HER2 Gene Amplification Research Articles

Abstract P2-06-02: Breast cancer HER2 epigenetic intratumoral heterogeneity results from lack of HER2 protein translation

Abstract Research objective Previously, we reported the negative correlation between pathological complete response (pCR) and HER2 positive breast cancer exhibiting amplified HER2 gene tumor cells without HER2 protein overexpression (HER2 epigenetic intratumoral heterogeneity) among trastuzumab-based neoadjuvant chemotherapy treated patients. Our objective in this study was to elucidate if tumor cells with HER2 epigenetic intratumoral heterogeneity express HER2 RNA using a HER2 RNA in situ hybridization (ISH) method. Materials and methods Formalin-fixed, paraffin-embedded (FFPE) sections of breast cancer biopsy samples were investigated for HER2 RNA expression at the individual cell level using a HER2 RNA ISH assay. RNA preservation in tissue sections was examined using a peptidylprolyl isomerase B (PPIB) RNA ISH assay. Three groups of cases were examined: 1) HER2 negative breast cancer cases (HER2 RNA ISH negative control group). 2) HER2 positive breast cancer cases with homogeneous HER2 positive tumor cells (HER2 RNA ISH positive control; pCR group) 3) HER2 positive breast cancer cases with HER2 epigenetic intratumoral heterogeneity (a mixture of HER2 gene and protein positive tumor cells and HER2 gene positive tumor cells without HER2 protein expression; incomplete response group) Consecutive sections of HER2 RNA ISH slides were stained for HER2 gene and protein concurrently on the same tissue section using HER2 gene-protein assay (GPA) which is a combination of FDA-approved HER2 immunohistochemical (HER2 protein) and HER2 dual ISH (HER2 gene and chromosome 17 centromere) assays. Analyses of HER2 RNA expression in individual cells was microscopically evaluated and matched to HER2 GPA slides. Results RNA preservation was confirmed in tissue sections of all three groups by a PPIB RNA ISH assay. Tumor cells of HER2 negative breast cancer cases (negative control group) lacked HER2 RNA ISH signal while HER2 gene and protein positive tumor cells of homogeneous breast cancer cases (positive control group) demonstrated high HER2 RNA expression levels. HER2 gene and protein positive tumor cells of HER2 positive intratumoral heterogeneity cases showed high HER2 RNA expression. However, amplified HER2 gene breast cancer cells without HER2 protein overexpression of HER2 positive intratumoral heterogeneity cases also showed high levels of HER2 RNA expression. Thus, revealing in cases with intratumoral heterogeneity, transcription of HER2 RNA occurs, but translation of HER2 protein is altered by some mechanism(s) in tumor cells. Conclusions Transcription of HER2 RNA was observed in breast tumor cells with amplified HER2 gene but absence of HER2 protein overexpression (HER2 epigenetic intratumoral heterogeneity) of patients who showed incomplete response to neoadjuvant trastuzumab therapy. Our study suggests that inconsistent HER2 protein translation in breast cancer with HER2 epigenetic heterogeneity might be the primary resistance mechanism to trastuzumab-based neoadjuvant chemotherapy. Citation Format: Nitta H, Horii R, Murillo A, Portier B, Akiyama F. Breast cancer HER2 epigenetic intratumoral heterogeneity results from lack of HER2 protein translation [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-06-02.

Simulated ablation for detection of cells impacting paracrine signalling in histology analysis.

Intra-tumour phenotypic heterogeneity limits accuracy of clinical diagnostics and hampers the efficiency of anti-cancer therapies. Dealing with this cellular heterogeneity requires adequate understanding of its sources, which is extremely difficult, as phenotypes of tumour cells integrate hardwired (epi)mutational differences with the dynamic responses to microenvironmental cues. The later comes in form of both direct physical interactions, as well as inputs from gradients of secreted signalling molecules. Furthermore, tumour cells can not only receive microenvironmental cues, but also produce them. Despite high biological and clinical importance of understanding spatial aspects of paracrine signaling, adequate research tools are largely lacking. Here, a partial differential equation (PDE)-based mathematical model is developed that mimics the process of cell ablation. This model suggests how each cell might contribute to the microenvironment by either absorbing or secreting diffusible factors, and quantifies the extent to which observed intensities can be explained via diffusion-mediated signalling. The model allows for the separation of phenotypic responses to signalling gradients within tumour microenvironments from the combined influence of responses mediated by direct physical contact and hardwired (epi)genetic differences. The method is applied to a multi-channel immunofluorescence in situ hybridisation (iFISH)-stained breast cancer histological specimen, and correlations are investigated between: HER2 gene amplification, HER2 protein expression and cell interaction with the diffusible microenvironment. This approach allows partial deconvolution of the complex inputs that shape phenotypic heterogeneity of tumour cells and identifies cells that significantly impact gradients of signalling molecules.

Abstract P3-07-03: Targeting low voltage-activated calcium channels in HER2-positive breast cancer

Abstract Under physiological conditions low voltage-activated calcium channels (LVA, also called T-type channels) occur mostly in the brain, peripheral nervous system, smooth muscles, and pacemaker of the heart. However, LVA are often aberrantly expressed in various cancers and their overexpression is inversely correlated with outcomes. Active LVA contribute to proliferation, progression and resistance to therapy in several human cancers, including breast cancer (BC). The molecular mechanism of this activity is still not fully understood, and there is a lack of pre-clinical studies that target LVA in animal models of BC.Data from the Cancer Genome Atlas (TCGA) show a positive correlation between HER2 gene amplification and LVA channel overexpression, particularly of Cav3.1 (CACNA1G) subunit. The goal of this study is to identify functional links between HER2 receptor signaling and LVA channels, and validate LVA Ca2+ channels as a potential target for BC therapy. Inhibition of LVA, by small molecule antagonist mibefradil (MIB) or shRNA, significantly decreased the activity of PI3K/mTOR/AKT pathway in HER2-positive BC cell lines, blocked cell cycle progression, and at higher concentrations induced apoptotic cell death. Furthermore, LVA antagonist, mibefradil, sensitized cells to trastuzumab, lapatinib and paclitaxel, commonly used standard treatments for HER2-positive BC. To test the effects of LVA inhibition in vivo, the MMTV-PyMT mouse model of BC was used. In this model several features of HER2-positive BC are recapitulated, such as activation of PI3K/AKT/mTOR pathway, overexpression of HER2, BIRC5 and cyclin D1, and low expression of ERα, PGR and FOXA1. In PyMT animals palpable tumors can be detected at the age of 10-12 weeks, and 100% of animals will develop high tumor burden, including lung metastases, at the age of 15-20 weeks. Expression of CACNA1G subunit was detected in breast epithelial cells of 7 week old mice, and throughout the adult life, in both wild-type and PyMT animals, but CACNA1G was not expressed in lung or skeletal muscles. In in vitro experiments, cell lines derived from PyMT tumors were sensitive to MIB. In in vivo experiment, 5 to 7 weeks-old PyMT mice were fed either control or MIB-containing diet ad lib. Analysis of mouse serum revealed stable MIB concentration of ˜1500 ng/mL achieved within one week of treatment. In MIB-fed animals tumor appearance was delayed by 2 weeks, and the tumors were smaller as compared to control, thus suggesting a supporting role for LVA channels in breast tumor development and progression. Together, our observations provide new insights into the role of Ca2+ channels in HER2 driven BC and posit a future use of LVA channel antagonists for the treatment HER2-positive breast tumors. Citation Format: Dziegielewska B, Dunlap-Brown ME, Warnock WT, Dillon PM, Bouton AH, Dziegielewski J. Targeting low voltage-activated calcium channels in HER2-positive breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-07-03.

Abstract P5-20-02: HER2 mutations detected by ctDNA in ER+/HER2- metastatic breast cancer patients: Incidence and impact on clinical outcomes

Abstract Background: While the human epidermal growth factor receptor 2 (HER2) gene has long been linked with the pathogenesis and prognosis of breast cancer, its significance has been recognized only when the receptor has been amplified. However, rare, but actionable, somatic mutations in HER2, without HER2 gene amplification, have been described in breast cancer based on molecular analysis of metastatic specimens (frequency ˜ 1%). Little is known about the incidence based on blood-based genotyping assays, as well as impact of HER2 mutations on clinical outcomes in patients with ER+/HER2- metastatic breast cancer (MBC). Methods: We evaluated the presence of HER2 mutations based on routine tissue and blood-based genotyping assays, ordered by treating oncologists at our institution, for patients with estrogen receptor positive (ER+)/HER2 negative MBC. The tissue analysis was based on SNAPSHOT-NGS, an anchored multiplex polymerase chain reaction assay that detects single nucleotide variants (SNV) and insertions/deletions (indel), and the blood-based genotyping analysis was based on circulating tumor DNA (ctDNA) detection using the Guardant 360 panel, a next-generation sequencing (NGS) assay capable of detecting mutations with comparable sensitivity to SNAPSHOT. Patients with acquired HER2 mutations were identified, and multivariate analysis was performed to evaluate the hazard ratio (HR) for the association between HER2 mutations and progression free survival (PFS), adjusting for age and number of prior therapies. Results: Among the ER+/HER2- MBC patients (N=118), 11% (N= 13) were found to have acquired HER2 mutation by ctDNA analysis, but no HER2 mutations were identified in any of patients based on tissue-based molecular analysis of archival specimens. Among patients with HER2 mutant, ER+/HER2- MBC, the median age at metastatic diagnosis was 57.34 (range 51.5-67.1) years, 7.7% had de-novo metastatic disease, and 30.8% had prior CDK 4/6 inhibitor therapy. In terms of outcomes, in the multivariate model, patients with HER2 mutant breast cancer had similar PFS when treated with endocrine and targeted therapy combination (HR = 0.24; p = 0.21), and trended towards worse PFS with chemotherapy (HR = 2.69; p = 0.06), as compared to non-HER2 mutant group, albeit duration of follow up was limited (median duration = 6.7 months). Of note, most of the detected HER2 mutations were activating or deleterious, but not all were clonal. Updated outcome data, including overall survival, will be presented at the meeting. Conclusions: A much larger subset of patients with ER+/HER2- MBC have HER2 mutations detectable by ctDNA, but not by tissue, which highlights the need for blood-based biomarker monitoring for identification of actionable mutations, as well as the potential clinical utility in development of genotype driven trials for patients with HER2 mutant, ER+/HER2- MBC. Citation Format: Medford A, Juric D, Niemierko A, Malvarosa G, Park H, Shellock M, Spring L, Moy B, Isakoff S, Ellisen L, Bardia A. HER2 mutations detected by ctDNA in ER+/HER2- metastatic breast cancer patients: Incidence and impact on clinical outcomes [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-20-02.

Abstract P2-03-03: A multicenter clinical study of Xpert® breast cancer STRAT4 demonstrates high concordance with central lab ER, PgR, HER2, and Ki67 IHC and HER2 FISH tests in FFPE breast tumor tissues

Abstract The Xpert® Breast Cancer STRAT4 (STRAT4) is a CE-IVD marked, semi-quantitative, cartridge-based RT-qPCR assay for the detection of ESR1, PGR, ERBB2 (HER2), and MKi67 mRNAs from formalin fixed, paraffin embedded (FFPE) breast tumors. The assay is fast (< 2 hrs), reproducible, robust, and easy to perform. The aim of this multicenter clinical study was to assess the performance characteristics of the STRAT4 assay relative to central lab immunohistochemistry (IHC) for ER, PgR, HER2, and Ki67 and to fluorescence in situ hybridization (FISH) for HER2 gene amplification. Methods: A total of 200 archived primary invasive breast cancer FFPE blocks were sourced from Indivumed for this study. From each block, twelve (12) adjacent tissue sections (4-µm thickness) on slides were prepared for pathological H&E confirmation to define tumor area, and for testing by STRAT4, IHC (ER, PgR, HER2,Ki67), and HER2 FISH. Standard STRAT4 lysate preparation using a single unstained slide per specimen and testing on N=84, N=68, and N=48 samples was performed at 3 independent sites, respectively (2 US and 1 EU). A single slide from each specimen was also processed using the recommended concentrated lysate procedure for STRAT4 testing at Cepheid. All IHC and FISH testing was performed by a central academic reference laboratory in the US. For a given sample, STRAT4 data generated using the standard lysate procedure was included for concordance analysis when all target gene test results were valid. In cases where the standard lysate preparation yielded indeterminate test results for any target, data from the concentrated lysate preparation was used for the data analysis. Receiver Operating Characteristic (ROC) analysis, overall percent agreement (OPA), positive percent agreement (PPA), and negative percent agreement (NPA) between STRAT4 and IHC (IHC/FISH for HER2) were determined for ESR1,PGR, ERBB2, and MKi67. Results: Of the 200 samples tested by STRAT4, all samples generated valid results for ESR1 and ERBB2, 199 of 200 samples were valid for PGR, and 198 of 200 samples were valid for MKi67 using the standard or concentrated lysate preparation protocol. One sample failed to generate results for both ER and PgR IHC. Twelve samples failed to yield HER2 FISH results. The STRAT4 success rate and results concordance with IHC were comparable across study sites. OPA between STRAT4 and IHC was 97% for ESR1, 88.9% for PGR, 93.3% for HER2 (92.4% for IHC and FISH), and 90.7% for MKi67 (excluding IHC 10-20% staining). Areas under the ROC curves were 0.9922 for ESR1, 0.9509 for PGR, 0.9958 for ERBB2, and 0.9395 for MKi67. Conclusion: STRAT4 measurements for ESR1, PGR, ERBB2 and MKi67 mRNA expression are robust and highly concordant with IHC (IHC/FISH for HER2). The technical portion of the assay is easily performed in < 2 hrs including hands-on time using standard FFPE tissue sections. Xpert STRAT4 offers local pathology labs an alternative to centralized, subjective IHC/FISH tests that require a higher level of expertise. Further investigations correlating STRAT4 markers directly with clinical outcomes in independent cohorts are in progress. Citation Format: Wu NC, Wong E, Acca B, Birkmeier J, Tran L, Zhao S, Wong W, Chu VC, Ho K, Malek M, Lu C, Ge G, David K, Quigley NB, Beqaj SS, Davenport S, Weidler J, Bates M, Press M. A multicenter clinical study of Xpert® breast cancer STRAT4 demonstrates high concordance with central lab ER, PgR, HER2, and Ki67 IHC and HER2 FISH tests in FFPE breast tumor tissues [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-03-03.

Methods and significance of the combined detection of HER2 gene amplification and chemosensitivity in gastric cancer.

This study aims to investigate the significance of combined detection of HER2 gene amplification and chemosensitivity in gastric cancer. Immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and fluorescence reverse-transcription polymerase chain reaction (RT-PCR) were used to analyze the expression of HER2 protein, HER2 gene amplification and the mRNA expression of ERCC1, TUBB3 and TYMS genes in 135 cases of gastric carcinoma. The expression rate of HER2 protein was 39.3% (53/135). Among these positive cases, patients with HER2 protein (3+) accounted for 9.6% (13/135), patients with HER2 protein (2+) accounted for 13.3% (18/135), and patients with HER2 protein (1+) accounted for 16.3% (22/135). The amplification rate of the HER2 gene was 35.8% (19/53). In the detection of the mRNA expression of ERCC1, TUBB3 and TYMS, 45 patients had low and moderate single gene expression, 50 patients had low and moderate double gene expression, 22 patients had low and moderate mRNA expression for ERCC1, TUBB3 and TYMS, and 18 patients had no low and moderate expression. Among the 53 patients with HER2 protein expression and 22 patients with low and moderate mRNA expression of ERCC1, TUBB3 and TYMS, 12 patients received chemotherapy and trastuzumab. Follow-up results revealed that HER2 gene status was positively correlated with the therapeutic effect of the combined treatment in patients with low mRNA expression of ERCC1, TUBB3 and TYMS. Among these patients, five patients with extensive HER2 (3+), HER2 cluster-specific amplification, and low mRNA expression of ERCC1, TUBB3 and TYMS had a total survival of up to 19.1 months. The detection of HER2 in gastric cancer is highly heterogeneity, and the combined detection of HER2 protein expression, HER2 gene amplification and chemosensitivity can provide important reference markers for the benefit of antitumor drugs.

Clinical significance of quantitative HER2 gene amplification as related to its predictive value in breast cancer patients in neoadjuvant setting.

BackgroundThe aims of this study were to determine whether the quantitative HER2 gene amplification level is related to the key clinicopathological features that represent the aggressiveness of breast cancer (BC) and to determine whether the quantitative HER2 gene amplification level could predict the treatment response in the subset of HER2-positive patients who received neoadjuvant targeted therapy.Materials and methodsPatients treated with weekly cisplatin- and paclitaxel-based neoadjuvant chemotherapy, who had undergone both immunohistochemistry and the fluorescence in situ hybridization test for HER2, were included in the study (n=103). For HER2-positive patients, defined as immunohistochemistry score 3+ or fluorescence in situ hybridization ratio ≥2.0, trastuzumab was recommended with neoadjuvant chemotherapy (n=45). Pathological complete response was defined as complete pathological remission of tumor cells both in breast and axillary lymph nodes postoperation.ResultsIn all patients enrolled in the study, a higher HER2 amplification level was significantly correlated with larger tumor size and the absence of ER and PR expression. In HER2-positive patients treated with neoadjuvant trastuzumab concurrent with chemotherapy, both univariate and multivariate logistic regression showed that a higher HER2/CEP17 ratio and HER2 gene copy number were associated with a higher pathological complete response rate. When calculated by receiver operating characteristics analysis, an optimal cutoff of 4.5 for the HER2/CEP17 ratio was expected to distinguish the most sensitive candidate for treatment with a combination of trastuzumab and neoadjuvant chemotherapy.ConclusionA higher HER2 amplification level was correlated with larger tumor size and reduced ER and PR expression, which may indicate more aggressive tumor behavior. For HER2-positive patients, the HER2/CEP17 ratio and HER2 gene copy number may be good predictive factors for concurrent neoadjuvant trastuzumab and chemotherapy.

The Role of Molecular Testing in the Differential Diagnosis of Salivary Gland Carcinomas.

Salivary gland neoplasms are a morphologically heterogenous group of lesions that are often diagnostically challenging. In recent years, considerable progress in salivary gland taxonomy has been reached by the discovery of tumor type-specific fusion oncogenes generated by chromosome translocations. This review describes the clinicopathologic features of a selected group of salivary gland carcinomas with a focus on their distinctive genomic characteristics. Mammary analog secretory carcinoma is a recently described entity characterized by a t(12;15)(p13;q25) translocation resulting in an ETV6-NTRK3 fusion. Hyalinizing clear cell carcinoma is a low-grade tumor with infrequent nodal and distant metastasis, recently shown to harbor an EWSR1-ATF1 gene fusion. The CRTC1-MAML2 fusion gene resulting from a t(11;19)(q21;p13) translocation, is now known to be a feature of both low-grade and high-grade mucoepidermoid carcinomas associated with improved survival. A t(6;9)(q22-23;p23-34) translocation resulting in a MYB-NFIB gene fusion has been identified in the majority of adenoid cystic carcinomas. Polymorphous (low-grade) adenocarcinoma and cribriform adenocarcinoma of (minor) salivary gland origin are related entities with partly differing clinicopathologic and genomic profiles; they are the subject of an ongoing taxonomic debate. Polymorphous (low-grade) adenocarcinomas are characterized by hot spot point E710D mutations in the PRKD1 gene, whereas cribriform adenocarcinoma of (minor) salivary glands origin are characterized by translocations involving the PRKD1-3 genes. Salivary duct carcinoma (SDC) is a high-grade adenocarcinoma with morphologic and molecular features akin to invasive ductal carcinoma of the breast, including HER2 gene amplification, mutations of TP53, PIK3CA, and HRAS and loss or mutation of PTEN. Notably, a recurrent NCOA4-RET fusion has also been found in SDC. A subset of SDC with apocrine morphology is associated with overexpression of androgen receptors. As these genetic aberrations are recurrent they serve as powerful diagnostic tools in salivary gland tumor diagnosis, and therefore also in refinement of salivary gland cancer classification. Moreover, they are promising as prognostic biomarkers and targets of therapy.

Clinicopathologic study of10 cases ofgastric adenocarcinoma with hepatoid or enteroblastic differentiation.

Gastric adenocarcinoma with hepatoid or enteroblastic differentiation (GAHED), known also as AFP-producing carcinoma, is arare neoplasm. Ten cases with GAHED and 209 cases without GAHED were selected. Clinicopathological features ofGAHED were investigated. Thedisease-free survival (DFS) oftheGAHED group was compared with that ofthenon-GAHED group. Grossly, thetumours consisted oftwo early types and eight advanced types. Histologically, all tumours were composed ofvarious proportions oftubular, cribriform, papillary, solid, and/or trabecular growth patterns ofclear to slightly eosinophilic tumour cells. Hyaline globules were observed in all tumours. AFP and Hep-Par1 were immunoreactive in all tumours. In fluorescence in situ hybridisation ofHER2 gene/chromosome 17, theamplification ofHER2 gene was observed in two cases that showed positive reaction for HER2 protein. Clinical follow-up was available in nine cases. Regarding theclinical outcome, 3 and 6 patients were alive without disease and alive with disease, respectively. In astatistical analysis, theDFS oftheGAHED group was significantly worse than that ofthenon-GAHED group. GAHED is morphologically characterised by various growth patterns ofclear to slightly eosinophilic tumour cells and intracytoplasmic possession ofhyaline globules. This tumour may have thepotential to behave in anaggressive clinical fashion.

Sequential HER2 blockade as effective therapy in chemorefractory, HER2 gene-amplified, RAS wild-type, metastatic colorectal cancer: learning from a clinical case

BackgroundConstitutive activation of HER2-dependent intracellular signalling by HER2 gene amplification or by HER2 mutations has been demonstrated as a mechanism of primary and secondary cancer resistance to cetuximab or panitumumab in preclinical and clinical models of metastatic colorectal cancer (mCRC). Both HER2 Amplification for Colorectal Cancer Enhanced Stratification (HERACLES) cohort A and My Pathway clinical trials provided clinical evidence that anti-HER2 therapies could be active in these patients.Patient and methodsHER2 gene amplification and HER2 protein overexpression analysis were performed in tumour tissue by fluorescence in situ hybridisation and immunohistochemistry. HER2 positivity was defined according to HERACLES CRC-specific HER2 scoring criteria. DNA analysis for multiple assessment of gene mutations or amplifications was carried out with the next-generation sequencing (NGS) Ion AmpliSeq Colon and Lung Cancer Panel and by using a more extensive targeted high-multiplex PCR-based NGS panel (OncoMine Comprehensive Assay).ResultsWe report the clinical case of a patient with HER2 gene amplified and RAS/BRAF wild-type mCRC who experienced a long lasting and relevant clinical efficacy from sequential anti-HER2 therapies (trastuzumab plus lapatinib, pertuzumab plus trastuzumab, trastuzumab emtansine, trastuzumab plus capecitabine) achieving a cumulative clinical benefit of 29 months, after failure of the first three lines of standard treatments, which included all the potentially active drugs in mCRC, and which accounted for only 14 months of disease control. HER gene amplification was confirmed by NGS on two different metastatic lesions during the evolution of the disease.ConclusionThe clinical case highlights the role of HER2 gene amplification as a key genetic driver of cancer development and progression in mCRC and suggests that sequential HER2 blockade could be a potential therapeutic strategy.

A decision tree-based prediction model for fluorescence in situ hybridization HER2 gene status in HER2 immunohistochemistry-2+ breast cancers: a 2538-case multicenter study on consecutive surgical specimens.

Objective: To investigate the proportion of HER2 gene amplifications and the association between the HER2-IHC-staining pattern and gene status in IHC-2+ breast cancers according to 2013 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines.Methods: We retrospectively analyzed and re-evaluated the IHC-staining pattern of 2538 IHC-2+ surgical specimens of breast cancer from November 2014 to October 2015 in 12 institutions. All cases used for building a prediction model of HER2 gene amplification according to the IHC-staining pattern and were randomly divided into a training set (n = 1914) or validation set (n = 624).Results: The overall HER2 fluorescence in situ hybridization (FISH) amplification, non-amplification and equivocation rates in HER2 IHC-2+ cases were 17.8%, 76.2% and 6.0%, respectively. In the training set, cases that had ≤ 10% of cells with intense, complete and circumferential membrane staining or had > 85% of cells with complete membrane staining of any staining intensity tended to be HER2 gene amplified (77.0% and 60.5%, respectively). And cases with weak and incomplete membrane staining had the lowest amplification rate of 6.1%. The prediction model was constructed based on IHC-staining pattern in the training set and validated using a validation set. The positive and negative prediction values were 51.6% and 79.2%, respectively, in the validation set. Moreover, the HER2 copy number per cell was much higher in cases with amplification-associated staining patterns (7.84 and 8.75) than in cases with non-amplification-associated staining patterns (2.97 to 4.41, P < 0.05).Conclusions: In HER2 IHC-2+ breast cancers, the staining pattern is associated with the HER2 gene status. This finding is compatible with recommendations of 2013 ASCO/CAP guidelines.

Predictive Marker: HER2 in Esophageal Adenocarcinoma.

HER2 positivity is based on the fundamental principle of amplification of the human epidermal growth factor receptor 2 (HER2) gene resulting in overexpression of the protein products . Arising from that a "HER2-positive cancer" is one that shows HER2 gene amplification and resultant protein expression as demonstrated by in situ hybridization and immunohistochemistry, respectively. Testing of the HER2 status is crucial to ensure selection of the correct patient who may benefit from target therapy for esophageal adenocarcinoma. Accurate testing is dependent on several pre-analytical and analytical factors including sample selection, laboratory techniques, and accurate interpretation of HER2 test results.

Study of HER-2 Gene Amplification by Chromogenic in-situ hybridization and HER-2 Protein Amplification by IHC in Breast Cancer using Manual Tissue Microarray Sections

Study of HER-2 Gene Amplification by Chromogenic in-situ hybridization and HER-2 Protein Amplification by IHC in Breast Cancer using Manual Tissue Microarray Sections

Amplification of HER-2 Gene in Benign and Malignant Breast Lesions in a Sample of Iraqi Women

Amplification of HER-2 Gene in Benign and Malignant Breast Lesions in a Sample of Iraqi Women

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.

FORMULATION AND EVALUATION OF DOCETAXEL TRIHYDRATE LOADED SELF-ASSEMBLED NANOCARRIERS FOR TREATMENT OF HER2 POSITIVE BREAST CANCER

Human Epidermal Growth Factor Receptor 2-positive (HER2-positive) breast cancer tend to be more aggressive among all breast cancers. Breast cancers with HER2 gene amplification or HER2 protein overexpression are called HER2-positive. It tends to grow faster and are more likely to spread and come back compared to HER2-negative breast cancers. HER2 positive breast cancer is often treated with chemotherapy drugs called anthracylines (e.g. doxorubicine, epirubicine), taxanes (e.g. Docetaxel, peclitaxel) as well as some others, usually in combination of two or more chemotherapy drugs1 but the development of potential drug delivery system by using nanotechnology is also the important aspect for the proper treatment of HER2 positive breast cancer. The aim of this study was to develop DTX-loaded self assembled nanocarriers (SANs) for treatment of HER2 positive breast cancer and also to evaluate their efficacy to release the drug by controlled manner. SANs were prepared from a glyceryl monooleate, Pluronic® F127 (0.5 – 1.5 % w/v) and Pluronic® F68 (0.25-1.0 %w/v) in different concentration with or without docetaxel trihydrate (DTX) (2.0%w/v) by high pressure homogenization, before preparation compatibility of drug and polymers was studied by differential scanning calorimetry (DSC) and FTIR spectroscopy. Prepared SANs was then subject to different evaluation test particle size, zeta potential, % entrapment efficiency, drug content, in vitro drug release study, measurement of pH, and stability study. Particle size of SANs prepared with Pluronic® F127 was found in the range of 170 nm to 280 nm whereas SANs with Pluronic® F68 was found between 200 to 240 nm and it shows more negative zeta potential value than -30 mV. More than 90 % of DTX was found to be entrapped in SANs formulations loaded with DTX 2.0% w/v. Drug released study revealed that formulation F9 containing 0.25% PF68 shows 89.59 % release after 12 h and F5 containing 1.0% PF127 releases 96.56% drug after 12 h. Results of one month stability study shows that the SANs formulations were found to be stable over a one month. Hence, the DTX-loaded SANs was act as an potential drug carrier to fulfill the demand of cancer therapeutics.

Gastric cancer: immunohistochemical classification of molecular subtypes and their association with clinicopathological characteristics

Gastric cancer is traditionally divided into intestinal and diffuse histological subtypes, but recent molecular analyses have led to novel classification proposals based on genomic alterations. While the intestinal- and diffuse-type tumours are distinguishable from each other at the molecular level, intestinal-type tumours have more diverse molecular profile. The technology required for comprehensive molecular analysis is expensive and not applicable for routine clinical diagnostics. In this study, we have used immunohistochemistry and in situ hybridisation in molecular classification of gastric adenocarcinomas with an emphasis on the intestinal subtype. A tissue microarray consisting of 244 gastric adenocarcinomas was constructed, and the tumours were divided into four subgroups based on the presence of Epstein-Barr virus, TP53 aberrations and microsatellite instability. The intestinal- and diffuse-type tumours were separately examined. The distribution of EGFR and HER2 gene amplifications was studied in the intestinal-type tumours. Epstein-Barr virus positive intestinal-type tumours were more common in male patients (p = 0.035) and most often found in the gastric corpus (p = 0.011). The majority of the intestinal-type tumours with TP53 aberrations were proximally located (p = 0.010). All tumours with microsatellite instability showed intestinal-type histology (p = 0.017) and were associated with increased overall survival both in the univariate (p = 0.040) and multivariate analysis (p = 0.015). In conclusion, this study shows that gastric adenocarcinomas can be classified into biologically and clinically different subgroups by using a simple method also applicable for clinical diagnostics.

Negative genic switch of HER-2 in the primary tumor instead of the synchronous metastatic nodal lesions after neoadjuvant chemotherapy in a patient with primary HER2-positive breast cancer

BackgroundA few retrospective studies have indicated that neoadjuvant chemotherapy (NAC) in breast cancer may change biomarker profiles of the primary tumor. Little is known about the status of HER-2 gene of the synchronous nodal metastases when that of the residual tumor undergoes negative conversion in a neoadjuvant setting.Case presentationWe describe a female patient with left breast cancer (T2N2M0) who underwent negative conversion of HER-2 in the primary tumor instead of the synchronous nodal lesions after NAC. Core needle biopsy showed invasive ductal carcinoma with HER2 immunohistochemistry (IHC) (2+) and amplified HER-2 gene determined by fluorescence in situ hybridization (FISH). Then, the patient underwent 4 cycles of anthracycline- and taxane-based NAC and subsequent left modified radical mastectomy. Postoperative pathology showed invasive ductal carcinoma involving 4 of 12 surgically excised axillary lymph nodes with HER2 IHC (1+) and FISH negative (HER2 gene not amplified) in the residual tumor of the breast specimen. Due to the negative genic switch of HER2 after NAC, the patient rejected to accept trastuzumab. Under the patient’s consent, the synchronous nodal lesions were further investigated and showed HER2 IHC(−) but FISH positive (HER-2 gene amplified). Therefore, the patient agreed to accept adjuvant trastuzumab treatment every 3 weeks for 1 year.ConclusionsWe propose further assessment of HER2 gene in the synchronous nodal metastases, especially when negative genic switch of HER-2 occurs in the primary tumor after NAC in order to tailor the systemic regimens for breast cancer patients.

Breast cancer stem cells are involved in Trastuzumab resistance through the HER2 modulation in 3D culture.

Breast cancer human cells culture as spheroids develop autophagy and apoptosis, which promotes Trastuzumab resistance in HER2 overexpressing cells. Our aim was to study the association of the hostile environment developed in 3D with the breast cancer stem cells population and the HER2 modulation. Human mammary adenocarcinoma cell lines were cultured as spheroids using the hanging drop method. We generated hypoxia conditions by using a hypoxic chamber and CoCl2 treatment. Breast cancer stem cells were measured with mammosphere assays, the analysis of CD44 + CD24low population by flow cytometry and the pluripotent gene expression by RT-qPCR. HER2 expression was evaluated by flow cytometry and Western blot. MTS assays were conducted to study cell viability. Hostil environment developed in spheroids, defined by hypoxia and autophagy, modulated the response to Trastuzumab. In HER2+ cells with acquired resistance, we observed an increase in the breast cancer stem cell population. In BT474 spheroids, Trastuzumab induced the acquisition of resistance, along with an increase in breast cancer stem cells. Also, in 3D culture conditions we determined a modulation in the HER2 expression. Moreover, breast cancer stem cells showed enhanced HER2 expression. Finally, cells without HER2 gene amplification cultured as spheroids were sensitive to Trastuzumab, diminishing HER2 expression and cancer stem cells. Our findings show that 3D architecture is able to modulate breast cancer stem cell population and HER2 distribution, modifying the cell response to Trastuzumab.