described in the following statistical section, a cut point of 50% was used for some analyses. For D-PCR, DNA was extracted from 10-μm sections cut from the same block used for IHC and FISH. Stringent criteria was used to define a case as amplified: unequivocal amplification of two reference genes (as an assay control) and unequivocal HER2 gene amplification relative to a normal spleen control and an amplified standard (SKBR3 cell line).22
Statistical Analyses
Power calculations determined that we needed approximately 500 cases with assessable FISH assays. To arrive at that number, we estimated that approximately 30% of cases selected would not yield informative assays, mostly due to concerns that blocks on file would have insufficient tissue remaining for FISH assays (since these blocks had previously been sectioned for several assays) and to a lesser degree to account for assay failures. Therefore 750 cases were originally selected randomly from the total group of 992. Characteristics of patients included in the current FISH study were compared with those of patients not included in the study: age, menopausal status, tumor size, number of positive lymph nodes, ER status, PR status, proportion of patients who received tamoxifen, S-phase fraction, proportion of cells staining positive for p53 and HER2 by IHC, and assessment of HER2 gene amplification by D-PCR. 2 tests were carried out for dichotomous variables and t-tests were used to compare continuous variables. We also compared these same characteristics across the three dose schedules for patients in the present study, using 2 tests for dichotomous variables and F tests for continuous variables.
Correlations were calculated between assessment of HER2 by FISH and various clinical and tumor characteristics, including assessment of HER2 using other methods (IHC and D-PCR). 2 tests were carried out for dichotomous variables and t-tests were used to compare continuous variables. For tests of dichotomous variables the cutpoints for FISH and IHC were as follows: amplification by FISH used HER2:CEP17 ratio, where ratios 2 were considered positive; and overexpression by IHC, where cases with 50% invasive cells staining were considered positive.1,2 All analyses presented for D-PCR were dichotomized as amplified (positive [+]) or not amplified (negative [–]) based on the definition in the Methods section.
Comparisons were made based on overall agreement among the three measures of HER2. statistics (with their SEs) were used to quantify level of agreement.23 The Spearman rank correlation was applied to comparisons of continuous data between IHC and FISH methods.
The interaction between HER2 and dose was evaluated in predicting DFS and OS. This interaction relates to the therapeutic importance of HER2. The principal analysis of the interaction between dose and HER2 was multivariate proportional hazards modeling with 2 tests, as previously described.1,2,24 As in our previous reports1,2 the covariates considered were square root of the number of positive axillary lymph nodes (SQRT_NPN), tumor size (TSIZE = 2 for > 2 cm and TSIZE = 1 otherwise), menopausal status (PREMENO = 1 for premenopausal and PREMENO = 0 otherwise), dose of CAF (high v low and moderate v low), HER2 status (positive v negative), and interaction of HER2 with dose of CAF. For comparison, the three different methods for assessing HER2 (IHC, FISH, and D-PCR) were used, one set of models for each technique. The actual value of HER2:CEP17 ratio for FISH and the percentage of cells staining positive by IHC were used, whereas gene amplification by D-PCR was treated as a dichotomous variable, (amplified v not amplified). The most important question in these proportional hazards models was the existence of the "CAF by HER2" interaction. The strength of this interaction may suggest that some patients should be treated differently from other patients. Multivariate analyses were illustrated with Kaplan-Meier DFS and OS curves for the various CAF/HER2 groups.1,2,25
We also performed hazard ratio analysis for DFS for patients receiving high-dose CAF versus patients receiving moderate or low dose for each paired combination of methods (eg, IHC+/FISH+, IHC+/FISH–, IHC+/PCR–, and so on).
RESULTS
FISH Assessable Cases
Of the 569 cases assayed by FISH, 524 (92%) had assessable results. Technical reasons for unassessable cases included the following: 41 cases (7%) did not show appropriate hybridization signals even after repeat assay (ie, no orange [HER2] or green [CEP 17] signal detected or only one signal [orange or green] detected); in two cases (0.3%), tissue fell off the slide; and in three cases (0.5%), fewer than 60 nuclei showed both hybridization signals.
HER2 Status by Method
Assessable data were obtained for FISH and IHC assays from 524 and 523 cases, respectively. D-PCR results were obtained from 491 of these cases. HER2 protein, evaluated by IHC, was overexpressed in 24% of cases (127 of 523). HER2 gene copy, determined by FISH, was amplified in 17% of cases (90 of 524); and amplification by D-PCR was observed in 18% of cases (91 of 491).
Tables 1 through 3 show the 2 x 2 contingency tables comparing FISH, IHC, and PCR. The statistic, applied to dichotomous data, demonstrated a moderate level of agreement among the methods: 74% agreement between the amplification methods FISH and PCR; 65% agreement between IHC and FISH; and 53% agreement between IHC and PCR. We observed a high concordance rate, 92.3%, between the two measures of amplification (Table 1, FISH compared with PCR; n = 491). Thirty-eight cases (7.7%) were discordant in the FISH/PCR comparison. The concordance rate for FISH compared with IHC was 88.3% and for IHC compared with PCR was 83.9%. In the IHC/PCR pairing (n = 491) discordance was observed in 79 (16.1%) cases.
the statistic FISH and IHC are clearly related; however, we observed discordance in 61 cases (11.6%; Table 2). All 12 FISH+ cases were also positive by D-PCR. For this analysis, IHC+ cases were defined as those having 50% invasive cancer cells staining. Compared with other studies in the literature, many of which use 10% as a cut point for IHC positivity, most of the FISH+/IHC– cases might not be considered discordant (eg, only three cases showed < 10% cells staining in this group). Of the 49 FISH–/IHC+ cases, 42 cases were also PCR–, supporting a true lack of amplication. The small number of discordant cases overall precluded any definitive conclusions regarding the superiority of any one method to predict outcome.
We also evaluated the relationship between level of amplification by FISH and level of overexpression by IHC and found that tumors with a high percentage of HER2-positive (HER2+) cells by IHC were more likely to have a high FISH amplification ratio (r = 0.46; P < .001; data not shown). Nearly every case with more than 80% cells positive by IHC was amplified by FISH.
Clinical Correlations
Table 4 compares patient characteristics of cases included in the present study (n = 524) versus the remaining patients in CALGB 8541 (n = 469). Patient age; menopausal status; hormone-receptor status; HER2 status by IHC; tumor size; number of positive nodes; and percentage of patients receiving low (n = 178), moderate (n = 167), or dose-escalated (n = 179) CAF were similar between the current study of 524 patients and the remaining 469 patients not eligible or not assayed by FISH. However, we did observe differences in two parameters in the current study population compared with those patients not included. In the current study, a higher proportion of patients received tamoxifen (38.2% v 30.5%; P = .012). This was the result of a change in management of breast cancer patients in 1988 when the National Cancer Institute (NCI) recommended that postmenopausal ER-positive (ER+) breast cancer patients be considered for tamoxifen therapy. (The majority of patients in the current study entered the trial after 1988). In addition, the median S-phase fraction was higher in the current study (14% v 10%; P = .0001). Median S-phase values from the current study and those not included, however, are similarly categorized as representing a high proliferative capacity.
Comparison of FISH, IHC, and PCR to Predict Outcome After Dose-Intensive CAF Therapy
We considered proportional hazards models for DFS and OS using the three different HER2 methods (FISH, IHC, and PCR). For these models, we considered continuous measurement of FISH and IHC, whereas gene amplification by D-PCR was entered as a dichotomous variable (amplified v nonamplified). The covariates used are the same as those in our previous publications.1,2 Results of the multivariate analyses are summarized in Table 5 for DFS. Similar results were obtained for OS (data not shown). Of particular interest is the therapeutic implication of HER2 status: the interaction between HER2 status and dose schedule of CAF by likelihood ratio test was statistically significant for all three methods of HER2 assessment (FISH, P = .033; IHC, P = .0003; PCR, P = .043).
Hazard ratios for the multivariate Cox proportional hazards model for prediction of DFS are presented in Table 6. The hazard ratios for high-dose CAF versus moderate or low dose are 0.822, 0.834, and 0.732 for FISH, IHC, and PCR, respectively. This indicates that the high-dose arm has better DFS than the moderate and low-dose arms combined. The numerical values of the hazard ratios for the interaction of high-dose CAF compared to low and moderate dose with HER2 status are 0.919, 0.418, and 0.585, for FISH, IHC, and PCR, respectively. Each of the three methods for measuring HER2 indicates that patients receiving high-dose CAF tend to have better disease-free survival in the subset of HER2-positive cases, but no differences are apparent in HER2-negative cases. Hazard ratios of interaction terms are difficult to interpret. The inferential strength is provided by the overall interaction P values in Table 5. In addition, quantifications of the interactions are shown graphically using Kaplan-Meier survival curves in Figure 1.
The hazard ratio for DFS for those patients receiving high-dose CAF compared with patients receiving moderate or low-dose for the various pairs of methods are reported in Table 7. Although the categories of IHC+/FISH+ and IHC+/PCR+ show higher hazards ratios compared to the other combinations, as judged by the 95% CI, no combination of methods tested was found to be statistically superior.
Kaplan-Meier curves for DFS by HER2 status and CAF dose are shown for each of the three methods in Figure 1. By any method of assessment, for patients whose tumors are HER2 negative (HER2–), there was little or no effect of dose of CAF. Five-year DFS probabilities for patients with FISH-negative (FISH–) tumors are 60%, 64%, and 68% for low, moderate, and high (dose-escalated) doses, respectively. Similarly, patients with IHC– tumors had comparable DFS probabilities: 59%, 65%, and 64%, for low, moderate, and high doses, respectively. For patients whose tumors were HER2+, all three methods showed a benefit from dose-intense CAF compared with low or moderate doses. In the HER2+ subset, there is a significant difference between high versus low and high versus moderate CAF doses, whereas there is no difference between low and moderate doses. Patients with FISH+ tumors had a 5-year DFS of 43%, 42%, and 77% in the low, moderate, and high-dose groups, respectively. The corresponding 5-year DFS estimates for patients with IHC+ tumors were 53%, 47%, and 84%.
Kaplan-Meier plots for overall survival by HER2 status by each method are provided in Figure 2. There was no apparent difference between the three CAF doses for patients with HER2– tumors. However, similar to DFS curves, all three methods indicate a benefit with high-dose (dose-intense) compared with low- or moderate- dose doxorubicin-based therapy for patients with HER2+ tumors.
DISCUSSION
HER2 is a clinically important tumor marker with the potential to influence treatment decisions in the breast cancer patient, a role among tumor markers that previously has been achieved only with hormone receptors. Abnormal HER2 (gene amplification or protein overexpression) is also important in selecting metastatic breast cancer patients who are candidates for anti-HER2 therapy with trastuzumab.7,8,26,27 In the adjuvant setting,1-3,5,28 and recently in the neoadjuvant setting,29 several studies have suggested that HER2 status may be useful in determining a patient's benefit from doxorubicin-based chemotherapy. In prior CALGB reports,1,2 we observed a significant interaction between HER2 status and CAF dose in predicting outcome. The relative benefit of high-dose CAF was greater in patients with HER2+ (amplified by PCR or overexpressed by CB11 IHC) compared with HER2– (not amplified or overexpressed) tumors. This benefit however, was revealed only when patients were stratified by HER2 status. Looking at 9-year median follow-up results from the clinical trial, which evaluated all patients but ignored HER2 status, showed no difference in DFS or OS between the moderate- and high-dose arms.17
The National Surgical Adjuvant Breast and Bowel Project (NSABP), assessed HER2 status by IHC using a cocktail of two antibodies, mab-1 and pab-1, in 637 node-positive, ER-negative (ER–) breast cancer patients who were randomly assigned to melphalan and fluorouracil (FU) versus melphalan, FU, and doxorubicin.3 Patients with HER2+ tumors benefited from doxorubicin while those with HER2– tumors did not. In a Southwest Oncology Group study (SWOG 8814), HER2 status was determined by a third IHC system (antibody TAB250) in 595 postmenopausal, ER+, axillary node-positive patients who were randomly assigned to tamoxifen alone or tamoxifen plus CAF.5 Preliminary analyses of this study suggest that CAF offered a substantial advantage for HER2+ patients but little, if any, advantage for HER2– patients.5
Today, although HER2 is considered a clinically useful marker, there is no consensus regarding which method(s) should be used for clinical decision making. Optimally, the method should be reproducible and reliable, and validated for its clinical and biologic accuracy.9,30 Few methods meet these criteria. In the present study we have used clinical outcome to compare three different methods to measure HER2 status: HER2 gene amplification was measured by D-PCR and FISH using the Pathvysion FISH kit; protein overexpresssion was measured by IHC using CB-11 and AO-11-854 antibodies. We evaluated the relationship of each of these method systems to predict DFS and OS in breast cancer patients following treatment with CAF. We found that the three methods used in this study are not identical in assignment of HER2 status, nor are they identical in predicting outcome following CAF therapy. However, there is concordance among PCR, FISH, and IHC in determining HER2 status, and all three methods predict outcome following dose-intense CAF. Though it is tempting, from the multivariate analysis, to assign a greater predictive value for one method versus another in relation to interaction with dose, none of the methods is more clearly predictive than the others.
When making individual patient care decisions, one test method does not always provide all the necessary information. For example, knowing IHC, does FISH give us any further information Does the answer depend on whether IHC was positive or negative From the present study we may be misclassifying up to 12% of patients depending on the method selected (eg, IHC by CB11 v Vysis FISH). From our hazard ratio analysis for DFS of the different paired combination of methods (eg, IHC+/FISH– or FISH+/IHC–), many of the subgroups were small, and we could not identify a paired combination that was clearly superior in predicting outcome.
Much attention has focused recently on the use of FISH as an adjunct to the IHC assay to confirm ambiguous IHC results or as a substitute for IHC assays.10-12,14,31-34 The present study has demonstrated that FISH is a reliable method to predict clinical outcome; however, similar to IHC methods, not all FISH methods are alike. For example, among the two United States Food and Drug Administration–approved HER2 FISH kits, the criteria for defining a case as amplified differs. The Vysis Pathvysion kit used in this study incorporates two probes, the HER2 gene probe and a chromosome 17 centromeric probe to exclude polyploidy of chromosome 17 as a source of increased copy number of HER2. For a case to be considered amplified by this kit, the ratio of the HER2 copy number to chromosome 17 copies must be greater than or equal to 2.00, similar to the definition of amplification used in classical cytogenetics. The Ventana/Oncor FISH kit (Ventana; Tucson, AZ) uses a single probe system, where copy number of the HER2 gene alone is evaluated. For a case to be amplified, an average of 4 or more copies of HER2 must be observed in cancer nuclei. Clinically, it is uncertain whether an increase in HER2 gene copy number alone, or an increase of HER2 gene copies relative to copies of chromosome 17 is more useful in predicting outcome. Therefore, it was important, in the current study, to consider the influence of measuring chromosome 17 in the FISH assay. Chromosome 17 polyploidy, defined in this study as an average of three or more CEP17 signals in cancer nuclei, was observed in 8.5% or 45 of 523 cases. Due to the low rate of chromosome 17 polyploidy in this study, the use of the chromosome 17 probe to correct for HER2 amplification accuracy was not a major determinant of HER2 status or predicting outcome following treatment in this population. Kaplan-Meier curves were similar (data not shown) regardless of whether or not amplification was defined relative to chromosome 17. Use of the chromosome 17 probe in the clinical laboratory is still warranted, however, as an internal assay control for hybridization and to correct for differences in signals resulting from sectioning through nuclei. In other patient populations (eg, patients with metastatic breast cancer), which may have a higher rate overall of chromosome polyploidy, the use of CEP17 probe may prove to be more clinically useful.
In summary, the three HER2 methods tested, IHC by CB11, FISH by PathVysion, and PCR by D-PCR are similar but not identical in assigning a tumor the same HER2 status. All three methods showed a significant interaction between HER2 status and dose of CAF. All three methods result in similar but not identical Kaplan-Meier plots for disease-free survival and overall survival. Current data from this study do not support that one method or a paired combination of methods is statistically superior to the other in predicting clinical outcome following adjuvant doxorubicin therapy.
Appendix
The following institutions participated in the study: CALGB Statistical Office, Durham, NC: Stephen George, PhD, supported by CA33601; Christiana Care Health Services, Inc CCOP, Wilmington, DE: Irving M. Berkowitz, DO, supported by CA45418; Community Hospital-Syracuse CCOP, Syracuse, NY: Jeffrey Kirshner, MD, supported by CA45389; Dana-Farber Cancer Institute, Boston, MA: George P. Canellos, MD, supported by CA32291; Dartmouth Medical School—Norris Cotton Cancer Center, Lebanon, NH: Marc S. Ernstoff, MD, supported by CA04326; Duke University Medical Center, Durham, NC: Jeffrey Crawford, MD, supported by CA47577; Eastern Maine Medical Center CCOP, Bangor, ME: Philip L. Brooks, MD, supported by CA35406; Kaiser Permanente CCOP, San Diego, CA: Jonathan A. Polikoff, MD, supported by CA45374; Massachusetts General Hospital, Boston, MA: Michael L. Grossbard, MD, supported by CA12449; McGill Department of Oncology, Montreal, Quebec, Canada: Brian Leyland-Jones, MD, supported by CA31809; Memorial Sloan-Kettering Cancer Center, New York, NY: George Bosl, MD, supported by CA77651; Mount Sinai Medical Center, Miami, FL: Enrique Davila, MD, supported by CA45564; Mount Sinai School of Medicine, New York, NY: Lewis R. Silverman, MD, supported by CA04457; North Shore—Long Island Jewish Health System, Manhasset, NY: Daniel Budman, MD, supported by CA35279; Rhode Island Hospital, Providence, RI: Louis A. Leone, MD, supported by CA08025; Roswell Park Cancer Institute, Buffalo, NY: Ellis Levine, MD, supported by CA02599; South New Jersey CCOP, Camden, NJ: Jack Goldberg, MD, supported by CA54697; Southeast Cancer Control Consortium Inc CCOP, Goldsboro, NC: James N. Atkins, MD, supported by CA45808; Southern Maine Medical Center, Scarborough, ME: Thomas J. Ervin, MD, supported by CA37447; Southern Nevada Cancer Research Foundation CCOP, Las Vegas, NV: John Ellerton, MD, supported by CA35421; SUNY Maimonides Medical Center, Brooklyn, NY: Samuel Kopel, MD, supported by CA25119; SUNY Upstate Medical University, Syracuse, NY: Stephen L. Graziano, MD, supported by CA21060; University of Alabama, Birmingham, AL: Robert Diasio, MD, supported by CA47545; University of California at San Diego, San Diego, CA: Stephen L. Seagren, MD, supported by CA11789; University of Chicago Medical Center, Chicago, IL: Gini Fleming, MD, supported by CA41287; University of Iowa Hospitals, Iowa City, IA: Gerald Clamon, MD, supported by CA47642; University of Maryland Cancer Center, Baltimore, MD: David Van Echo, MD, supported by CA31983; University of Massachusetts Medical Center, Worcester, MA: F. Marc Stewart, MD, supported by CA37135; University of Missouri/Ellis Fischel Cancer Center, Columbia, MO: Michael C. Perry, MD, supported by CA12046; University of North Carolina at Chapel Hill, Chapel Hill, NC: Thomas C. Shea, MD, supported by CA47559; University of Tennessee Memphis, Memphis, TN: Harvey B. Niell, MD, supported by CA47555; Wake Forest University School of Medicine, Winston-Salem, NC: David D. Hurd, MD, supported by CA03927; Walter Reed Army Medical Center, Washington, DC: John C. Byrd, MD, supported by CA26806; Washington University School of Medicine, St Louis, MO: Nancy Bartlett, MD, supported by CA77440; Weill Medical College of Cornell University, New York, NY: Michael Schuster, MD, supported by CA07968.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
Acknowledgment
We thank the participating CALGB institutions, pathologists, and patients for their help, support, and contribution to this study.
NOTES
Supported in part by grants from the National Cancer Institute (National Institutes of Health, Bethesda, MD; CA31946) to the Cancer and Leukemia Group B (Richard L. Schilsky, MD, Chairman) and by NCI-U01-CA64061-05, the T.J. Martell Foundation for Leukemia, Cancer and AIDS Research, and Vysis Inc, Downers Grove, IL.
The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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