HER2 Amplicon Research Articles

Abstract 4719: Dynamic changes in gene expression after one dose of trastuzumab (T) reveal novel biomarkers of response to combination therapy in HER2 positive early stage breast cancer

Abstract Introduction: The use of a ‘brief exposure’ to single agent T allows the measurement of dynamic changes in the transcriptome that may predict response to T-based combinations. We have shown that AKT and IGF pathway signatures are significantly perturbed in HER2 positive tumors treated with T, only in tumors that ultimately respond to therapy (ASCO 2010). We now demonstrate that transcriptional changes in responding tumors are unique, and uncover unanticipated mechanisms of action of T in early stage breast cancer. Patients/Methods: Fresh tumor core biopsies were taken at a 2 week timepoint after a single dose of T (8mg/m2) from 80 HER2-overexpressing, early breast cancer patients enrolled on a clinical trial of T>T+C. Nucleic acids were extracted using Qiagen AllPrep and were analyzed with Illumina HT12v3 Beadchip and Illumina 610QUADv1 SNP arrays. RNA was also processed for sequencing using the Ovation® RNA-Seq System (NuGEN Technologies Inc). The Illumina RNA-Seq Library Preparation and sequencing was performed at the Sequencing Core of the Yale Center for Genome Analysis (West Haven, CT) using the Genome Analyzer IIx sequencer. Gene expression was analyzed in Bioconductor using LIMMA analysis. Gene expression levels from RNA-Seq data was computed using Cufflinks taking mapping results from tophat in SAM format using the Human hg18 RefSeq reference gene set. Genomic biomarkers were compared pathologic complete response (pCR) vs objective response (CR+PR=OR) and non-response (SD+PD=NOR) by RECIST criteria at the surgical timepoint. Pathway analysis was performed using pre-defined gene signatures (IGF-1, AKT) and using DAVID bioinformatics resource. Results: Gene expression before and after one dose of trastuzumab showed significant changes only in tumors that ultimately achieved pCR. Specifically, AKT and IGF-1 pathways were downregulated in pCR tumors compared with NOR and OR tumors (p=0.04 and p=0.01 respectively). In addition, HER2 and GRB7 were significantly downregulated only in pCR tumors (p=0.003) by both Illumina HT12 arrays and by RNA-sequencing for 17q12 amplicon genes. Analysis of differential expression showed significant changes only in pCR tumors(adjusted pvalue <0.05), compared with NOR and OR tumors. Functional annotation clustering showed enrichment for SH3 domain genes (Enrichment score 2.24) apoptosis pathway genes (ES 1.85;) and differentiation genes (ES 1.3). Conclusions: Early changes in gene expression can predict pathologic complete response to trastuzumab-containing chemotherapy regimens. Changes in HER2 amplicon genes, AKT, IGF-1 pathways, and genes associated with both apoptosis and differentiation may predict pCR. Brief exposure to trastuzumab may uncover novel mechanisms of action of this life-saving therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4719. doi:10.1158/1538-7445.AM2011-4719

A gene on the HER2 amplicon, C35, is an oncogene in breast cancer whose actions are prevented by inhibition of Syk

Background:C35 is a 12 kDa membrane-anchored protein endogenously over-expressed in many invasive breast cancers. C35 (C17orf37) is located on the HER2 amplicon, between HER2 and GRB7. The function of over-expressed C35 in invasive breast cancer is unknown.Methods:Tissue microarrays containing 122 primary human breast cancer specimens were used to examine the association of C35 with HER2 expression. Cell lines over-expressing C35 were generated and tested for evidence of cell transformation in vitro.Results:In primary breast cancers high levels of C35 mRNA expression were associated with HER2 gene amplification. High levels of C35 protein expression were associated with hallmarks of transformation, such as, colony growth in soft agar, invasion into collagen matrix and formation of large acinar structures in three-dimensional (3D) cell cultures. The transformed phenotype was also associated with characteristics of epithelial to mesenchymal transition, such as adoption of spindle cell morphology and down-regulation of epithelial markers, such as E-cadherin and keratin-8. Furthermore, C35-induced transformation in 3D cell cultures was dependent on Syk kinase, a downstream mediator of signalling from the immunoreceptor tyrosine-based activation motif, which is present in C35.Conclusion:C35 functions as an oncogene in breast cancer cell lines. Drug targeting of C35 or Syk kinase might be helpful in treating a subset of patients with HER2-amplified breast cancers.

Abstract LB-267: GRB7: A novel target in basal breast cancer

Abstract Basal breast cancers lack expression of estrogen and progesterone receptors and HER2, rendering them non-responsive to the most commonly used targeted therapies in breast cancer. These tumors are typically more aggressive, more locally invasive and more likely to metastasize to the brain than other breast cancer subtypes and, consequently, have a poor prognosis. GRB7 is a cytoplasmic adaptor protein that interacts with receptor tyrosine kinases, relaying information to their downstream signaling cascades. It also plays a role in integrin signaling and cell migration by binding focal adhesion kinase (FAK) and ephrin receptors. It is a part of the HER2 amplicon and is frequently overexpressed in HER2-amplified breast tumors. Breast tumors in which HER2 and GRB7 are both co-amplified and co-overexpressed have a poor prognosis. We and others have recently determined that high levels of GRB7 are associated with recurrence in women with triple-negative breast cancer treated with anthracyclines (Sparano et al. J Clin Oncol 27(15s) 500). In this study, we have tested the hypothesis that triple-negative breast cancer cell lines are dependent on GRB7 for proliferation, migration and invasion. We have utilized a highly specific cell-penetrating peptide inhibitor of GRB7 (G7-18NATE) and tested its effects on a panel of 5 basal breast cancer cell lines in a series of culture assays: monolayer wound healing assays (motility on 2D substrates), transwell invasion assays (in vitro invasiveness) and 3D Matrigel cultures (morphogenesis in 3D environment). In all cases evaluated, the Grb7 inhibitory peptide impaired the proliferation, migration and invasion of these cell lines, even though these cells express low levels of GRB7 compared to HER2-amplified cell lines. These data implicate GRB7 as a key mediator of several important phenotypes of basal breast cancer cells and suggest that GRB7 itself, or GRB7-dependent pathways, may prove to be important targets for the development of specific therapies for this disease. This work was supported in part by Susan G. Komen for the Cure (KG091136). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-267.

FISH Versus IHC as the Primary Test for HER-2 Analysis: Cost Analysis, Aneusomy Significance and Gene Heterogeneity in a Consecutive Series.

Abstract Objective: To utilize a test algorithm for HER-2 that has the lowest cost per reportable test and establishes the most accurate results for trastuzumab therapy decisions.By restricting hybridization area, we have reduced the probe quantity required without altering sensitivity and specificity. Our FISH reagent cost per test is $32.20. The cost per reportable test is $62.30 for FISH and $56.86 for IHC (Ventana). FISH cost/result is slightly costlier than IHC cost/result. However, reflex to FISH (20-25%) after 2+ IHC analyses is a more expensive test algorithm than primary FISH analysis with reflex to IHC (5.6%). The total cost of the FISH first algorithm/100 cases is $6548 and for IHC first algorithm is $6932. In a recent study of patients being considered for trastuzumab treatment, primary FISH analysis was the most cost-effective diagnostic approach.Studies have shown greater accuracy and precision of FISH vis a vis IHC in predicting response to trastuzumab: Our institution has selected FISH as the primary method for assessing HER-2 status, rather than IHC.HER-2 gene amplification is observed in 15-30% of breast cancers. FISH positivity predicts the response to trastuzumab. Increased gene copies may be due to repeated duplication of HER-2 amplicon or inherent chromosomal instability due to mitotic mis-segregation of cancer cells. Aneusomy is frequently found in cancers and may complicate the interpretation of HER-2 testing results. Tumors with aneusomy of 17 without amplification have a high and uncertain response rate to trastuzumab. There is no clear consensus on how to report tumor heterogeneity in cases due to 17 aneusomy.We report here the MGH experience in 90 consecutive cases using FISH as the primary test for assessing HER-2 status. Using CAP/ASCO guidelines for reporting HER-2 FISH, we determined 15.6% cases amplified, 5.6% equivocal and 78.9% normal. Chromosome 17 and Her2 aneusomy was observed in 27.8% of cases. Our percent aneusomy may be higher than some have reported due to preselection of cases in our reference lab setting. Most of the Her-2 amplified cases had HER-2/CEN-17 ratio >10.In one case the HER 2 amplicon extended widely to chromosome 17 centromeric region. FISH studies using Topoisomerase II alpha probes showed probable deletion of this gene putatively located within the HER-2 amplicon. Even though HER gene copy number was > 20 HER-2/Chromosome17 according to ASCO/CAP was normal (< 1.8). In instances of reported co-amplification of topoisomerase II alpha gene, its status may influence the treatment decision.The incidence of chromosome 17 aneusomy has varied from as low as 4% to as high as >30% in studies of invasive breast cancer influencing the reporting of amplification. There are no existing guidelines on how to report such tumor heterogeneity. We hope this analysis will be useful to other centers evaluating the use of primary FISH algorithm for HER-2 testing. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6037.

Abstract C34: Amplified loci on chromosomes 8 and 17 predict Tamoxifen resistance in ER+ breast tumors

Abstract Most ER+ breast cancers are treated with anti-estrogens like Tamoxifen which disrupt the Estrogen growth pathway. However, not all ER+ cases respond to this therapy. This study aims to identify genetic markers associated with increased sensitivity or resistance to Tamoxifen using outlier analysis. We collected 268 ER+ samples of gene expression data from three separate published studies on Tamoxifen treated patients with follow-up information on distant metastasis-free survival time. Genes were associated with Kaplan-Meyer survival curves based on their outlier profile and filtered, using the long-rank test, for differential survival distributions at p-value<0.05. Additional correlation analysis and PCA clustering were used to identify pathways and chromosomal regions enriched with gene markers correlated with Tamoxifen response. We find significantly over-expressed cell cycle genes correlated with poor survival together with an enrichment of chromosomal regions 17q21.33–q25.1, 17q12, 8p11.2 and 8q24.3. These amplicons were previously documented in breast cancer and contain known oncogenes ERBB2, LSM1, WHSC1L1 and HSF1 associated with more aggressive forms of the disease. Since the majority of samples enriched in the cell cycle pathway also have at least one of the amplicons, this suggests the amplifications on chromosomes 8 and 17 are associated with increased proliferation. In addition to this, a relative Oncotype DX™ score was calculated for samples using normalized expression levels and published weights. This analysis showed that high Oncotype DX™ scores are generated by tumors having either the HER2 amplicon (highest Oncotype DX™ scores), or one of the other amplicons on chromosomes 17 and 8. In contrast, low Oncotype DX™ scores were found only for tumors that do not exhibit any of the identified chromosomal aberrations. This implies that amplicons 17q21.33–q25.1, 8p11.2 and 8q24.3 may be responsible for higher proliferation and poor outcome in the setting of Tamoxifen treatment clinically observed in a subset of ER+, HER2− breast cancers. These results have significant implications for the development of targeted treatments for Tamoxifen-resistent ER+ breast cancers as well as in improving current methods of drug response prediction. Citation Information: Cancer Res 2009;69(23 Suppl):C34.

Cytogenetic characterization and gene expression profiling of the trastuzumab-resistant breast cancer cell line JIMT-1

Resistance to the HER-2 targeting drug trastuzumab can be observed clinically, but the lack of suitable experimental models hampers studies of resistance mechanisms. We characterized a HER-2–positive carcinoma cell line (JIMT-1) derived from a 62-year-old breast cancer patient which was clinically resistant to trastuzumab. Multicolor fluorescence in situ hybridization revealed a complex hyperdiploid karyotype with numerous marker chromosomes and unbalanced translocations. Comparative genomic hybridization (CGH) revealed numerous regions of copy number aberration (CNA). Further analysis by array CGH identified 27 regions of CNA (16 amplified, 11 deleted). Thirty-eight percent of the genes in the amplified regions were overexpressed, compared to only 9% in regions of normal copy number ratios (CNR). Accordingly, 26% of the genes in the deleted regions were underexpressed, compared to 10% in regions of normal CNR. Most amplified and overexpressed genes were located on chromosome 1 as well as on 8q, 12q14.1, 17q11∼q21, and 20q13. In 17q11∼q21, we identified two separate amplicons, the HER-2 amplicon and a previously unreported amplicon at 17q21.31. Several aberrant genes are implicated in cancer development (e.g., JUN, CDK4, and SLUG protooncogenes, as well as the drug/hormone–metabolizing genes GSTM1 and CYP24). We conclude that cytogenetic and expression profiling of JIMT-1 revealed several new features that need further characterization and may shed light on trastuzumab resistance.

Simultaneous Amplification of HER-2 (ERBB2) and Topoisomerase IIα (TOP2A) Genes - Molecular Basis for Combination Chemotherapy in Cancer

The HER-2 (also known as ERBB2/ErbB2/c-erbB2/HER-2/neu) oncogene is the most frequently amplified oncogene in breast cancer and is also amplified in other forms of cancer. Beside its important role in tumor induction, growth and progression, HER-2 is also a target for new therapeutic approaches such as Herceptin (trastuzumab), a recombinant antibody designed to block signaling through the HER-2 receptor. In addition to Herceptin, which is in a wide clinical use for HER-2 amplified breast cancer, a number of various HER-2 directed immunological and genetic strategies, either targeting the HER-2 receptor, its signaling pathways or both HER-2 and epidermal growth factor receptor (EGFR) simultaneously, have demonstrated promising pre-clinical activity in HER-2 amplified carcinomas. Moreover, the HER-2 amplicon is known to contain more than 30 genes with altered copy numbers that could be therapeutic targets for chemotherapy. The topoisomerase IIalpha gene, TOP2A, is located adjacent to the HER-2 oncogene at the chromosome location 17q12-q21 and is either amplified or deleted (with equal frequency) in a great majority of HER-2 amplified primary breast tumors and also in tumors without HER-2 amplification. Recent experimental as well as numerous, large, multi-center trials suggest that amplification (and/or deletion) of TOP2A may account for both sensitivity or resistance to commonly used cytotoxic drugs, i.e. topoII-inhibitors (anthracyclines etc.), depending on the specific genetic defect at the TOP2A locus. The understanding of HER-2 amplification and its role in the pathogenesis of cancer is expanding, and a number of therapeutic strategies targeting either the HER-2 or its signaling pathways in cancer therapy are being investigated. Combining HER-2 targeting therapies with conventional forms of cytotoxic chemotherapy, where additional diagnostic tests such as those ascertaining TOP2A status, may be helpful for the ideal selection of patients for the combination therapy of an HER-2 targeting drug together with a cytotoxic drug such as topoII-inhibitor especially in the case of TOP2A amplification.

Topoisomerase IIalpha gene (TOP2A) amplification and deletion in cancer--more common than anticipated.

In solid tumours the predominant genetic mechanism for oncogene activation is through amplification of genes. The HER-2 (also known as ErbB2/c-erbB2/HER-2/neu) oncogene is the most frequently amplified oncogene in breast cancer and is also commonly amplified in other forms of cancer. The HER-2 amplicon also contains other biologically relevant genes with altered copy numbers, among these genes is the topoisomerase IIalpha (TOP2A). TOP2A gene is located adjacent to the HER-2 oncogene at the chromosome location 17q12-q21 and is either amplified or deleted, with equal frequency, in almost 90% of HER-2 amplified primary breast tumours. Recent data suggest that amplification and deletion of TOP2A may account for both sensitivity and resistance to topoII-inhibitor-chemotherapy, depending on the specific genetic defect at the TOP2A locus. In this issue of the Cytopathology, Bofin et al. present preliminary evidence for high prevalance of TOP2A amplification and deletion not only in the HER-2 amplified breast tumours, but also in the primary breast tumours without the HER-2 amplification. This finding together with the concept that TOP2A gene amplification and deletion seem to account for both relative chemosensitivity and resistance to topoII-inhibitor therapy further highlights the importance of screening for TOP2A gene copy number aberrations when topoII-inhibitors are considered either alone or in combination of other chemotherapeutic drugs for the treatment of cancer patients.

HER-2/neu and topoisomerase IIalpha--simultaneous drug targets in cancer.

In solid tumors the predominant genetic mechanism for oncogene activation is through amplification of genes. The HER-2 (also known as ErbB2/c-erbB2/HER-2/neu) oncogene is the most frequently amplified oncogene in breast cancer and is also commonly amplified in other forms of cancer. Alongside its important role in tumor induction, growth and progression, HER-2 is also a target for a new form of chemotherapy. Since 1998, breast cancer patients have been treated with considerable success with Herceptin (trastuzumab), a recombinant antibody designed to block signaling through the HER-2 receptor. In addition to Herceptin, a large number of various HER-2 directed immunological and genetic approaches, either targeting the HER-2 receptor, its signaling pathways or both HER-2 and epidermal growth factor receptor (EGFR) together, have demonstrated promising pre-clinical potential towards HER-2 amplified carcinomas. Moreover, the HER-2 amplicon contains other genes with altered copy numbers that could be used as targets for chemotherapy. The topoisomerase IIalpha (topoIIalpha) gene (TOP2A) is located adjacent to the HER-2 oncogene at the chromosome location 17q12-q21 and is either amplified or deleted, with equal frequency, in almost 90% of HER-2 amplified primary breast tumors. Recent data suggest that amplification or deletion of TOP2A may account for both sensitivity or resistance to topoII-inhibitor-chemotherapy, depending on the specific genetic defect at the TOP2A locus. The understanding of HER-2 amplification and its role in the pathogenesis of cancer is expanding. The number of therapeutic strategies targeting HER-2 signaling pathways will most probably be introduced in the treatment of HER-2 amplified tumors within the next few years. Combining HER-2 targeting therapies with conventional forms of cytotoxic chemotherapy, where additional diagnostics tests such as those ascertaining topoIIalpha status, may be helpful for the ideal selection of patients for the combination therapy of a HER-2 targeting drug together with a cytotoxic drug. The clinical and therapeutic importance of the HER-2 and TOPO2A status of tumor cells in cancer management will only increase within the next few years.