CEP17 Copy Number Research Articles

Assessment of HER2 gene status in breast carcinomas with polysomy of chromosome 17

The current study was performed to determine the impact of polysomy 17 on the interpretation of HER2 testing of invasive breast carcinomas using fluorescent in situ hybridization methods. Current American Society of Clinical Oncology/College of American Pathologists guidelines define HER2-positive tumors as those with >6 HER2 genes per nucleus or those with HER2/CEP17 (chromosome 17) ratio >2.2. These guidelines are potentially contradictory in tumors with polysomy of chromosome 17. Seventy-two breast carcinoma cases with reported polysomy of chromosome 17 (≥ 3 CEP17 signals on average) by fluorescent in situ hybridization were identified, and the corresponding HER2 immunohistochemistry was obtained. The HER2 status of the archived samples was reviewed, and the tumors were recategorized according to the 2007 American Society of Clinical Oncology/College of American Pathologists guidelines. The average CEP17 copy number for the group was 4.5 (range, 3.0-10.4). Thirty-three (45.8%) cases had >6 copies of HER2 per nucleus. Twenty-one cases (29.2%) qualified as HER2 gene amplified using the HER2/CEP17 ratio (>2.2) guideline. All these cases had >6 HER2 signals, which represented 63.6% of all cases with >6 HER2 signals. HER2 protein expression showed significant positive correlations with both HER2 gene copy number and HER2/CEP17 ratio (P < .01, r(s) = 0.56 and 0.64, respectively). Increased CEP17 signals detected in invasive breast carcinomas may lead to discordant interpretation of gene amplification in a significant proportion of the cases, depending on which criterion (ratio vs absolute number) is used for interpretation. However, increased gene dosage (>6 HER2 genes or HER2/CEP17 ratio >2.2), regardless of the evaluation method, is positively correlated with HER2 protein expression.

Determining true HER2 status in breast cancers with polysomy using alternative chromosome 17 reference genes: Implications for trastuzumab therapy.

10549 Background: Detection of HER2 gene amplification by FISH is a key test to predict response of breast cancer to trastuzumab. The ratio of HER2 to CEP17 signals is often used to determine HER2 status, based on the assumption that centromeric probe to CEP17 indicates the chromosome 17 copy number. This study tests the hypothesis that increased copy numbers of CEP17 may not reflect the presence of polysomy and that the use of other genes on chromosome 17 might more accurately serve as reference genes to determine HER2 gene status. Methods: 122/5683 (2%) of HER2 FISH cases performed between October 2007 and December 2009 with CEP17 copy numbers 2.6 and above were selected for additional gene mapping studies using probes for other chromosome 17 genes, including SMS, RARA, and TP53. Revised HER2/chromosome-17-reference ratios utilizing these alternative loci were calculated. Results: 95/122 cases (78%) demonstrated at least one chromosome 17 gene locus with eusomic copy number. Using these new reference genes, 63 of 95 cases (66%) would have HER2 amplification status changed (negative to positive, equivocal to positive or negative to equivocal), with 46 cases (48%) now considered eligible for trastuzumab therapy. The cases were further divided into three groups based on level of HER2 signals (high [>6], intermediate [4-6], borderline/low [<4]) for analysis. The number of cases having HER2 status upgraded in these groups (high, intermediate and border/low) were 14/45 (31.1%), 43/50 (86%) and 4/27 (14.8%) respectively. Conclusions: Our results support recent findings of CGH studies suggesting that true polysomy 17 is rare in breast cancer, and that the CEP17 region is subject to chromosomal gains similar to other regions. Performing additional FISH using probes to the SMS, RARA and TP53 genes in order to obtain a HER2/chromosome-17-reference ratio is an effective way to determine the true HER2 amplification status in these

Advancing Breast Cancer HER2 FISH Quality by Image Analysis.

Abstract Background: A critically important factor in the correlation of Her2 amplification and clinical outcome in breast cancer is the quality of histologic Her2 amplification detection. FISH is the “gold standard” for HER2 amplification detection but presently most labs use manual methods which are labour intensive and restricted by analysis time to small samples (60 cells or less if amplification status appeared clear to the observer).An ideal FISH detection system should be rapid (less than 30 minutes per sample), identify HER2/neu and CEP17 copy number objectively in each cell, count sufficient cells to be representative statistically, count Her2/neu in cell clusters objectively, and have a permanent record of each cell counted. Our objective is to develop an image analysis system for Her2/neu FISH that is superior to current manual and automated methods in all the above criteria.Materials and Methods: The Visiopharm Integrator System software and a Leica DM6000B fluorescence microscope equipped with a Prior 8-slide capacity motorized stage (Mac 5000 ps system stage control) and Hamamatsu camera (Model CA4742-80-12AG) were used for image analysis. Forty breast cancer biopsies, 15 core biopsies included, were examined for Her2 FISH previously assessed manually on the same slide. For manual detection, at least 3 representative fields were selected by the observer. For image analysis, unbiased tumour sampling, typically 16 fields, was assessed within a region of interest previously identified. Methods were compared by ASCO/CAP amplification criteria and by assessment of technical time, cells counted and objectivity of counting criteria.Results:Comparison manual vs image analysisMethodManualImage AnalysisTechnical time: minutes, average, range50 (45-135)25 (20-40)Cells counted: average, range58 (20-217)238 (18-1151)HER2 in clusterssubjectiveobjective and reproducibleASCO/CAP amplified/equivocal19/217/3ASCO/CAP not amplified/uninterpretable18/120/0 Seven discordant cases were observed. Five cases were downgraded by image analysis. Two cases, one uninterpretable manually and one seen as not amplified manually, were seen as amplified by image analysis.Discussion: Image analysis FISH with Visiopharm software allows for establishment of finite cell inclusion criteria reflecting size, circularity and other measurable cell features. Image analysis facilitates higher cell counts without observer selection bias in less time, and with smaller increases in technical time as more cells are counted. Discordance may be attributable to heterogeneity with larger sample of cells assessed and objective assessment of clusters in the image analysis method. Our image analysis protocol demonstrated successfully the quantification of HER2 in separate signals, in clusters and in split signals. HER2/CEP17 copy numbers were determined for each cell and for more cells in much less time while providing a permanent image record of all cells assessed.Image analysis has promise to improve substantially the quality of Her2 FISH assessment in breast cancer biopsies.Supported in part by an unrestricted grant from Hoffman-LaRoche Ltd. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6015.

Absence of chromosome 17 polysomy in breast cancer: analysis by CEP17 chromogenic in situ hybridization and multiplex ligation-dependent probe amplification

Amplification of the HER2 gene, present in 15-30% of breast carcinomas, correlates with poor outcome and is an indication for treatment with trastuzumab. Standard testing methods for HER2 amplification are fluorescence (FISH) or chromogenic in situ hybridization (CISH). In FISH/CISH scoring, correction for chromosome 17 polysomy is believed to be critical for determination of true HER2 amplification as opposed to increased chromosome 17 copy number. The term "polysomy 17" is widely used and defined as > or =3 copies of the chromosome 17 centromere (probe CEP17, D17Z1). Thus, the centromere is assumed to be representative for the entire chromosome. This study aimed to investigate the frequency of polysomy 17 and its association with HER2 amplification in 111 invasive breast cancer patients by CEP17 CISH and by copy number analysis of a set of 17 genes along chromosome 17 using multiplex ligation-dependent probe amplification (MLPA).Chromosome 17 usually showed a complex pattern of gains and losses by MLPA, unrelated to the copy number status of the centromere. Increase in centromere 17 copy number (denoted "polysomy 17"), as assessed by CEP17 CISH, was found in 19% of the patients. Of these patients, 60% also showed amplification of HER2 measured by MLPA. However, none of the 111 patients showed a true polysomy of chromosome 17 by MLPA. Only two patients (1.8%) had a possible gain of 17q. Amplification of 17p was not found in any of the patients, although a possible loss of 17p was found in one patient. In conclusion, this extensive analysis of amplicons along chromosome 17 shows that true polysomy of chromosome 17, either of the whole chromosome or of the short or the long arm, is very rare in invasive breast cancer. Abnormal CEP17 copy numbers may therefore actually stem from high level gains or amplification of CEP17 regardless of copy number gains of the short and long arms of chromosome 17 and, at least in some cases, correction with CEP17 probes may provide misleading HER2 gene status assessment results.

283 Delineating the cellular pathways and molecular determinants of normal and leukemic hematopoiesis

Increased copy number of chromosome enumeration probe (CEP) targeting centromere 17 is frequently encountered during HER2 in situ hybridization (ISH) in breast cancer. The aim of this study was to clarify the clinicopathologic significance of CEP17 copy number gain in a relatively large series of breast cancer patients. We analyzed 945 cases of invasive breast cancers whose HER2 fluorescence ISH reports were available from 2004 to 2011 at a single institution and evaluated the association of CEP17 copy number gain with clinicopathologic features of tumors and patient survival. We detected 186 (19.7%) cases of CEP17 copy number gain (CEP17 ≥ 3.0) among 945 invasive breast cancers. In survival analysis, CEP17 copy number gain was not associated with disease-free survival of the patients in the whole group. Nonetheless, it was found to be an independent adverse prognostic factor in the HER2-negative group but not in the HER2-positive group. In further subgroup analyses, CEP17 copy number gain was revealed as an independent poor prognostic factor in HER2-negative and hormone receptor-positive breast cancers, and it was associated with aggressive histologic variables including high T stage, high histologic grade, lymphovascular invasion, p53 overexpression, and high Ki-67 proliferative index. In conclusion, we found that elevated CEP17 count can serve as a prognostic marker in luminal/HER2-negative subtype of invasive breast cancer. We advocate the use of the dual-colored fluorescence ISH using CEP17 rather than the single-colored one because it gives additional valuable information on CEP17 copy number alterations.

Does chromosome 17 centromere copy number predict polysomy in breast cancer? A fluorescence in situ hybridization and microarray‐based CGH analysis

Approximately 8% of breast cancers show increased copy numbers of chromosome 17 centromere (CEP17) by fluorescence in situ hybridization (FISH) (ie average CEP17 >3.0 per nucleus). Currently, this pattern is believed to represent polysomy of chromosome 17. HER2-amplified cancers have been shown to harbour complex patterns of genetic aberrations of chromosome 17, in particular involving its long arm. We hypothesized that aberrant copy numbers of CEP17 in FISH assays may not necessarily represent true chromosome 17 polysomy. Eighteen randomly selected CEP17 polysomic cases and a control group of ten CEP17 disomic cases, as defined by dual-colour FISH, were studied by microarray-based comparative genomic hybridization (aCGH), which was performed on microdissected samples using a 32K tiling-path bacterial artificial chromosome microarray platform. Additional FISH probes were employed for SMS (17p11.2) and RARA (17q21.2) genes, as references for chromosome 17 copy number. Microarray-based comparative genomic hybridization revealed that 11 out of the 18 polysomic cases harboured gains of 17q with involvement of the centromere, one displayed 17q gain sparing the centromeric region, and only one could be defined as polysomic. The remaining five cases displayed amplification of the centromeric region. Among these, one case, showing score 2+ by immunohistochemistry and 8.5 HER2 mean copy number, was classified as not amplified by HER2/CEP17 ratio and as amplified by HER2/SMS ratio. Our results suggest that true chromosome 17 polysomy is likely to be a rare event in breast cancer and that CEP17 copy number greater than 3.0 in FISH analysis is frequently related to gain or amplification of the centromeric region. Larger studies investigating the genetic profiles of CEP17 polysomic cases are warranted.

Effect of High Copy Number of HER2 Associated With Polysomy 17 on HER2 Protein Expression in Invasive Breast Carcinoma

HER2 gene amplification by fluorescence in situ hybridization and protein expression by immunohistochemistry (IHC) have been used for prognosis and guiding treatment of invasive ductal carcinoma of the breast with trastuzumab. Accurate evaluation of HER2 status is important in the management of patients with candidacy for the HER2-targeting therapy. Despite previous studies, effects of polysomy of chromosome 17, at which HER2 is located, on HER2 protein expression remains controversial. In this study, we calculated the average copy numbers of HER2 and chromosome 17 (CEP17) per nucleus in 109 cases of invasive breast carcinoma and analyzed their correlations with the HER2/CEP17 ratio and protein expression. As expected, there were close correlations between HER2 protein expression and the HER2/CEP17 ratio (CC: 0.49, P<0.001), along with the HER2 copy number per nucleus (CC: 0.48, P<0.001) and between the CEP17 copy number per nucleus and the HER2 copy number per nucleus (CC: 0.45, P<0.001). Correlation between the CEP17 copy number per nucleus and the HER2/CEP17 ratio was not significant (CC: 0.2, P>0.05). There was a weak, but statistically insignificant, correlation between the CEP17 copy number per nucleus and HER2 protein expression by IHC (CC: 0.26, P>0.05). The cases were then grouped on the basis of the amplification of the HER2 gene by fluorescence in situ hybridization. In the cases that showed no amplification, there was a significantly higher CEP17 copy number per nucleus in cases with strong HER2 protein expression (2.99) when compared with cases with weak (2.39) or absent (1.86) expression. In conclusion, a high HER2 gene copy number-associated polysomy 17 is a significant contributing factor in HER2 protein overexpression in unamplified invasive breast carcinomas. Cases without HER2 amplification, but carrying polysomy 17, should be further evaluated for HER2 protein overexpression by IHC. Those with polysomy 17-associated HER2 protein overexpression, like any other IHC+ ones, should be eligible candidates for trastuzumab therapy.

Amplification involving the CEP17 region may lead to false negative results of HER2 gene amplification by FISH.

Abstract Abstract #1079 Background: Detection of HER2 gene amplification by fluorescence in-situ hybridization (FISH) is one of the key tests predicting clinical response of breast cancer to trastuzumab. According to the recent CAP/ASCO guidelines for HER2 testing, the tumor is considered positive for HER2 gene amplification when the HER2/CEP17 ratio is greater than 2.2, negative if less than 1.8, and equivocal if the ratio is between 1.8 and 2.2. The need to use CEP17 as reference for HER2 evaluation is supported by the observation that an increased HER2 gene copy number as a result of chromosome 17 polysomy may not bear the same clinical significance as HER2 gene amplification. However, response to trastuzumab has been observed in some cases with polysomy 17. This study aims to examine the reliability of using CEP17 for chromosome 17 enumeration.&amp;#x2028; Material and methods:&amp;#x2028; 40 cases of HER2 FISH performed at the PhenoPath Laboratories between October 2007 and May 2008 having a mean CEP17/cell of over 2.5 were selected. In order to confirm the elevated chromosome 17 copy number, FISH mapping studies were performed using probes for the Smith-Magenis syndrome (SMS) and retinoic acid receptor alpha (RARA) loci which reside on the short arm and long arm of chromosome 17 respectively and are telomeric to the centromere/HER2 loci. Chromosome 17 status was determined by correlating the mean copy numbers of HER2, CEP17, SMS and RARA. A gene locus with signal number/cell between 1.4 and 2.4 was considered consistent with eusomy. Additional studies of the TP53 locus which is telomeric to SMS on 17p was performed on a subset of cases having only one gene locus in the eusomic range.&amp;#x2028; Results:&amp;#x2028; The CEP17/cell of the 40 cases ranges from 2.61 to &amp;gt;10 with a median of 3.92. 32 cases (80%) show at least one gene locus with copy numbers consistent with eusomy (17 [42.5%] have one gene locus and 15 [37.5%] have two gene loci showing eusomy). The TP53 locus was studied in 7 cases having only one gene locus in the eusomic range. Six of these cases also showed gene copy numbers consistent with eusomy. Overall, 9 cases (22.5%) would have the HER2 amplification status upgraded (negative to positive, negative to equivocal or equivocal to positive) if a gene locus showing eusomy was used as reference for HER2 evaluation.&amp;#x2028; Discussion:&amp;#x2028; The result of the present study shows that CEP17 may not be a reliable internal reference standard in HER2 FISH studies in a subset of cases. A majority of the apparent polysomy cases based on CEP17 copy number likely represent low-level amplification of the chromosome 17 centromere and would have been shown to be eusomic if another gene locus were used as a reference. The HER2/CEP17 ratio could be falsely low in as high as 22% of the cases with apparent polysomy 17. Additional FISH studies of other gene loci on the chromosome 17 are recommended in all HER2 FISH negative/equivocal cases with apparent polysomy 17. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1079.

The Influence of Polysomy 17 on HER2 Gene and Protein Expression in Adenocarcinoma of the Breast

Breast carcinomas with amplification of HER2 on chromosome 17 are associated with HER2 protein overexpression, adversely affecting prognosis and predicting response to Herceptin therapy. Chromosome 17 polysomy is encountered in assessing HER2 gene status, and its impact on HER2 gene and protein expression remains unclear. This impact was investigated in breast carcinomas identified by fluorescence in situ hybridization (FISH) to have a gain of chromosome 17 (CEP17+; n = 56), using a dual probe assay, which detects HER2 gene copy number and enumerates chromosome 17 (HER2/CEP17; Vysis). Cases were immunostained for HER2 protein (CB-11, Ventana), and scored blinded to FISH. A subgroup was evaluated by isotopic in situ hybridization for HER2 mRNA expression. Controls included ten HER2 amplified and ten nonamplified tumors, eusomic for chromosome 17. Immunohistochemistry (IHC) for HER2 protein was negative (0 or 1+) in 69% (39 of 56), 2+ in 27% (15 of 56), and 3+ in 3% (2 of 56) of CEP17+ cases. The mean CEP17 copy number among the three groups was similar (3.1, 3.0, and 3.1 for IHC 0/1+, 2+, and 3+, respectively). Isotopic in situ hybridization for HER2 mRNA performed on 26 CEP17+ cases (16 IHC 0-1+, 10 IHC 2+ or 3+) showed no increased HER2 mRNA expression (normalized to beta-actin mRNA). The mRNA expression and the IHC staining of the HER2-amplified and nonamplified controls was concordant with their FISH status. These results suggest that chromosome 17 polysomy in the absence of HER2 amplification does not have a significant biologic influence on HER2 gene expression in breast carcinoma.