Summary
Background
The 2013 testing guidelines for determining HER2 status included new cutoff points for HER2/CEP17 ratio and average HER2 copy number/cell and recommended using a reflex test with alternative chromosome 17 probes (Ch17P) to resolve equivocal HER2 results. We sought to determine the clinical utility of alternative Ch17P in equivocal cases and the effect of equivocal results and/or the change in HER2 status on patients’ outcome.
Methods
Our institution’s database of HER2 dual-probe fluorescent in situ hybridization results between 2000 and 2010 was searched for cases of invasive breast cancer with HER2/CEP17 ratio of <2 and average HER2 copy numbers of <6/cell. Cases with HER2 copy number between 4 and <6 (the definition of equivocal HER2 results) were analyzed using alternative Ch17P for SMS (Smith-Magenis syndrome) and RARA (retinoic acid receptor alpha) genes. Disease-free survival (DFS) and overall survival (OS) were evaluated in relation to HER2 copy number using multivariable Cox proportional hazards regression.
Findings
Of the 3630 patients meeting inclusion criteria, 137 (4%) had an equivocal HER2 results. Using alternative Ch17P, 36 of 57 (61%) equivocal HER2 cases were upgraded to positive HER2 status, and 22 (39%) cases remained unchanged. The 5-year DFS and OS adjusted hazard ratios (HRs) with 95% confidence intervals (CIs) for copy number of 4 to <6 vs < 4 were 0.6 (0.3–1.2) and 0.5 (0.2–1.0) with p = 0.16 and 0.66, respectively. Compared to HER2-negative cases, these CIs indicate that equivocal HER2 results are associated with either a protective effect (HR < 0.5) or no effect (HR = 1.0).
Interpretation
Our findings rule out a significant deleterious effect of equivocal HER2 results. Alternative Ch17P may erroneously upgrade HER2 status, and therefore they cannot be relied upon in clinical practice.
Introduction
HER2, a member of the epidermal growth factor receptor family, is encoded by a gene (HER2, or ERBB2) located on the long arm of chromosome 17 (17q12). HER2 amplification and/or overexpression is noted in about 15% to 20% of invasive breast cancers1 and is associated with a poor prognosis. Most importantly, breast cancers with HER2 overexpression or gene amplification can be targeted with agents that are remarkably effective in both the metastatic and adjuvant settings.1 Because HER2 status predicts whether HER2-targeting agents would provide benefit, precise assessment is essential for treatment decisions.2
Two diagnostic techniques, immunohistochemistry (IHC) and in situ hybridization (ISH), are currently approved for assessing HER2 status in clinical practice.3 Fluorescence in situ hybridization (FISH) is the most commonly used ISH technique. FISH is conducted using either a single probe to enumerate HER2 copies per nucleus or dual probes--a HER2 probe and a chromosome 17 centromere probe (chromosome enumeration probe 17, CEP17)--allowing determination of the HER2/CEP17 ratio. The use of the HER2/CEP17 ratio has long been regarded as a better reflection of HER2 gene status than the mean HER2 copy number, since it compensates for the loss of signals by tissue sectioning and adjusts for the natural increase in the number of chromosomes during replication.4
In 2007, the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) developed guidelines for HER2 testing in breast cancer and advocated that results be reported as positive, negative, or equivocal.2 Equivocal HER2 results have been attributed to chromosome 17 polysomy since the number of CEP17 signals in tumor nuclei has been generally considered a surrogate of the number of copies of chromosome 17.
Recent studies, however, have shown that true polysomy 17 is extremely rare in breast cancer and that increased CEP17 copy number often results from amplification or copy number gain in the centromeric or pericentromeric region.4–12 Consequently, in 2013, ASCO/CAP released new guidelines for HER2 testing that included cutoff points that differed from those in the 2007 guidelines for ISH HER2/CEP17 ratio and average HER2 copy number/cell; the guidelines also recommend the use of a reflex test with alternative chromosome 17 probes for resolving equivocal HER2 ISH results.13
FISH probes that have been used as surrogates for chromosome 17 in cases with a high CEP17 copy number have included SMS (Smith-Magenis syndrome, also called RAI1), RARA (retinoic acid receptor alpha), and TP53.5, 11, 12 The SMS probe targets sequences at 17p11.2, and the RARA probe hybridizes with RARA and neighboring genes at 17q21.2. While some studies have advocated the use of these alternative chromosome 17 genes as surrogates,4, 5, 13 others 7, 12 have cautioned against their use in daily practice due to frequent heterozygous deletions. Furthermore, the clinical utility of these tests in relation to the clinical course of disease has not been determined.
Although the goal of the new ASCO/CAP guidelines is to ensure maximal accuracy of HER2 testing, the numbers of equivocal cases have been noted to increase in a significant number of cases.12, 14 The increase in the number of equivocal cases compounded by the lack of a uniform approach to resolving such problems make treatment recommendation for this group of patients difficult. We therefore undertook this study to determine the relationship between an equivocal HER2 copy number (4 to <6) and patient outcome. We also determined whether equivocal HER2 results could be resolved using alternative chromosome 17 genes (RARA and SMS) and whether a change in HER2 status based on the use of alternative chromosome 17 probes was associated with patients’ outcomes.
Materials and Methods
Patient Population
This study was approved by MD Anderson’s Institutional Review Board. Our institution’s database of HER2 dual-probe FISH results was searched for cases of invasive breast cancer. For the period between 2000 and 2010, there were a total of 7862 patients with invasive breast cancer and clinical and pathologic data available for analysis. Of these, 5157 had an HER2 assay within six months of diagnosis. Of these, 3651 had stage I–III disease, were not treated with trastuzumab, and had a HER2/CEP17 ratio <2. Of these, 3630 had complete disease –free survival (DFS) and overall survival (OS) data and a minimum of 5 years follow-up. Of these, 3493 (96%) cases had a HER2 copy number of <4 signals/cell, and 137 (4%) cases had a HER2 copy number between 4 and <6 signals/cell (cases meeting the new ASCO/CAP definition of “equivocal”).
Sixty-three of the 137 cases with equivocal HER2 results had available tissue sections for additional testing of the alternative SMS/RARA genes.
FISH
HER2/CEP17 analyses were performed using the Vysis PathVysion probe kit, which includes a SpectrumGreen-conjugated probe for the alpha satellite DNA located at the centromeric region of chromosome 17 (17p11.1-q11.1) and a SpectrumOrange-conjugated probe for the HER2 gene locus (Abbott Molecular/Abbott Laboratories, Abbott Park, IL). Briefly, 4-μm paraffin sections were deparaffinized and hybridized according to the manufacturer’s instructions. Fluorescence was visualized with an Olympus microscope with multiple filters and correlated directly with hematoxylin and eosin-stained tissue sections to ensure scoring of invasive carcinoma. HER2 and CEP17 signals were manually counted in 60 tumor cells, and a total HER2/CEP17 ratio was calculated.
Corresponding tissue sections (available in 63 cases) were analyzed for SMS and RARA gene copy number by using Vysis probe sets (Abbott Molecular/Abbott Laboratories, Abbott Park, IL) for the Smith-Magenis syndrome (SMS) critical region at 17p11.2 (encompassing genes SHMT1, TOP3A, FLII, and LLGL1 and labelled with SpectrumOrange) and retinoic acid receptor (RARA) at 17q21.2 (encompassing GRB7, MLN51, SHGC -146999,THRA, and RARA exons 2–6, labeled with SpectrumGreen). The FISH procedure was performed according to the manufacturer’s instruction. Manual counting was performed on all samples. Gene signals per cell in 20 tumor nuclei were evaluated for each case. Criteria used to calculate a revised HER2-to-chromosome-17 ratio were as those used previously.4, 12 Briefly, if any of SMS or RARA signals were found to be less than 2.6 per cell, the gene with the highest signal count was chosen as the chromosome 17 reference gene instead of CEP17 for the calculation of the HER2 ratio. Cases displaying <1.5 signals/cell were considered to be hypodisomic. The revised HER2 status based on HER2/Ch17P ratio was stratified as positive or unchanged.
Statistical Analysis
DFS and OS were computed from the date of diagnosis to the first event. DFS and OS of patients with HER2 copy number <4 vs 4 to <6 were estimated using Kaplan-Meier curves. Hazard ratios were estimated using Cox proportional hazards regression models. These results were then adjusted as to the following variables: age (<40 years, 40–49 years, 50–59 years, and above), race (white vs others), menopausal status (pre vs post), nodal status (negative vs positive), tumor grade (well, moderately, and poorly differentiated), tumor type (ductal, lobular, tubular, mucinous, others), pathologic tumor size (>2 cm vs <2 cm), and estrogen/progesterone receptor status (positive vs negative). The proportional hazards assumptions of the Cox models were verified using rescaled Schoenfeld residual analysis.
DFS and OS of the patients with equivocal HER2 results were also calculated based on the revised HER2 ratio obtained after the use of alternative probes.
The immunohistochemical score distribution of HER2 protein expression (3+, 2+, 1+, or 0) was compared between patients with and without a change to from equivocal to positive HER2 status using an exact Wilcoxon rank sum test.
Role of the funding source
The funder had no role in the data collection, data analysis, data interpretation, or writing of the manuscript. NS and KH had full access to all the data in the study and had final responsibility for the decision to submit for publication.
Results
Clinical outcomes
Of the total 7862 patients with invasive breast cancer and available clinical and pathologic data for analysis, 3630 met our inclusion criteria and had a HER2/CEP17 ratio <2 and an average HER2 copy number <6 signals/cell). Of these, 137 (4%) had a HER2 copy number between 4 and <6 signals/cell (“equivocal results”), and 3493 (96%) had a HER2 copy number <4 signals/cell. All 137 cases with HER2 equivocal results had a CEP17 copy number gain (range 2.4–8.9 signals/cell), with the majority of cases (131/137) having an average CEP17 copy number per nucleus > 2.6.
Kaplan-Meier DFS and OS curves stratified by HER2 copy number (4 to <6 vs <4 signals/cell) are shown in figures 1 and 2. Further analysis of this phenomenon using multivariable Cox proportional hazards regression models adjusting for age, race, menopausal status, number of positive lymph nodes, tumor histologic type and grade, tumor size, and hormone receptor status (total 2864 patients with complete data) show that the 5-year DFS for patients with HER2 copy number of 4 to <6 signals/cell, when compared with patients with copy number of <4 signals/cell, did not significantly differ (HR 0.6; 95% CI 0.3 to 1.2; p = 0.16); the same was true for 5-year OS (HR 0.5; 95% CI 0.2 to 1.0; p = 0.66). Compared to HER2-negative cases, both of these CIs indicate that equivocal HER2 copy number (4 to <6) is associated with either a protective effect (HR = < 0.5) or no effect (HR = 1.0). Importantly, these findings rule out a significant deleterious effect of elevated HER2 copy number in the absence of targeted therapy.
Figure 1.
Kaplan-Meier disease-free survival curves according to HER2 copy number (4 to <6 signals/cell versus <4 signals/cell).
Figure 2.
Kaplan-Meier overall survival curves according to HER2 copy number (4 to <6 signals/cell versus <4 signals/cell).
FISH results using SMS and RARA probes
Of the 137 equivocal cases scored on the basis of HER2/CEP17 ratios of <2 and mean HER2 copy number of 4 to <6, 63 had available tissue for analyses. Six of the 63 cases were excluded because residual invasive cancer was absent in the sections. The results of the average copy number of HER2, CEP17, and alternative Ch17 genes (SMS and RARA) per nucleus, and the revised HER2 ratio are shown in Table-1. Of the 57 cases, 35 (61%) cases had their HER2 gene status upgraded from equivocal to positive (figure 3), and the remaining 22 (39%) cases remained unchanged. The distribution of the FISH results on chromosome 17 for the 57 patients is shown in figure 4.
Table 1.
Average copy number of HER2, CEP17, and alternative chromosome 17 genes (SMS, and RARA) per nucleus and corresponding ratios of HER2 to SMS or RARA (revised HER2 ratio) in 56 cases with equivocal HER2 results. Corresponding HER2 protein expression is also shown.
| Case# | HER2 | CEP17 | SMS | RARA | Revised HER2 ratio | HER2-protein expression |
|---|---|---|---|---|---|---|
| 1 | 4.67 | 3.5 | 1.5 | 1.5 | 3.1 | 2 |
| 2 | 4.08 | 2.5 | 3.5 | 4.5 | <2 | 1 |
| 3 | 4.02 | 2.33 | 2.4 | 2.6 | <2 | 2 |
| 4 | 4.31 | 3.96 | 1.6 | 2.6 | 2.7 | 1 |
| 5 | 4.88 | 2.78 | 2 | 2 | 2.4 | 1 |
| 6 | 4.65 | 4.28 | 2.7 | 5.1 | <2 | NA |
| 7 | 4.62 | 3.68 | 1.3 | 2 | 2.4 | NA |
| 8 | 4.32 | 2.97 | 1.5 | 1.5 | 2.9 | NA |
| 9 | 4.35 | 3 | 1.5 | 4.1 | 2.9 | 2 |
| 10 | 4.98 | 2.97 | 1.5 | 4.5 | 3.1 | 1 |
| 11 | 4.5 | 3.93 | 2.8 | 2.8 | <2 | 2 |
| 12 | 5.27 | 3.18 | 1.8 | 1.8 | 2.9 | 1 |
| 13 | 4.3 | 3.93 | 3 | ? | <2 | NA |
| 14 | 4.68 | 2.8 | 2.9 | 2.2 | 2.1 | 1 |
| 15 | 5.35 | 3.18 | 4.5 | 5.1 | <2 | 3 |
| 16 | 4.5 | 2.5 | 1.6 | 1.6 | 2.8 | 2 |
| 17 | 4.02 | 3.33 | 2.5 | 2.7 | <2 | 1 |
| 18 | 4.73 | 3.58 | 1.8 | 1.8 | 2.6 | 2 |
| 19 | 4.22 | 3.83 | 1.7 | 1.6 | 2.5 | 1 |
| 20 | 4.5 | 3.03 | 1.5 | 1.5 | 3 | 1 |
| 21 | 4.48 | 3.48 | 1.6 | 1.6 | 2.8 | 1 |
| 22 | 4.15 | 3.65 | 2.5 | 3.5 | <2 | 2 |
| 23 | 4.1 | 3.05 | 1.7 | 1.7 | 2.4 | 2 |
| 24 | 4.1 | 3.45 | 2.7 | 2.7 | <2 | 1 |
| 25 | 4.38 | 3.68 | 2.7 | 2.9 | <2 | 1 |
| 26 | 4.57 | 3.63 | 1.8 | 1.8 | 2.5 | 2 |
| 27 | 4.38 | 3.23 | 2.5 | 3 | <2 | 2 |
| 28 | 5.97 | 4.87 | 2.2 | 2.6 | 2.7 | 2 |
| 29 | 4.8 | 3.8 | 2.17 | 3.5 | 2.2 | 2 |
| 30 | 5.95 | 4.23 | 3.5 | 3.5 | <2 | NA |
| 31 | 4.15 | 3.38 | 1.5 | 3.8 | 2.8 | 2 |
| 32 | 4.73 | 2.46 | 1.6 | 1.6 | 2.95 | 1 |
| 33 | 4.43 | 3.52 | 1.5 | 1.5 | 2.95 | 1 |
| 34 | 4.83 | 3.6 | 2.6 | ? | <2 | 3 |
| 35 | 4.63 | 3.18 | 1 | 1 | 4.6 | NA |
| 36 | 4.35 | 3.75 | 2.9 | 4.1 | <2 | NA |
| 37 | 4.19 | 4.79 | 1.3 | 1.5 | 2.7 | NA |
| 38 | 5.63 | 4.35 | 2.6 | 5.3 | 2.2 | NA |
| 39 | 5.15 | 3.12 | 1.6 | 1.6 | 3.2 | 1 |
| 40 | 5.07 | 3.58 | 2 | 5.3 | 2.5 | NA |
| 41 | 4.28 | 2.78 | 3 | 3.8 | <2 | 0 |
| 42 | 4.2 | 2.65 | 1.5 | 2.4 | <2 | 1 |
| 43 | 4.27 | 4.48 | 1.7 | 1.5 | 2.5 | 2 |
| 44 | 4.12 | 2.78 | 1.5 | 5 | 2.7 | 1 |
| 45 | 5.35 | 3.83 | 1.6 | 1.6 | 3.3 | 2 |
| 46 | 4.7 | 4.55 | 3.5 | 4.1 | <2 | NA |
| 47 | 4.07 | 3.02 | 2.4 | 2.4 | <2 | 2 |
| 48 | 5.7 | 5.25 | 3 | 2.6 | 2.2 | 2 |
| 49 | 4.1 | 3.93 | 2.8 | 4.3 | <2 | NA |
| 50 | 4.63 | 3.93 | 1.5 | 2 | 2.4 | 0 |
| 51 | 4.27 | 2.42 | 2.39 | 3 | <2 | 1 |
| 52 | 4.75 | 4.27 | 1.5 | 5.1 | 3.2 | 2 |
| 53 | 5.28 | 3.37 | 1.9 | 6 | 2.8 | 1 |
| 54 | 4.22 | 2.68 | 1.5 | 1.5 | 2.8 | 2 |
| 55 | 4.17 | 2.92 | 3.1 | 4.2 | <2 | NA |
| 56 | 4.22 | 2.95 | 1.5 | 1.6 | 2.6 | 2 |
| 57 | 4.73 | 2.89 | 3.4 | 3.4 | <2 | 1 |
Figure 3.
An example of FISH analysis (Case # 44, table-1) showing increased signals for the RARA (retinoic acid receptor alpha) gene (average copy number per nucleus: 5, green signals), while the SMS (Smith-Magenis syndrome) gene shows a disomic signal (average copy number per nucleus: 1.5, red signals). Based on previously published criteria, these findings resulted in a change in HER2 status from equivocal to positive (see discussion for further comments).
Figure 4.
Schematic illustration of chromosome 17 showing the mapping position of the SMS and RARA genes in relation to the HER2 gene and CEP17. Below, red bars indicate gain or amplification of the corresponding gene, and white bars indicate a disomic copy number. The numbers in the bars indicate the number of patients corresponding to each group based on FISH results (RARA signals in two of the three cases shown in the top bar were not clearly visualized, cases #13 and 34 in table-1. CEP17, centromere portion of chromosome 17; HER2, human epidermal growth factor receptor 2; RARA, retinoic acid receptor alpha; SMS, Smith-Magenis syndrome.
Kaplan-Meier DFS and OS curves stratified by the ratio of HER2 to alternative chromosome 17 genes are shown in figures 5 and 6. Both the DFS and OS results were inconclusive. The 95% confidence intervals for the hazard ratios include values < 0.5, indicating a significant protective effect for elevated ratios as well as values > 2.0, indicating a significant deleterious effect for elevated ratios. Since the data are consistent with both of these hypotheses (protective as well as deleterious effects for elevated ratios), we must conclude that the data are inconclusive rather than negative because of the small numbers of patients included.
Figure 5.
Kaplan-Meier disease-free survival curves according to the ratio of HER2/alternative chromosome 17 genes in 57 patients with equivocal HER2 results (22 with ratios < 2 and 35 with ratios >= 2).
Figure 6.
Kaplan-Meier overall survival curves according to the ratio of HER2/alternative chromosome 17 genes in 57 patients with equivocal HER2 results (22 with ratios < 2 and 35 with ratios >= 2)
We compared the FISH results of the 57 cases before and after the use of alternative Ch17P with HER2 protein overexpression by IHC. Forty-six of these 57 cases had available IHC data (Table-1). Of the 46 cases, 43.5% showed 2+ HER2 protein expression (equivocal results), and 47.8% were negative (1+). Only 2 cases were positive (3+), and these cases remained equivocal/unchanged with the use of SMS/RARA genes. No correlation was found between the distribution of IHC score and HER2 FISH results before and after the use of alternative Ch17P (Table-2) (p = 0.24).
Table 2.
Comparison between immunohistochemical (IHC) score of HER2 protein overexpression and HER2 equivocal FISH results in a total of 46 cases before and after the use of alternative chromosome 17 probes (SMS and RARA). No significant correlation between the distribution of IHC score and the change in HER2 status, P=0.24
| IHC score of HER2 protein-overexpression | Cases with HER2 equivocal results based on CEP17 ratio (%) | Cases changed to HER2 positive using SMS/RARA probes (%) | Remaining unchanged HER2 equivocal cases (%) |
|---|---|---|---|
| 0 | 2 (4.3) | 1 (0.5) | 1 (0.5) |
| 1+ | 22 (47.8) | 15 (68.2) | 7 (31.8) |
| 2+ | 20 (43.5) | 15 (75) | 5 (25) |
| 3+ | 2 (4.3) | 0 (0) | 2 (~) |
| Total # of cases | 46 | 31 (67.4) | 15 (32.6) |
P=0.24
Discussion
In our study, equivocal HER2 results approached 4% of the total cases with HER2/CEP17 ratio <2. This rate is similar to those reported by others.12, 14 Compared to HER2-negative cases, we demonstrated that HER2-equivocal results (HER2 copy number 4 to <6) (in the absence of trastuzumab-based therapy) have no deleterious effect on patient outcome. In addition, we demonstrated that the “revised HER2 status” due to the use of alternative Ch17 probes is not reflective of patient outcome.
The impact of equivocal HER2 results on patient outcome has not been fully evaluated. Most published reports have dealt with “CEP17/so-called polysomy 17” and were rarely in direct relationship to the increase in HER2 copy numbers. Since equivocal HER2 results by ISH are often associated with increased CEP17 copy number, a review of clinical studies that focused on elevated CEP17/polysomy 17 is relevant and may reflect on the impact of equivocal HER2 results. Elevated CEP17 count (CEP17 > 3 signals/cell) was reported to be associated with higher nuclear and histologic grade and estrogen receptor negativity,15 high proliferative rate,16 and nodal involvement17 compared with non-polysomic tumors; however, other studies18, 19–21 found no significant differences between tumors with a CEP17 copy number ≥ 3 and tumors with CEP17 <3 in clinicopathologic variables.
Studies that have directly focused on HER2 gene amplification or copy number or HER2 protein expression have demonstrated that intermediate HER2 amplification or low-level gene copy number or protein expression influence breast cancer behavior.18, 22–24 In one study of 1400 breast cancer cases with more than 15 years of follow-up,22 the authors reported that cases with intermediate HER2 ratios between 1.5 and 2.2 (which is often a reflection of a slight increase in HER2 copy number) had a significantly better outcome than the conventional “amplified” group (HER2 ratio >2.2) and a significantly worse outcome than cases with FISH ratios less than 1.5. In the study by Vanden Bempt and colleagues,18 equivocal HER2 gene status, in contrast with HER2 gene amplification, was not associated with reduced DFS, although survival was intermediate between HER2-negative and HER2-positive patients. Similarly, in our study, the DFS and OS adjusted hazard ratios for patients with HER2 copy number of 4 to <6 vs < 4 were 0.6 and 0.5 (p=0.16 and 0.66, respectively), which indicate that elevated HER2 copy number is not associated with significant deleterious effect on patient outcome (in the absence of adjuvant or neoadjuvant trastuzumab). Conversely, in another study of 91 node-positive patients with invasive breast carcinoma treated with mastectomy and doxorubicin-based chemotherapy without trastuzumab with a median follow-up of 12.5 years, the authors found that even a low level of HER2 expression (≥ 1+ using IHC) in the primary tumor was significantly associated with decreased locoregional recurrence-free survival, DFS, and OS.23
Despite the recent findings that true polysomy 17 in breast cancer is rare and that increased CEP17 copy number often results from amplification or copy number gain in the centromeric or pericentromeric region,4, 6, 7, 9, 10 the 2013 ASCO/CAP guidelines advocate for additional testing in equivocal cases.13 This can be done by testing for additional genes on chromosome 17 that are not expected to coamplify with HER2.13, 25 The selective use of alternative chromosome 17 probes for RARA, SMA, and TP53 genes in some studies was due to their commercial availability and ease of testing in the clinical setting. In our study, the use of SMS and RARA genes resulted in change of HER2 status from equivocal to positive in 61% of patients with HER2 copy numbers of 4 to <6. These findings are concordant with those reported previously.4, 5, 12 In a study by Tse et al.,4 using three different chromosome 17 genes, SMS, RARA, and TP53, 48% (41/86) of cases with mean HER2 copy number of 4 to 6 and increased CEP17 signals had their HER2 gene status upgraded from nonamplified to amplified/positive. Another study by Jang et al.12 reported more than 75% of cases with equivocal HER2 status were upgraded to amplified after additional FISH analyses for the same three probes. In these studies, however, no clinical outcome data were available. Although our results in regard to the change in HER2 status due to the use of alternative Ch17P are similar to those reported previously, the criteria used in the interpretation of the published data are questionable. Recent studies have demonstrated the presence of frequent complex structural alterations of chromosome 17 in breast cancer cases including losses and gains of genetic material in both the long and short arms, as well as gains and losses of centromeric regions, 6, 7, 26 and proved that the use of additional FISH probes such as SMS and RARA are not sufficient in correcting the HER2 gene status. 7, 12 Furthermore, in a comprehensive review by Jang et al.,12 the authors highlighted many of the limitations associated with these genes. For example, the RARA gene, which is located at 17q21, maps very close to HER2 and is amplified in up to 51% of HER2-amplified breast cancers. In our series, although RARA copy gain or amplification was seen in 30 of 55 HER2-equivocal cases, in 24 cases, a disomic copy number was present. On the other hand, the SMS gene, which is located at 17p11.2, is rarely amplified or coamplified; however, its loss is frequently identified due to heterozygous deletions. In our study, only three of 57 (5.4%) cases demonstrated genetic loss for SMS (defined as <1.5 mean copy number). Although TP53, which is located at 17p13.1, is relatively far from HER2, it is one of the most frequently mutated genes in breast cancer.12 As advocated by Jang et al.,12 the use of more stable genes could be considered; however, based on The Cancer Genome Atlas data, at least a quarter had a copy number deletion and as a result, their use may be limited as alternative reference genes for chromosome 17. In our study, the findings that these patients had an outcome that was no worse than the HER2 negative cases, as would not be expected for a cohort containing a substantial numbers (61%) of up-graded (HER2-positive), coupled by the lack of associations with HER2 overexpression (IHC3+) among the “upgraded HER-2 positive”, indicate that these alternative Ch17 genes may overestimate HER2 positive cases due to unrecognized reduction in signal number due to heterozygous deletions, thus leading to erroneous upgrade of HER2 status to “positive”.
Whether this new category of patients with equivocal HER2 results “upgraded” to HER2 positive will have more aggressive disease or will have a favorable response to targeted therapy has not been fully determined. Previous studies, however, have indicated that tumors with extra copies of HER2 (which used to be attributed to polysomy 17) represent a distinct group of cases and are fundamentally different from tumors with extra copies resulting from HER2 gene amplification.19, 27, 28 For example, in contrast to what occurs with gene amplification, these cases are not associated with increased HER2 mRNA and do not overexpress protein at the significant IHC 3+ level. Only 2 of our 46 cases showed 3+ protein overexpression, and the remaining cases were distributed among 1+ and 2+ protein expression, irrespective of the changes in HER2 gene status as a result of the use of alternative Ch17p (p= 0.24). Furthermore, treatment response to targeted therapy may vary depending on HER2 copy numbers or HER2/CEP17 ratio. 29–33 In one study, pathologic complete response rate to trastuzumab-based neoadjuvant therapy in high-amplification tumors (defined as >10 signals per nucleus vs 6–10 signals for low amplification) was significantly higher compared with low-amplification tumors (56% vs 22%; p= 0.005). 29 Other studies have shown that a high HER2/CEP17 ratio is a significant predictor of progression-free survival30 in patients with metastatic breast cancer treated with chemotherapy and trastuzumab, and a ratio of ≥ 7.0 was also a predictor of pathologic complete response in patients with locally advanced breast cancer who received neoadjuvant chemotherapy and trastuzumab.31 However, in National Surgical Adjuvant Breast and Bowel Project (NSABP) trial B-31, no significant association between HER2 copy number and benefit from adjuvant trastuzumab was found, and even patients with normal gene copy numbers appeared to benefit from trastuzumab.32 In a subsequent analysis 33 and using a gene expression profile, the authors developed a model that could predict the degree of benefit from trastuzumab and also demonstrated that that patients with HER2- negative tumors belong to the moderate benefit group. This finding is being tested through a randomized clinical trial (NSABP protocol B-47). 33 Future clinical trials should be extended to cases with equivocal HER2 results to determine the degree of benefit of HER2 targeted therapy in a fashion similar to that of HER2 negative cases.
In summary, considering the extremely low incidence of true chromosome 17 polysomy, the lack of consensus on the use of alternative genes, and our clinical data, although from a limited sample size, that tumors with equivocal HER2 results more closely resemble HER2-negative than the conventional HER2-amplified cases, we believe the use of alternative chromosome 17 genes in determining HER2 status is of limited value and perhaps not needed and that management of patients with equivocal HER2 results (HER2 copy 4 to <6) should not rely upon the use of alternative chromosome 17 probe testing.
Acknowledgments
Funding
National Institutes of Health
This research was supported in part by National Institutes of Health/National Cancer Institute through MD Anderson’s Cancer Center Support Grant, CA016672, and used the Clinical Trials Support Resource. Sunita Patterson, Department of Scientific Publications, MD Anderson Cancer Center, provided editorial assistance.
Footnotes
Disclosures
KR Hess is an unpaid consultant for Angiochem Inc.
The other authors have nothing to disclose.
Contributors
Nour Sneige, Ken R Hess and Nuhad K Ibrahim contributed to the conception and design of the study. Nour Sneige, Ken R Hess, Nuhad K Ibrahim and Asha S Multani contributed to the collection and assembly of data and data analysis and interpretation. All authors, including Dr. Yun Gong, contributed to the writing of the report and critical revision of the report for important intellectual content. All authors read and approved the final manuscript.
References
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