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PubMed | Targos Molecular Pathology GmbH, Royal Marsden Hospital, University of Chicago, Frontier Science Foundation Hellas and 6 more.
Type: Journal Article | Journal: JAMA oncology | Year: 2016

A number of studies suggest that response to antihuman epidermal growth factor receptor-2 (currently known as ERBB2, butreferred to asHER2 in this study) agents differs by estrogen receptor (ER) level status. The clinical relevance of this is unknown.To determine the magnitude of trastuzumab benefit according to quantitative levels of ER and HER2 in the HERceptin Adjuvant (HERA) trial.The HERA trial was an international, multicenter, randomized trial that included 5099 patients with early-stage HER2-positive breast cancer, randomized between 2001 and 2005 to receive either no trastuzumab or trastuzumab, after adjuvant chemotherapy. This is a secondary analysis of the HERA study. Local ER immunohistochemical (IHC) analyses, HER2 fluorescence in situ hybridization (FISH) ratio, and copy number results were available for 3037 patients (59.6%) randomized to observation and trastuzumab (1 or 2 years) (cohort 1). Transcript levels of ESR1 and HER2 genes were available for 615 patients (12.1%) (cohort 2).Patients were randomized to receive either no trastuzumab or 1 year vs 2 years of trastuzumab. Endocrine therapy was given to patients with hormone receptor-positive disease as per local guidelines.Disease-free survival (DFS) and overall survival (OS) were the primary and secondary end points in the intent-to-treat population (ITT). Analyses adjusting for crossover (censored and inverse probability weighted [IPW]) were also performed. Interactions among treatment, ER status, and HER2 amplification using predefined cutoffs were assessed in Cox proportional hazards regression models.Median follow-up time was 8 years. Levels of FISH and HER2 copy numbers were significantly higher in ER-negative patients (P<.001). In cohort 1, for DFS and OS, a significant treatment effect was found for all ER, IHC, and FISH levels, except for the ER-positive/HER2 low FISH ratio (2 to <5) group (DFS: 3-way ITT Pvalue for interaction=.07; censored=.02; IPW=.03; OS ITT Pvalue for interaction=.007; censored=.04; IPW=.03). In cohort 2, consistent with cohort 1, a significant predictive effect of the ESR1 gene for both end points was also observed (DFS Pvalue for interaction=.06; OS=.02), indicating that breast cancers with higher ESR1 levels also derive less benefit from trastuzumab.Patients with HER2-positive breast cancers that are ER-positive by IHC analyses with low FISH ratio (2 to <5), or with higher ESR1 levels derive significantly less benefit from adjuvant trastuzumab after chemotherapy. These data may explain heterogeneity in response to anti-HER2 agents in HER2-positive, ER-positive breast cancers as some may be more luminal-like than HER2 driven.clinicaltrials.gov Identifier: NCT00045032.


Van Cutsem E.,Catholic University of Leuven | Bang Y.-J.,Seoul National University | Feng-yi F.,Cancer Institute and Hospital | Xu J.M.,Affiliated Hospital 307 Hospital Cancer Center | And 14 more authors.
Gastric Cancer | Year: 2015

Background: In the Trastuzumab for GAstric cancer (ToGA) study, trastuzumab plus chemotherapy improved median overall survival by 2.7 months in patients with human epidermal growth factor receptor 2 (HER2)-positive [immunohistochemistry (IHC) 3+/fluorescence in situ hybridization-positive] gastric/gastroesophageal junction cancer compared with chemotherapy alone (hazard ratio 0.74). Post hoc exploratory analyses in patients expressing higher HER2 levels (IHC 2+/fluorescence in situ hybridization-positive or IHC 3+) demonstrated a 4.2-month improvement in median overall survival with trastuzumab (hazard ratio 0.65). The ToGA study provides the largest screening dataset available on HER2 overexpression/amplification in this indication. We further analyzed correlation(s) of HER2 overexpression/amplification with clinical and epidemiological factors. Methods: HER2-positivity was analyzed by histological subtype, tumor location, geographic region, and specimen type. Exploratory efficacy analyses were performed. Results: The HER2-positivity rate was 22.1 % across analyzed tumor samples. Rates were similar between European and Asian patients (23.6 % vs. 23.9 %), but higher in intestinal- vs. diffuse-type (31.8 % vs. 6.1 %), and gastroesophageal junction cancer versus gastric tumors (32.2 % vs. 21.4 %). Across all IHC scores, variability in HER2 staining (≤30 % stained cells) was observed in almost 50 % of cases, with increasing rates in lower IHC categories, and did not affect treatment outcome. The polysomy rate was 4 %. Conclusions: HER2 expression varies by tumor location and type. All patients with advanced gastric or gastroesophageal junction cancer should be tested for HER2 status, preferably using IHC initially. Due to the unique characteristics of gastric cancer, specific testing/scoring guidelines should be adhered to. © 2014, The Author(s).


Zabaglo L.,Royal Marsden NHS Trust | Zabaglo L.,Breakthrough Breast Cancer Research Center | Stoss O.,Targos Molecular Pathology GmBH | Ruschoff J.,Targos Molecular Pathology GmBH | And 10 more authors.
Annals of Oncology | Year: 2013

Background: Trastuzumab treatment improves survival of HER2-positive primary breast cancer. HER2 staining intensity varies widely in HER2-positive tumours. Patients and methods: We investigated whether differences in immunohistochemical (IHC) staining intensity for HER2 in HER2-positive tumors (IHC 3+ or FISH ratio ≥2.0) was associated with prognosis or benefit from trastuzumab treatment in patients randomized to 1 year or no trastuzumab in the HERceptin Adjuvant (HERA) trial. Median follow-up was 2 years. The nested case-control analysis, included 425 patients (cases) with a disease-free survival (DFS) event and two matched controls (no DFS event) per case. Tissue sections stained for HER2 were assessed for HER2 staining intensity by image analysis. Results: HER2 staining intensity varied widely and correlated with HER2 gene copy number (Spearman, r = 0.498, P < 0.001) or less closely with HER2/CEP17 FISH ratio (r = 0.396, P < 0.001). We found no significant difference in DFS in the observation arm according to staining intensity (odds ratio [OR] change per 10 unit change in intensity: 1.015, 95% confidence interval [CI] 0.930-1.108) and no impact of staining intensity on benefit derived from 1-year trastuzumab (OR: 1.017, 95% CI 0.925-1.120). Conclusions: Variability in HER2 staining in HER2-positive tumours has no role in clinical management with adjuvant trastuzumab. © The Author 2013. Published by Oxford University Press on behalf of the European Society for Medical Oncology All rights reserved.


Grant
Agency: Cordis | Branch: FP7 | Program: CP-FP | Phase: HEALTH-2009-1.2-2 | Award Amount: 3.96M | Year: 2010

Cancer is one of the major causes of mortality. Epithelial cells become malignant after accumulating genetic mutations followed by morphological changes in the epithelium. DNA Alterations include stable genetic changes in oncogenes, tumor suppressor genes and reversible epigenetic changes. Different forms of epigenetic mechanisms have been shown to modify the expression of key genes during tumour progression. Promoter DNA hypermethylation of tumour suppressor genes or DNA repair genes, and covalent histone modifications appear in early stages of neoplasia. Methods to identify early markers in different types of cancer are being developed, although very few are specific and sensitive enough to be applied in the clinic. The aim of the present proposal is to develop sensitive and specific methodologies to identify early epigenetic markers for major types of cancer, like prostate and colorectal cancer. This project is based on recent findings that selected covalent histone modifications and their modifying enzymes can be early markers of tumourigenesis. For this purpose, the following will be applied. a) selected covalent histone modification like acetylation, methylation, phosphorylation, ubiquitination among others b) their modifying enzymes, like DNA (de)methylases and histone (de)acetylases, (de)methyltransferases c) appropriate diagnostic methods and tests for detection of selected markers in clinical samples. These markers are revealed from studies in cell and animal models as well from patient cohorts. Non-invasive diagnostic methods based on technologies developed in the participating organizations will be tested in clinical samples. Appropriately selected clinical samples will be utilized according to EU and national ethical procedures. The major task of this consortium will be the development of methods to be applied in the clinic for the immediate benefit of cancer patients.


PubMed | Targos Molecular Pathology GmbH, University of Cologne, Genentech and University of Gottingen
Type: Journal Article | Journal: PloS one | Year: 2015

Soft tissue sarcomas are a heterogeneous group of tumors with many different subtypes. In 2014 an estimated 12,020 newly diagnosed cases and 4,740 soft tissue sarcoma related deaths can be expected in the United States. Many soft tissue sarcomas are associated with poor prognosis and therapeutic options are often limited. The evolution of precision medicine has not yet fully reached the clinical treatment of sarcomas since therapeutically tractable genetic changes have not been comprehensively studied so far. We analyzed a total of 484 adult-type malignant mesenchymal tumors by MET fluorescence in situ hybridization and MET and hepatocyte growth factor immunohistochemistry. Eleven different entities were included, among them the most common and clinically relevant subtypes and tumors with specific translocations or complex genetic changes. MET protein expression was observed in 2.6% of the cases, all of which were either undifferentiated pleomorphic sarcomas or angiosarcomas, showing positivity rates of 14% and 17%, respectively. 6% of the tumors showed hepatocyte growth factor overexpression, mainly seen in undifferentiated pleomorphic sarcomas and angiosarcomas, but also in clear cell sarcomas, malignant peripheral nerve sheath tumors, leiomyosarcomas and gastrointestinal stromal tumors. MET and hepatocyte growth factor overexpression were significantly correlated and may suggest an autocrine activation in these tumors. MET FISH amplification and copy number gain were present in 4% of the tumors (15/413). Two samples, both undifferentiated pleomorphic sarcomas, fulfilled the criteria for high level amplification of MET, one undifferentiated pleomorphic sarcoma reached an intermediate level copy number gain, and 12 samples of different subtypes were categorized as low level copy number gains for MET. Our findings indicate that angiosarcomas and undifferentiated pleomorphic sarcomas rather than other frequent adult-type sarcomas should be enrolled in screening programs for clinical trials with MET inhibitors. The screening methods should include both in situ hybridization and immunohistochemistry.


PubMed | Targos Molecular Pathology GmbH, University of Cologne, Nazarbayev University, University of Milan and 3 more.
Type: Journal Article | Journal: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc | Year: 2015

Recently the American Society of Clinical Oncology and the College of American Pathologists have updated their clinical practice guidelines for HER2 testing in breast cancer. In order to evaluate these new recommendations, we have re-assessed the HER2 status of 6018 breast cancer cases of the screening population for the HERceptin adjuvant (HERA) trial that were originally centrally tested by fluorescence in situ hybridization based on the FDA-released test guidelines. According to the most recent 2013 ASCO/CAP recommendations, 3380 (56.2%) cases were classified as HER2 positive compared with 3359 (55.8%) applying the HERA/FDA scheme and 3339 (55.5%) applying the 2007 ASCO/CAP guidelines. Twenty-one cases switched from negative (HERA/FDA scheme) to positive (2013 ASCO/CAP guidelines). This group is characterized by a mean HER2 gene copy number of 6.0, polysomy or co-amplification of CEP17 with an average CEP17 count of 5, and with HER2 receptor overexpression in 75% of cases. On the basis of the HER2 gene copy number alone, we observe 494 cases (8.2%) that are in the equivocal range. Most of these cases (>80%) were also nondecisive by immunohistochemistry (score 2+) irrespective of whether ratio was <2.0>. The number of equivocal cases that would require HER2 reflex testing decreases to 113 (1.9%) if in addition to the HER2 gene copy number also the ratio of HER2 and CEP17 copy numbers is considered via dual-color in situ hybridization. The combination of applying the HER2 mean gene copy number as well as the HER2/CEP17 ratio to define equivocal test decisions by fluorescence in situ hybridization as proposed by the current ASCO/CAP guidelines appears to be a more optimum approach to adopt in order to avoid or minimize reporting of false negative results. Using the mean HER2 gene copy number alone for decision making results in a significant increase of equivocal cases.


PubMed | TARGOS Molecular Pathology GmbH, Protagen, Cornell University, Innsbruck Medical University and Center for Personalized Cancer Medicine
Type: Journal Article | Journal: PloS one | Year: 2016

Chronic inflammation is frequently observed on histological analysis of malignant and non-malignant prostate specimens. It is a suspected supporting factor for prostate diseases and their progression and a main cause of false positive PSA tests in cancer screening. We hypothesized that inflammation induces autoantibodies, which may be useful biomarkers. We aimed to identify and validate prostate inflammation associated serum autoantibodies in prostate cancer patients and evaluate the expression of corresponding autoantigens.Radical prostatectomy specimens of prostate cancer patients (N = 70) were classified into high and low inflammation groups according to the amount of tissue infiltrating lymphocytes. The corresponding pre-surgery blood serum samples were scrutinized for autoantibodies using a low-density protein array. Selected autoantigens were identified in prostate tissue and their expression pattern analyzed by immunohistochemistry and qPCR. The identified autoantibody profile was cross-checked in an independent sample set (N = 63) using the Luminex-bead protein array technology.Protein array screening identified 165 autoantibodies differentially abundant in the serum of high compared to low inflammation patients. The expression pattern of three corresponding antigens were established in benign and cancer tissue by immunohistochemistry and qPCR: SPAST (Spastin), STX18 (Syntaxin 18) and SPOP (speckle-type POZ protein). Of these, SPAST was significantly increased in prostate tissue with high inflammation. All three autoantigens were differentially expressed in primary and/or castration resistant prostate tumors when analyzed in an inflammation-independent tissue microarray. Cross-validation of the inflammation autoantibody profile on an independent sample set using a Luminex-bead protein array, retrieved 51 of the significantly discriminating autoantibodies. Three autoantibodies were significantly upregulated in both screens, MUT, RAB11B and CSRP2 (p>0.05), two, SPOP and ZNF671, close to statistical significance (p = 0.051 and 0.076).We provide evidence of an inflammation-specific autoantibody profile and confirm the expression of corresponding autoantigens in prostate tissue. This supports evaluation of autoantibodies as non-invasive markers for prostate inflammation.


Ruschoff J.,Targos Molecular Pathology GmbH | Ruschoff J.,Institute of Pathology Nordhessen | Hanna W.,University of Toronto | Bilous M.,University of Sydney | And 5 more authors.
Modern Pathology | Year: 2012

Trastuzumab in combination with capecitabine or 5-fluorouracil and cisplatin is approved by the European Medicines Agency for the treatment of patients with human epidermal growth factor receptor 2 (HER2+)-positive (immunohistochemistry 3+ or immunohistochemistry 2/fluorescence in situ hybridization-positive or immunohistochemistry 2/silver in situ hybridization-positive) metastatic adenocarcinoma of the stomach or gastro-esophageal junction. Approvals are underway in other countries, with recent approvals granted in the United States and Japan. Experience and data from trastuzumab use in breast cancer have highlighted the importance of quality HER2+ testing and scoring to ensure accurate identification of patients eligible for treatment. HER2+ testing in gastric cancer differs from testing in breast cancer due to inherent differences in tumor biology; gastric cancer more frequently shows HER2+ heterogeneity (focal staining) and incomplete membrane staining. Consequently, gastric cancer-specific HER2+ testing protocols have been developed and standardized and it is imperative that these recommendations be adhered to. Given the predictive value of HER2+ protein levels with response in the trastuzumab for GAstric cancer study (ToGA), immunohistochemistry should be the initial testing methodology and fluorescence in situ hybridization or silver in situ hybridization should be used to retest immunohistochemistry 2+ samples. Wherever possible, bright-field methodologies should be used as these are considered to be superior to fluorescent methodologies at identifying heterogeneous staining. Specific training is required before embarking on HER2+ testing in gastric cancer, irrespective of the experience of HER2+ testing in breast cancer. This paper provides the most up-to-date practical guidance on HER2+ testing and scoring in patients with gastric and gastro-esophageal junction cancer, as agreed by a panel of expert pathologists with extensive experience of HER2+ testing particularly reflecting the European Medicines Agency-approved indication. It is anticipated that these recommendations should ensure accurate and consistent HER2+ testing, which will allow appropriate selection of patients eligible for treatment with trastuzumab. © 2012 USCAP, Inc. All rights reserved.


Derer S.,University of Kiel | Berger S.,University of Kiel | Schlaeth M.,University of Kiel | Schneider-Merck T.,University of Kiel | And 9 more authors.
Neoplasia | Year: 2012

Oncogenic KRAS mutations in colorectal cancer (CRC) are associated with lack of benefit from epidermal growth factor receptor (EGFR)-directed antibody (Ab) therapy. However, the mechanisms by which constitutively activated KRAS (KRAS G12V) impairs effector mechanisms of EGFR-Abs are incompletely understood. Here, we established isogenic cell line models to systematically investigate the impact of KRAS G12V on tumor growth in mouse A431 xenograft models as well as on various modes of action triggered by EGFR-Abs in vitro. KRAS G12V impaired EGFR-Ab-mediated growth inhibition by stimulating receptor-independent downstream signaling. KRAS G12V also rendered tumor cells less responsive to Fc-mediated effector mechanisms of EGFR-Abs-such as complementdependent cytotoxicity (CDC) and Ab-dependent cell-mediated cytotoxicity (ADCC). Impaired CDC and ADCC activities could be linked to reduced EGFR expression in KRAS-mutated versus wild-type (wt) cells, which was restored by small interfering RNA (siRNA)-mediated knockdown of KRAS4b. Immunohistochemistry experiments also revealed lower EGFR expression in KRASmutated versus KRAS-wt harboring CRC samples. Analyses of potential mechanisms by which KRAS G12V downregulated EGFR expression demonstrated significantly decreased activity of six distinct transcription factors. Additional experiments suggested the CCAAT/enhancer-binding protein (C/EBP) family to be implicated in the regulation of EGFR promoter activity in KRAS-mutated tumor cells by suppressing EGFR transcription through up-regulation of the inhibitory family member C/EBPβ-LIP. Thus, siRNA-mediated knockdown of C/EBPβ led to enhanced EGFR expression and Ab-mediated cytotoxicity against KRAS-mutated cells. Together, these results demonstrate that KRAS G12V signaling induced C/EBPβ-dependent suppression of EGFR expression, thereby impairing Fc-mediated effector mechanisms of EGFR-Abs and rendering KRAS-mutated tumor cells less sensitive to these therapeutic agents. © 2012 Neoplasia Press, Inc. All rights reserved.

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