Heterogeneity Of HER2 Gene Amplification Research Articles

HER2 Mutated and Nonmutated Non-small Cell Lung Carcinomas Can Harbor Heterogeneous HER2 Gene Amplification and HER2 Protein Expression.

Molecular analyses have become mandatory for treatment choices in patients with advanced non-small cell lung cancers (NSCLC). Among them, HER2 gene mutation, HER2 gene amplification, and HER2 protein expression consist in potential targets of various treatments. Tumor heterogeneity and overlapping of molecular alterations may cause dilemmas in treatment choices but to date there are few that reported about HER2 with discrepant data. We led a retrospective study evaluating HER2 protein expression and HER2 gene/chromosome 17 copy number variations across different tumor areas and samples from patients with advanced NSCLC harboring HER2 gene mutations and other oncogenic mutations. Among patients with HER2-mutated (10 patients) and nonmutated lung adenocarcinomas (10 patients), we observed frequent heterogeneous HER2 protein expression with no correlation with HER2 gene copy number variations. HER2 gene amplification was observed in 6 patients (3 HER2-mutated and 3 HER2-nonmutated), but with intrasample heterogeneity in 2 cases and intersample heterogeneity in another case. Our small case series emphasizes the potential overlapping and spatial heterogeneity of HER2 alterations in NSCLC, which must be taken into account as a limitation in building predictive strategies accompanying the development of anti-HER2 therapeutic strategies in patients with advanced NSCLC.

Somatic mutations and copy number variations in breast cancers with heterogeneous HER2 amplification.

Intratumour heterogeneity fuels carcinogenesis and allows circumventing specific targeted therapies. HER2 gene amplification is associated with poor outcome in invasive breast cancer. Heterogeneous HER2 amplification has been described in 5–41% of breast cancers. Here, we investigated the genetic differences between HER2‐positive and HER2‐negative admixed breast cancer components. We performed an in‐depth analysis to explore the potential heterogeneity in the somatic mutational landscape of each individual tumour component. Formalin‐fixed, paraffin‐embedded breast cancer tissue of ten patients with at least one HER2‐negative and at least one HER2‐positive component was microdissected. Targeted next‐generation sequencing was performed using a customized 53‐gene panel. Somatic mutations and copy number variations were analysed. Overall, the tumours showed a heterogeneous distribution of 12 deletions, 9 insertions, 32 missense variants and 7 nonsense variants in 26 different genes, which are (likely) pathogenic. Three splice site alterations were identified. One patient had an EGFR copy number gain restricted to a HER2‐negative in situ component, resulting in EGFR protein overexpression. Two patients had FGFR1 copy number gains in at least one tumour component. Two patients had an 8q24 gain in at least one tumour component, resulting in a copy number increase in MYC and PVT1. One patient had a CCND1 copy number gain restricted to a HER2‐negative tumour component. No common alternative drivers were identified in the HER2‐negative tumour components. This series of 10 breast cancers with heterogeneous HER2 gene amplification illustrates that HER2 positivity is not an unconditional prerequisite for the maintenance of tumour growth. Many other molecular aberrations are likely to act as alternative or collaborative drivers. This study demonstrates that breast carcinogenesis is a dynamically evolving process characterized by a versatile somatic mutational profile, of which some genetic aberrations will be crucial for cancer progression, and others will be mere ‘passenger’ molecular anomalies.

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers.

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast cancers. However, a minority of cases displays a heterogeneous distribution of HER2-positive cells, which generates major clinical challenges. To date, no reliable and standardized protocols for the characterization and quantification of HER2 heterogeneous gene amplification in large cohorts have been proposed. Here, we present a high-throughput methodology to simultaneously assess the HER2 status across different topographic areas of multiple breast cancers. In particular, we illustrate the laboratory procedure to construct enhanced tissue microarrays (TMAs) incorporating a targeted mapping of the tumors. All TMA parameters have been specifically optimized for the silver in situ hybridization (SISH) of formalin-fixed paraffin-embedded (FFPE) breast tissues. Immunohistochemical analysis of the prognostic and predictive biomarkers (i.e., ER, PR, Ki67, and HER2) should be performed using automated procedures. A customized SISH protocol has been implemented to allow a high-quality molecular analysis across multiple tissues that underwent different fixation, processing, and storage procedures. In this study, we provide a proof-of-principle that specific DNA sequences could be localized simultaneously in distinct topographic areas of multiple and heterogeneously processed breast cancers using an efficient and cost-effective method.

Clinicopathologic Significance of the Intratumoral Heterogeneity of HER2 Gene Amplification in HER2-Positive Breast Cancer Patients Treated With Adjuvant Trastuzumab.

Although intratumoral heterogeneity of human epidermal growth factor receptor 2 (HER2) gene amplification has been associated with a poor prognosis for primary HER2-positive breast cancer and metastatic HER2-positive breast cancer treated with trastuzumab, the clinicopathologic significance in a setting involving trastuzumab treatment as an adjuvant treatment has not been studied in patients. We retrospectively investigated 443 patients with HER2-positive breast cancer treated with surgery, adjuvant chemotherapy, and 1 year of trastuzumab. Three areas that showed different levels of HER2 protein expression were chosen, and silver in situ hybridization was performed. HER2 regional and genetic heterogeneity was found in 6.2% and 6.8% of tumors, respectively. Both types of heterogeneity were significantly associated with hormone receptor positivity, HER2 immunohistochemistry score of 2+, a low level of HER2 gene amplification, and absence of an extensive intraductal component. Genetic heterogeneity also showed strong correlation with a lower histologic grade. In the hormone receptor-positive group, the regional heterogeneity affected disease-free survival of patients (hazard ratio, 4.869; 95% confidence interval, 1.424-16.646; P = .005), whereas genetic heterogeneity did not. Evaluation of intratumoral heterogeneity, especially in cases with hormone receptor positivity, may be valuable for assessing the prognosis of HER2-positive patients anticipating treatment with adjuvant systemic therapy and trastuzumab.

Intra-tumor genetic heterogeneity and alternative driver genetic alterations in breast cancers with heterogeneous HER2 gene amplification.

BackgroundHER2 is overexpressed and amplified in approximately 15% of invasive breast cancers, and is the molecular target and predictive marker of response to anti-HER2 agents. In a subset of these cases, heterogeneous distribution of HER2 gene amplification can be found, which creates clinically challenging scenarios. Currently, breast cancers with HER2 amplification/overexpression in just over 10% of cancer cells are considered HER2-positive for clinical purposes; however, it is unclear as to whether the HER2-negative components of such tumors would be driven by distinct genetic alterations. Here we sought to characterize the pathologic and genetic features of the HER2-positive and HER2-negative components of breast cancers with heterogeneous HER2 gene amplification and to define the repertoire of potential driver genetic alterations in the HER2-negative components of these cases.ResultsWe separately analyzed the HER2-negative and HER2-positive components of 12 HER2 heterogeneous breast cancers using gene copy number profiling and massively parallel sequencing, and identified potential driver genetic alterations restricted to the HER2-negative cells in each case. In vitro experiments provided functional evidence to suggest that BRF2 and DSN1 overexpression/amplification, and the HER2 I767M mutation may be alterations that compensate for the lack of HER2 amplification in the HER2-negative components of HER2 heterogeneous breast cancers.ConclusionsOur results indicate that even driver genetic alterations, such as HER2 gene amplification, can be heterogeneously distributed within a cancer, and that the HER2-negative components are likely driven by genetic alterations not present in the HER2-positive components, including BRF2 and DSN1 amplification and HER2 somatic mutations.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0657-6) contains supplementary material, which is available to authorized users.

Non-small cell lung cancer: HER2 oncogene status

to study HER2 protein and HER2 gene, their heterogeneity in non-small cell lung cancer. 218 intraoperative non-small cell lung samples were examined using tissue matrix methods. HER2 protein was determined by immunohistochemistry (clone 4B5, <<Ventana>> and HER2 gene and CEP1 7 were evaluated by in situ hybridization (SISH, <<Ventana>>). Positive and indefinite statuses were found in 59 (27%) and 47 (22%) cases, respectively; intratumor heterogeneity was detected in 32 (30%) cases. Amplification of the HER-2 gene was found in 12 (6%) cases; that of the HER2 gene along with an increase in CEPI 7 was observed in 7 (3%) cases; elevated CEP1 7 levels were seen in 19 (9%) cases. Intratumor heterogeneity of HER2 gene amplification was not found; however, one case of adenocarcinoma showed high-level HER2 gene amplification in the gland-like areas and low-level HER2 gene amplification in the solid areas. HER2-positive status and amplification were more common in adenocarcinoma than in squamous cell carcinoma (p<0.001). There was a moderate correlation between HER2 immunohistochemical status and amplification (r=0.38; p<0.001). Thus, in non-small cell lung cancer, there is an elevated HER2 protein level and, well less frequently, altered activity in the HER2 gene (amplification) as a cause of enhanced protein synthesis.

HER2 heterogeneity affects trastuzumab responses and survival in patients with HER2-positive metastatic breast cancer.

Heterogeneity of HER2 gene amplification is found in a subset of breast cancers. We investigated the impact of HER2 heterogeneity on trastuzumab responses and clinical outcomes in 112 patients with HER2-positive metastatic breast cancer. Regional and genetic heterogeneity of HER2 gene amplification was determined in three different areas of each tumor by immunohistochemistry and silver in situ hybridization. We also assessed the overall levels of HER2 amplification and the proportion of tumor cells with a HER2/CEP17 ratio of more than 2.2 or strong and complete membranous (3+) expression of HER2 protein. HER2 regional and genetic heterogeneity based on the HER2/CEP17 ratio was confirmed in 8.7% and 2.7% of cases, respectively. Poor response to trastuzumab was associated with overall low-level or equivocal amplification, HER2 regional heterogeneity by the HER2/CEP17 ratio, the HER2/CEP17 ratio of more than 2.2 in less than 80% of tumor cells, and HER2 immunohistochemical expression of 3+ in less than 75% of tumor cells. In survival analyses, low-level or equivocal HER2 amplification, HER2 regional heterogeneity based on the HER2/CEP17 ratio, and the HER2/CEP17 ratio of more than 2.2 in less than 80% of tumor cells were associated with shorter time to progression and lower overall survival in univariate and multivariate analyses. These results suggest that accurate assessment of HER2 status, including HER2 heterogeneity, is important in predicting trastuzumab responses and outcomes in patients with HER2-positive metastatic breast cancer.

Intratumoral heterogeneity of HER2 gene amplification in occult breast cancer

Intratumoral heterogeneity of HER2 gene amplification in occult breast cancer

Abstract P1-08-38: HER2 heterogeneity affects trastuzumab responses and survival in patients with HER2-postive metastatic breast cancer

Abstract Purpose: Heterogeneity of HER2 gene amplification is found in a subset of breast cancers. However, it is not known whether breast cancers with heterogeneous HER2 amplification respond differently to HER2-targeted therapy than those with homogeneous amplification. In this study, we investigated the relationship between HER2 heterogeneity and trastuzumab resistance and clinical outcomes in patients with HER2-positive metastatic breast cancer. Patients and methods: We studied tumor tissues from 127 patients with HER2-positive metastatic breast cancers who had received trastuzumab-based chemotherapy. Regional and genetic heterogeneity of HER2 amplification was determined in three different areas of each tumor by immunohistochemistry (IHC) and silver in situ hybridization. We also assessed the overall levels of HER2 amplification, and the proportion of tumor cells with a HER2/CEP17 ratio &amp;gt;2.2 or HER2 overexpression (IHC score of 3+). HER2status including HER2 heterogeneity was correlated with trastuzumab responses and survival of the patients. Results: HER2regional and genetic heterogeneity was confirmed in 7.8% and 3.6% of cases, respectively. Poor response to trastuzumab was associated with overall low-level amplification, HER2regional heterogeneity, HER2/CEP17 ratio &amp;gt;2.2 in &amp;lt;80% of tumor cells, and HER2 IHC score of 3+ in &amp;lt;75% of tumor cells. In survival analyses, low-level HER2 amplification, HER2regional heterogeneity, and HER2/CEP17 ratio &amp;gt;2.2 in &amp;lt;80% of tumor cells were associated with shorter time to progression and lower overall survival in univariate and multivariate analyses. Conclusion: Accurate assessment of HER2 status including HER2 heterogeneity is important in predicting trastuzumab responses and outcomes in patients with HER2-positive metastatic breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-38.

Adverse prognostic impact of intratumor heterogeneous HER2 gene amplification in patients with breast cancer.

617 Background: There is increasing recognition of the existence of intratumoral heterogeneity of the human epidermal growth factor receptor (HER2), which affects interpretation of HER2 positivity in clinical practice and may have implications for patient prognosis and treatment. Only patients (pts) with HER2 positivity - defined as 3+ by IHC or FISH amplified defined as a HER2 gene to chromosome 17 (HER2/CEP17) ratio ≥ 2.0 - are eligible to receive trastuzumab treatment. Limited information is available on the prognosis of pts with HER2 2+ or FISH test with a HER2/CEP17 ratio &lt; 2.0. Methods: We retrospectively analyzed data from 455 consecutive early BC pts with HER 2+ and HER2/CEP17 ratio &lt; 2.0 who underwent surgery after 2007. The association between HER2/CEP17 ratio and other known prognostic factors was evaluated with multivariable linear regression models. The role of HER2/CEP17 ratio on recurrence free survival was assessed with multivariable Cox regression models. Results: Fifty-one percent of the evaluated pts were node negative, 51% were postmenopausal, 93% had ER positive BC and 85% had Ki-67 ≥14%. The mean HER2/CEP17 ratio was 1.27 (SD=0.3). A significant positive relationship between HER2/CEP17 ratio and Ki-67 was observed (p&lt;0.01). During a median follow-up time of 2.7 years, 40 recurrences were observed (15 locoregional events and 25 distant metastases). Overall, the association between HER2/CEP17 and the risk of recurrence was not significant. From subgroup analysis, a significant interaction between HER2/CEP17 ratio and nodal involvement emerged (p=0.02). Among pts with node-negative disease, pts with HER2/CEP17 ratio ≥1.27 were at higher risk of recurrence with respect to pts with HER2/CEP17 ratio &lt; 1.27 (adjusted HR 4.0, 95% CI 1.01-15.9). Conclusions: Among pts with BC and HER2 intratumoral heterogeneity, HER2/CEP17 ratio ≥1.27 could have a strong prognostic role in node negative HER2 2+ BC, thus suggesting potential future therapeutic approaches in this setting of pts.

Abstract PD05-08: Genomic characterisation of invasive breast cancers with heterogeneous HER2 gene amplification

Abstract Aims: HER2 gene amplification is observed in up to 15% of breast carcinomas. In a rare subset of breast cancers classified as HER2-positive by immunohistochemistry and in situ hybridisation, HER2 overexpression and gene amplification are restricted to a subset of &amp;gt;30% but not all cancer cells. Here we sought to characterise the repertoire of gene copy number aberrations and somatic mutations in the HER2-positive and HER2-negative components of cases with heterogeneous HER2 overexpression and gene amplification. Material and methods: Cases diagnosed as HER2 positive but with &amp;gt;30% but &amp;lt;100% of cells displaying HER2 overexpression were retrieved from the authors' institutions. HER2 heterogeneity status was re-assessed using immunohistochemistry and chromogenic and/ or fluorescence in situ hybridisation. For cases with confirmed HER2 gene amplification heterogeneity, HER2-positive and HER2-negative components were microdissected from tissue sections stained with the Herceptest antibody. DNA samples extracted from both components of each case were subjected to microarray-based comparative genomic hybridisation (aCGH), using a 32K BAC array platform with 50Kb resolution. The HER2-positive and HER2-negative components of cases with frozen material were also subjected to massively parallel targeted exome sequencing. Results: Twelve cases yielded sufficient DNA for aCGH analysis. Tumours were preferentially ER positive (83%) and of histological grade 3 (67%). The HER2-positive and HER2-negative components of all cases shared most of the copy number aberrations. A pairwise comparison of the genomic profiles of the two components from each case revealed that in ten of the twelve cases, copy number aberrations in addition to 17q12 amplification encompassing the HER2 gene locus were restricted to one of the two components. Exome sequencing of two cases suggested that the HER2-positive and HER2-negative components from each case harboured &amp;gt;30 somatic mutations in common, including identical TP53 somatic mutations in both components of each case. The HER2-negative component of one of the cases displayed a somatic mutation in NRG2, an ERBB receptor ligand, and the HER2-negative component of the other case harboured a mutation in PTTG1IP, a proto-oncogene with putative oestrogen receptor elements. Conclusions: Our results demonstrate that in HER2-positive breast cancers with heterogeneous HER2 gene amplification, the HER2-positive and HER2-negative components are clonally related. The distinct genomic profiles of HER2-positive and HER2-negative components, however, suggest that, at least in some of these cases, HER2 amplification may constitute a relatively late event in tumour evolution. Exome sequencing revealed mutations restricted to the HER2-negative components of HER2-positive tumours with heterogeneous HER2 overexpression/gene amplification, which may constitute potential drivers in the absence of HER2 overexpression/gene amplification. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD05-08.

Adverse Prognostic Impact of Intratumor Heterogeneous HER2 Gene Amplification in Patients With Esophageal Adenocarcinoma

There is increasing recognition of the existence of intratumoral heterogeneity of the human epidermal growth factor receptor (HER2), which affects interpretation of HER2 positivity in clinical practice and may have implications for patient prognosis and treatment. We determined the frequency and prognostic impact of heterogeneous HER2 gene amplification and polysomy 17 in patients with esophageal adenocarcinoma (EAC). HER2 amplification (by fluorescence in situ hybridization) was examined in surgical EAC specimens (n = 675). HER2 heterogeneity was defined according to consensus guidelines as gene amplification (HER2/CEP17 ratio ≥ 2.0) in more than 5% but less than 50% of cancer cells. No patient received neoadjuvant or HER2-targeted therapy. Cox models were used to assess disease-specific survival (DSS) and overall survival (OS). Overall, 117 EACs (17%) demonstrated HER2 amplification, of which 20 (17%) showed HER2 heterogeneity. All HER2-heterogeneous tumors were amplified. Among HER2-amplified tumors, heterogeneous tumors had significantly higher frequency of poor histologic grade and polysomy 17. In multivariable models that included number of metastatic lymph nodes, grade, tumor stage, and polysomy 17, only HER2 heterogeneity and node number were prognostic among HER2-amplified tumors, with heterogeneity showing worse DSS (hazard ratio, 2.04; 95% CI, 1.09 to 3.79; P = .025) and OS (P = .026). Among HER2-nonamplified EACs, polysomy 17 was independently associated with worse DSS (P = .012) and OS (P = .023). Among HER2-amplified EACs, 17% show HER2 heterogeneity, which independently predicts for worse cancer-specific death. Among HER2-nonamplified EACs, polysomy 17 is independently associated with worse survival. These novel findings demonstrate aggressive subgroups in HER2-amplified and -nonamplified EACs that have important implications for HER2 analysis and determination of benefit from HER2-targeted therapy.

Intratumoral heterogeneity of HER2 gene amplification in breast cancer: its clinicopathological significance

AbstractIntratumoral heterogeneity of human epidermal growth factor receptor 2 (HER2) gene amplification has been reported to occur with variable frequencies in breast cancers. However, there have been few studies of its clinicopathological significance. We used tissue microarrays to evaluate two aspects of intratumoral heterogeneity of HER2 gene amplification: regional heterogeneity and genetic heterogeneity. We examined 96 invasive breast cancers in which HER2 amplification had been diagnosed in whole sections, and determined the clincopathological characteristics of those tumors. HER2 regional heterogeneity, defined as the existence of amplification/negative or amplification/equivocal patterns in different tissue microarray cores of a tumor, was present in 17 (18%) of the 96 cases. HER2 genetic heterogeneity, defined as the presence of tumor cells with a HER2/chromosome enumeration probe 17 ratio higher than 2.2 in 5–50% of the tumor cells, was found in 11 cases (11%), all of which showed HER2 regional heterogeneity. The cases with intratumoral heterogeneity of HER2 gene amplification were characterized by low grade or equivocal HER2 amplification and equivocal (2+) HER2 expression in whole sections. The patients with intratumoral heterogeneity of HER2 gene amplification had significantly shorter disease-free survival times than those with homogeneous HER2 gene amplification, and this effect was also evident in subgroup analysis by hormone receptor status. In multivariate analysis, intratumoral HER2 heterogeneity retained its status as an independent prognostic factor for disease-free survival. In conclusion, intratumoral heterogeneity of HER2 gene amplification is present in a subset of HER2-amplified breast cancers, especially in cases with low-grade HER2 amplification and equivocal HER2 expression, indicating a need for HER2 testing on more representative, larger tumor samples for accurate assessment of HER2 status in such cases. The patients with this heterogeneity have decreased disease-free survival, suggesting that genetic instability, and hence aberrant HER2 amplification in subclones of such tumors, may be associated with breast cancer progression.

P3-05-07: Genetic Heterogeneity of Amplification Status in Breast Invasive Carcinoma with 2+ HER2 Immunostaining: What Can We Learn?

Abstract Background The genetic heterogeneity of HER2 gene amplification (GA) in breast cancer has previously been described, but the clinical significance of this phenomenon remains unknown. We studied the genetic categories of a series of consecutive 2+ IHC cases over 5 years, with a focus on cases with HER2 GA detected in minor clone(s). We compared the HER2 status in primary tumors and positive axillary lymph nodes (ALN) when available to test the hypothesis that HER2 amplified cells are more aggressive and metastase quicker than non amplified cells. Material and methods: From January 1st. 2006 to May 30. 2011, 4491 invasive breast carcinomas had HER2 immunohistochemical (IHC) and/or HER2 gene status evaluation on their tumor sample in Institut Gustave Roussy. The distribution according to their IHC was as follows: 0 in 2915 cases (65%), 1+ in 569 cases (12.6%), 2+ in 536 cases (11.8%) and 3+ in 471 cases (10.6%). All 2+ samples were checked by Fluorescence in situ hybridization (FISH). For each case we analysed 100 invasive cells in 10 microscopic fields. Heterogeneous amplification was defined as presence of 5–50% of amplified cells (ratio&amp;gt;2,2) within the tumors otherwise classified as not amplified or borderline (ratio for 100 cells: &amp;lt;2,2 according to the ASCO 2007 and CAP criteria). The IHC 2+ tumors show five main genetic categories. Results: FISH with HER2/cen17 probes can classify IHC 2+ tumors into 5 genetic categories: normal gene status (two chromosomes (chr) 17 and two HER2 genes): 90 cases; chr 17 “ polysomy “/17q gain: 145 cases; HER2 gain: 122 cases; HER2 amplification (major clone): 135 cases and chr 17 monosomy: 44 cases. We focused our interest to IHC 2+ cases where the major clone showed no HER2 amplification, but minority clone (cut off 5%) showed the HER2 amplification (ratio &amp;gt;2,2 or &amp;gt;6 HER2 copies per cell). We found 48 such cases (10% of 2+ cases). These cases had been reported as not amplified (42/48) or borderline (6/48). To find out if the amplified cells are those which metastase, we checked 10 involved ALN: they all showed the similar genetic heterogeneity as a primary tumor with a global ratio &amp;lt; 2,2. Other cases with involved ALN are in ongoing study by FISH. Conclusions: The genetic heterogeneity of IHC 2+ tumors is a common event and five different genetic categories can be detected. A part of the HER2 negative cases contained one or several HER2 amplified clones. Preliminary results show a similar genetic heterogeneity of metastatic population. Our results on the small series of cases do not confirm the hypothesis that the only amplified clones metastase, but all clones (amplified and not amplified) has a metastatic capacity. A multi-center collaboration is needed to collect the high number of cases with heterogeneous amplification to: 1) Find out a proportion of such patients in HER2 IHC 2+ group (10% in our study). 2) Study a status of the gene in relapse cases. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-05-07.

Abstract P6-05-03: Analysis of College of American Pathologists Recommendations for Reporting HER2 Intra-Tumoral heterogeneity (ITH) on 1329 Breast Cancers

Abstract Background: The College of American Pathologists (CAP) recently published recommendations for reporting intratumoral heterogeneity for HER2 gene amplification in breast cancers. These guidelines recommend reporting cases with between 5-50% of cells with HER2:CEP17 ratios &amp;gt; 2.2 (or &amp;gt; 6 HER2 signals/cell) as “heterogeneous for HER2 gene amplification.” Because these recommendations lack biological and clinical foundation, we applied them to a large number of historical breast cancers and compared the ITH classification to routine FISH Her2 results. Materials and Methods: We collected Her2 and CEP17 counts in 32,116 tumor cells from 1329 consecutive breast cancer cases analyzed by FISH. Data from Excel counting sheets were imported to a SQL Server database for analysis and statistics; histograms and analyses were also performed in Excel. We specifically computed concordances between guidelines for traditional and intra-tumoral heterogeneity (ITH) reporting. Results: 309 of 1,329 cases (23%) met the criteria for heterogeneous HER2 gene amplification by HER2:CEP17 ratio, but &amp;gt; 79% of these were non-amplified by standard FISH reporting criteria. Using Her2 signals per cell, ITH was found in 87 (6.5%) of cases and most of these were not amplified or equivocal by standard criteria. Discussion: A significant proportion of breast cancers contain ITC heterogeneity based on CAP/ASCO reporting recommendations. The majority of these cases are non-amplified without consideration of ITH. Because it is not known whether these patients (who would not currently be treated) would benefit from HER2 targeted therapies, it may be premature to use the ITH reporting recommendations for clinical decision-making. More validation of ITH criteria and biological significance are needed. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-05-03.

Intratumoral heterogeneity of HER-2 gene amplification and protein overexpression in breast cancer

We report a case of a patient with invasive breast carcinoma that demonstrated HER-2 gene amplification on core biopsy, who relapsed while on adjuvant trastuzumab therapy after her mastectomy and ultimately died 15 months after diagnosis. Surprisingly, analysis of multiple metastases harvested at rapid autopsy demonstrated no evidence of HER-2 gene amplification. Retrospective examination of the carcinoma in the patient's mastectomy specimen revealed only focal HER-2 amplification within the tumor, localized to the region of the prior core biopsy site. This case highlights several important issues in HER-2 testing of breast cancer, including core biopsy-excision specimen discordance, primary-metastasis discordance, and potential selection for unamplified portions of a heterogeneously amplified tumors by trastuzumab.

Genetic Heterogeneity in HER2 Testing in Breast Cancer: Panel Summary and Guidelines

Intratumoral heterogeneity of HER2 gene amplification has been well documented and represents subclonal diversity within the tumor. The reported incidence of intratumor HER2 amplification genetic heterogeneity ranges in the literature from approximately 5% to 30%. The presence of HER2 genetic heterogeneity may increase subjectivity in HER2 interpretation by the pathologist. To define HER2 genetic heterogeneity and to provide practice guidelines for examining and reporting breast tumors with genetic heterogeneity for improvement of HER2 testing in breast cancer. We convened an expert panel to discuss HER2 gene amplification testing by fluorescence in situ hybridization. Components addressed included a definition of HER2 amplification heterogeneity, practice guidelines for examination of the tissue, and reporting criteria for this analysis. Genetic heterogeneity for amplification of HER2 gene status in invasive breast cancer is defined and guidelines established for assessing and reporting HER2 results in these cases. These guidelines are additive to and expand those published in 2007 by the American Society of Clinical Oncology and the College of American Pathologists. Standardized methods for analysis will improve the accuracy and consistency of interpretation of HER2 gene amplification status in breast cancer.