Amplification In Breast Cancer Research Articles

Abstract P4-09-01: FGFR1 protein expression is associated with prognosis in primary breast cancer: A comprehensive analysis of gene copy number, mRNA and protein expression

Abstract Background: The Cancer Genome Atlas (TCGA) showed that copy number gain/amplification of FGFR1 was around 10% in primary breast cancer. FGFR1 gene amplification in breast cancer has been reported in some studies, more likely seen in ER-positive subtype. Several preclinical and clinical studies demonstrated that FGFR1 was one of novel targets of therapy for metastatic breast cancer. Previous studies suggested that aberrant FGFR1 expression was associated with poor prognosis, while there was no report that compared copy number aberration, mRNA and protein expression. The aim of this study is to analyze FGFR1 gene copy number, expression levels of FGFR1 mRNA and FGFR1 protein in ER-positive/HER2-negative primary breast cancer, and to examine the relationship between FGFR1 status and clinicopathological parameters including prognosis. Methods: The cohort of this study included 307 ER-positive/HER2-negative primary invasive breast cancer patients treated with standard care at Kumamoto University Hospital between June 2000 and January 2011. We performed a comprehensive analysis of FGFR1 at the levels of gene copy number, mRNA and FGFR1 protein expression analyzed by qPCR, qRT-PCR and immunohistochemistry, respectively. Results: FGFR1 gain/amplification was identified in 43 (14.0%) out of 307 patients. FGFR1 gain/amplification had significantly associated with higher nuclear grade (p=0.010). No correlations between FGFR1 mRNA expression levels and any clinicopathological factors were found. Expression levels of FGFR1 protein was positively associated with invasive tumor size (p=0.039). Modest positive correlations between these three (FGFR1 gene gain/amplification, expression levels of FGFR1 mRNA and FGFR1 protein) were found. The univariate analysis revealed that high FGFR1 protein expression was significantly related to poor prognosis (p=0.0019, HR: 2.63, 95%CI: 1.17-5.98) in terms of relapse-free survival (RFS) but not breast cancer-specific survival. The univariate analysis did not show that any factors except FGFR1 protein expression were significantly associated with RFS in this cohort. Conclusion: Expression levels of FGFR1 protein may be an independent prognostic factor in terms of RFS for ER-positive/HER2-negative breast cancer patients receiving standard care. Citation Format: Tomiguchi M, Yamamoto Y, Yamamoto-Ibusuki M, Yamaguchi R, Fujiki Y, Fujiwara S, Sueta A, Takeshita T, Inao T, Iwase H. FGFR1 protein expression is associated with prognosis in primary breast cancer: A comprehensive analysis of gene copy number, mRNA and protein expression. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-09-01.

Continuous quantification of HER2 expression by microfluidic precision immunofluorescence estimates HER2 gene amplification in breast cancer

Chromogenic immunohistochemistry (IHC) is omnipresent in cancer diagnosis, but has also been criticized for its technical limit in quantifying the level of protein expression on tissue sections, thus potentially masking clinically relevant data. Shifting from qualitative to quantitative, immunofluorescence (IF) has recently gained attention, yet the question of how precisely IF can quantify antigen expression remains unanswered, regarding in particular its technical limitations and applicability to multiple markers. Here we introduce microfluidic precision IF, which accurately quantifies the target expression level in a continuous scale based on microfluidic IF staining of standard tissue sections and low-complexity automated image analysis. We show that the level of HER2 protein expression, as continuously quantified using microfluidic precision IF in 25 breast cancer cases, including several cases with equivocal IHC result, can predict the number of HER2 gene copies as assessed by fluorescence in situ hybridization (FISH). Finally, we demonstrate that the working principle of this technology is not restricted to HER2 but can be extended to other biomarkers. We anticipate that our method has the potential of providing automated, fast and high-quality quantitative in situ biomarker data using low-cost immunofluorescence assays, as increasingly required in the era of individually tailored cancer therapy.

The relation between percentage of immunostained cells and amplification status in breast cancers with equivocal result for Her2 immunohistochemistry

We analyzed Her2 amplification in breast cancers with equivocal IHC staining to investigate the significance of percentage of Her2 immunostained tumor cells. We especially included the breast cancers with complete/incomplete membrane staining at moderate intensity in less than 10% of the invasive tumor cells.Of 1804 breast cases, 180 had moderate complete/incomplete membrane Her2 immunostaining. According to 2013 ASCO/CAP guideline, 124 were in IHC score 2+ and 56 in 1+ category. The percentage of Her2 immunostained invasive tumor cells were determined by visual estimation at the time of diagnosis and categorized into three groups; <10%, 10–50%, and >50% for this review. FISH was performed by dual DNA probe and data of Her2/cep17 ratio, Her2/cell and Cep17/cell were recorded applying 2013 ASCO/CAP criteria.There were 22 cases (12.2%) in >50% group, 102 (56.7%) in 10–50% group, and 56 (31.1%) in <10% group. Overall Her2 amplification was 25.5%. Her2 was amplified in 12 (54.5%) cases in >50% group; 26 (25.4%) in 10–50% and 8 (14.3%) in <10%.Her2 amplification was more frequent in cancers with >50% moderately immunostained cells in IHC equivocal group. However, most of Her2 IHC equivocal breast cancers contain moderate amount (10–50%) of immunostained cells. Relatively small but non-neglectable number (14%) of cases in <10% group was Her2 amplified by FISH which approaches to overall incidence of Her2 overexpression (15–20%) in all breast cancer. Breast cancers with moderate complete/incomplete membrane Her2 IHC staining in <10% of invasive tumor cells should be regarded as equivocal and reflex FISH should be performed in these cases.

An audit of frontline (F)ISH testing outside the Australian setting on HER2 positivity rates in breast cancer and the impact of the 2013 ASCO guidelines

Aim: To determine our HER2 positivity rates for invasive breast carcinoma using frontline FISH testing rather than CISH or SISH in a laboratory outside Australia, and to understand the impact of the 2013 ASCO guidelines on overall HER2 positivity rates.Data: The HER2 status in patients diagnosed with invasive breast carcinoma between 2009–2014 was determined on core or excision specimens sent to Canterbury Health Laboratories (n=797).Results: HER2 positivity rates varied from 12–21%, with an average of 15.5%. With the introduction of frontline FISH testing, the overall HER2 positivity rate was 13%, below the average for the preceding 4 years of 16%, achieving similar rates for positivity as Bilous et al.1 The 2013 ASCO guidelines resulted in an increase in overall Her2 positivity rates to 20.93%.Discussion: Discussion points will include:•Should New Zealand laboratories adopt the frontline ISH testing model as per College guidelines?•Does frontline ISH testing save money from a whole of systems perspective?•The impact of ASCO guidelines on HER2 positivity rates in breast cancer.•Is it time to change the testing algorithm to an ISH only model?•Suggested guidelines for dealing with equivocal FISH results.•Treatment implications for ISH negative, IHC positive results.•Interesting cases of IHC and FISH discordance.Reference1.Bilous M, Morey AL, Armes JE, et al. Assessing HER2 amplification in breast cancer: findings from the Australian In Situ Hybridisation Program. Breast Cancer Res Treat 2012; 134: 617–24. Aim: To determine our HER2 positivity rates for invasive breast carcinoma using frontline FISH testing rather than CISH or SISH in a laboratory outside Australia, and to understand the impact of the 2013 ASCO guidelines on overall HER2 positivity rates. Data: The HER2 status in patients diagnosed with invasive breast carcinoma between 2009–2014 was determined on core or excision specimens sent to Canterbury Health Laboratories (n=797). Results: HER2 positivity rates varied from 12–21%, with an average of 15.5%. With the introduction of frontline FISH testing, the overall HER2 positivity rate was 13%, below the average for the preceding 4 years of 16%, achieving similar rates for positivity as Bilous et al.1 The 2013 ASCO guidelines resulted in an increase in overall Her2 positivity rates to 20.93%. Discussion: Discussion points will include:•Should New Zealand laboratories adopt the frontline ISH testing model as per College guidelines?•Does frontline ISH testing save money from a whole of systems perspective?•The impact of ASCO guidelines on HER2 positivity rates in breast cancer.•Is it time to change the testing algorithm to an ISH only model?•Suggested guidelines for dealing with equivocal FISH results.•Treatment implications for ISH negative, IHC positive results.•Interesting cases of IHC and FISH discordance. Reference1.Bilous M, Morey AL, Armes JE, et al. Assessing HER2 amplification in breast cancer: findings from the Australian In Situ Hybridisation Program. Breast Cancer Res Treat 2012; 134: 617–24.

Abstract A32: MCL1 gene amplification in breast cancer is associated with TNBC status and can respond to a sorafenib/vorinostat regimen

Abstract Background: MCL1 encodes the induced myeloid leukemia cell differentiation protein Mcl-1, a member of the BCL-2 family which functions to inhibit apoptosis. Mcl-1 over-expression has been associated with high tumor grade and adverse prognosis in triple negative breast cancer (TNBC) but therapies specifically leading to inhibition of MCL-1 have not been identified. Methods: Comprehensive genomic profiling (CGP) using hybridization capture of 3,769 exons from 315 cancer-related genes and 47 introns of 19 genes commonly rearranged in cancer was applied to ≥50ng of DNA extracted from 2,824 consecutive BC and sequenced to high, uniform median coverage (&amp;gt;600X). The original primary BC was assayed in 44% of cases and a sample from a metastatic focus was assayed in 56% of cases. Results: Of 2824 consecutive BC cases, 200 (7.1%) cases harbored MCL1 amplification. Of these MCL1-amplified cases, 146 (73%) were TNBC and 54 were non-TNBC (p&amp;lt;0.0001). Twelve of the latter cases (22%) were ERBB2 (HER2) amplified and slide-based HER2 status concordance with CGP was 99%. MCL1 amplification was also observed in the TCGA dataset for 32/123 (26%) of TNBC (p=0.008). Of the MCL1 amplified TNBC cases, 88% were high grade and 98% were stage IV at the time of CGP. Genes co-altered within MCL1 amplified TNBC included TP53 (86%), MYC (41%), MYST3 (21%), LYN (20%), CCNE1 (19%), PIK3CA (18%), and AKT3 (15%). Two MCL1 amplified TNBC patients were treated with a multi-drug regimen based on sorafenib and vorinostat and experienced significant clinical benefit. Conclusions: MCL1 amplification is a frequent feature in advanced stage and high grade TNBC, and correspondingly such MCL1 amplified tumors very seldom harbor co-amplifications of ERBB2. Clinical observation suggests that treatment with sorafenib and vorinostat in heavily pre-treated MCL1 amplified patients may be correlated with clinical benefit, consistent with historic preclinical investigation. These preliminary findings suggest that MCL1 amplified TNBC may be able to benefit from combination targeted therapy, and warrant further systematic investigation. Citation Format: JS Ross, K Wang, A Johnson, J Watson, C Hatzis, L Pusztai, J Chmielecki, R Yelensky, D Lipson, JA Elvin, J Vergilio, J Suh, VA Miller, K Dicke, PJ Stephens, SM Ali. MCL1 gene amplification in breast cancer is associated with TNBC status and can respond to a sorafenib/vorinostat regimen. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A32.

Estrogen receptor alpha gene amplification in breast cancer: 25 years of debate.

Twenty-five years ago, Nembrot and colleagues reported amplification of the estrogen receptor alpha gene (ESR1) in breast cancer, initiating a broad and still ongoing scientific debate on the prevalence and clinical significance of this genetic aberration, which affects one of the most important genes in breast cancer. Since then, a multitude of studies on this topic has been published, covering a wide range of divergent results and arguments. The reported prevalence of this alteration in breast cancer ranges from 0% to 75%, suggesting that ESR1 copy number analysis is hampered by technical and interpreter issues. To date, two major issues related to ESR1 amplification remain to be conclusively addressed: (1) The extent to which abundant amounts of messenger RNA can mimic amplification in standard fluorescence in situ hybridization assays in the analysis of strongly expressed genes like ESR1, and (2) the clinical relevance of ESR1 amplification: Such relevance is strongly disputed, with data showing predictive value for response as well as for resistance of the cancer to anti-estrogen therapies, or for subsequent development of cancers in the case of precursor lesions that display amplification of ESR1. This review provides a comprehensive summary of the various views on ESR1 amplification, and highlights explanations for the contradictions and conflicting data that could inform future ESR1 research.

Abstract 52: QDNAseq: A bioinformatics pipeline for DNA copy number analysis from shallow whole genome sequencing with noise levels near the probabilistic lower limit imposed by read counting

Abstract The emerging field of precision medicine considers the patient's individual genetic make up, environment, and lifestyle to arrive at individualized treatment. For cancer patients, analysis of the tumour genome can reveal actionable alterations, including mutations (e.g., BRAF mutation in melanoma), translocations (e.g., t(9;22)(q34;q11) in CML) and copy number changes (e.g., HER2 amplification in breast cancer). Formalin-fixed paraffin embedded (FFPE) biopsy or surgical pathology samples are often the only materials available for analysis, highlighting the need for genomic analysis techniques that perform reliably with such materials. We present a complete pipeline for DNA copy number profiling by sequencing called quantitative DNA sequencing (QDNAseq), which offers a cost effective procedure for genome-wide DNA copy number analysis, facilitates correct biological interpretations from samples with a varying range of quality and is suitable for routine pathology diagnostics from fresh, frozen and FFPE materials. QDNAseq is implemented in R and made available through BioConductor and Galaxy. The pipeline requires as little as ∼0.1× genome coverage (∼6 million single-end 50nt reads per sample) and utilizes a depth of coverage (DOC) approach. A two-dimensional LOESS correction is applied for GC content and mappability and data from the 1000 genomes project are used to compile a blacklist of problematic genome regions. Downstream analyses have been included in QDNAseq by adapting algorithms previously developed for segmentation (DNAcopy) and the calling of gains and losses (CGHcall). QDNAseq is able to produce profiles with noise levels near the probabilistic lower limit imposed by read counting and yields a higher resolution than is available from microarrays. Some samples, however, have variances clearly above the probabilistic lower limit and are characterized by a wave pattern. This pattern originates in the sample, as it is reproducible, both in magnitude and in shape along the genome. Much of this wave bias can be reduced by regression with profiles from matched normal FFPE DNA samples using the algorithm implemented in NoWaves. The QDNAseq procedure is highly cost effective and robust. We have processed over 1500 clinical samples obtained from the FFPE archives of more than 25 institutions from Europe and North America. Citation Format: Daoud Sie, Ilari Scheinin, Stef Lieshout, van, Martijn Cordes, Daniel Pinkel, Donna G. Albertson, Mark A. Wiel, van de, Bauke Ylstra. QDNAseq: A bioinformatics pipeline for DNA copy number analysis from shallow whole genome sequencing with noise levels near the probabilistic lower limit imposed by read counting. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 52.

FGFR1 is an adverse outcome indicator for luminal A breast cancers.

Fibroblast growth factor receptor 1 (FGFR1) has been suggested to be the candidate gene for 8p11–12 amplification in breast cancer and its therapeutic/prognostic value is explored. Most previous studies focused on FGFR1 gene amplification, which may not necessarily lead to protein expression. Therefore, analysis of protein level may have more clinical relevance. We evaluated FGFR1 expression in a large cohort of breast cancer by immunohistochemistry, correlated with the tumor clinic-pathologic features, biomarkers expression, and patient's survival. FGFR1 expression was associated mainly with luminal cancers, particularly luminal B subtype (23.5%; p < 0.001), and it also showed adverse prognostic impact on luminal A cancers. FGFR1 expression was associated with higher pN (p = 0.023), pT (p = 0.003) stages, lymphovascular invasion (p = 0.010), p-cadherin (p = 0.028), synaptophysin (p = 0.009) and SOX2 expression (p = 0.034) in luminal A cancers. FGFR1 expressing luminal A cancers showed a similar outcome as luminal B cancers. Multivariate Cox regression analysis demonstrated FGFR1 positive luminal A cancers to be an independently poor prognosticator for disease free survival in luminal cancers (hazard ratio = 3.341, p = 0.008). Thus FGFR1 could be useful in identifying the aggressive cases amongst heterogeneous luminal A cancers. Given the relevance of FGFR pathway in treatment resistance in luminal cancers, FGFR1 could be an important tumor biomarker and adverse prognostic factor potentially exploitable in the clinical management of luminal cancers.

Economical and Organizational Impact of Adopting Different in Situ Hybridization Technologies to Assess HER2 Gene Amplification in Breast Cancer

Human epidermal growth factor receptor 2 (HER2) status identification is established by immunohistochemistry and in situ hybridisation (ISH). Silver in situ hybridisation (SISH) is an alternative technique to the fluorescence in situ hybridization (FISH). Both methods are recommended by American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) 2013 guidelines. The primary aim of this analysis is to evaluate the economical and organizational impact by adopting FISH or SISH test for HER2 testing. We conducted an analysis to evaluate costs, times (laboratory personnel working hours) and laboratory-space to process a sample using the manual method (FISH) compared to the automated system (SISH). The analysis was performed from the pathology laboratory’s perspective. We analysed the complete workflow (laboratory personnel and medical resource used) to perform the two tests (FISH and SISH) during pathology laboratory standard working days. The time required to test one sample was timed for each methodology separately and the pertaining costs were calculated. The working dimension of the two technologies and of the instruments needed was also measured. The hands-on time for SISH was 76.25% lower than the implementation of FISH and the dedicated spaces needed to carry out the SISH test was 45.27% less than FISH. Lastly, the costs of using an automated SISH system were lower than FISH. As a consequence of a significant reduction of laboratory personnel working hours, medical resources used and pathology laboratory spaces, the use of SISH technique to identify HER2 status is a cost-saving alternative to FISH from the perspective of the pathology laboratory.

Abstract 630: Detection of copy number variations in breast cancer using targeted sequencing without normal tissue controls

Abstract Cancer exome sequencing including recent studies of TCGA and ICGC consortia revealed the landscapes of somatic mutations and copy number variations (CNVs) in multiple cancers. CNVs represent an important genetic change that may contribute to tumorigenesis, tumor growth and metastatic spread. HER2 amplification in breast cancer represents a paradigm of a molecular target and personalized cancer therapy. As part of the breast cancer routine diagnostics, HER2 status is determined by immunohistochemistry (IHC) and subsequent in situ hybridization (ISH) when necessary. HER2 breast cancer patients are known to benefit from blockage of HER2 signaling, for example by treatment with the therapeutic antibody trastuzumab. A large cohort of frozen breast cancer tissues (184 patients) was investigated by semiconductor sequencing. A customer-designed panel of 154 amplicons was used including regions of mutations and CNVs known to be recurrent from preceding exome-wide studies. After sample-wise normalization, a null distribution of copy numbers was estimated using outlier robust statistics and the sequencing data of all patients and amplicons. Significance of gene amplifications was assessed by comparison with the null distribution leading to cutoff values of 2.77 copies for single hypothesis testing (p &amp;lt; 0.05) and 3.59 copies for multiple hypotheses testing (p &amp;lt; 0.05/154). First, we evaluated the accuracy of targeted sequencing and the new algorithm compared to the gold standard of IHC and ISH. The amplicon panel included three regions of HER2 located in exons 19, 20 and 21. Based on the target region in exon 20 and the single hypothesis cutoff, we obtained a sensitivity of 83% and a specificity of 95% in the detection of HER2 amplifications. Correlations between the copy number estimated from the coverage of exon 19, 20 and 21 where high (Pearson correlation R = 0.92, 0.96 and 0.97). Second, we did an unsupervised search for gene amplifications with the 154 amplicons of the sequencing panel using the multiple testing method. We detected gene amplifications that were abundant in at least 8 patients (&amp;gt;5% of the cohort) in the following 17 genes: CCND1, CDH1, CDKN1B, ERBB2, FGFR1, GRB7, MDM2, MED1, MYC, PAK1, PIK3CA, PIK3R3, RB1, STARD3, TBL1XR1, TSHZ2 and ZNF703. In conclusion, the new algorithm allowed detecting of CNVs from semiconductor sequencing without the need of normal tissue controls. Thus, using targeted sequencing, CNV detection and simple somatic mutation detection can be integrated in an easy and efficient manner. Citation Format: Jan Budczies, Volker Endris, Nikola Bangemann, Thomas Wolf, Jens-Uwe Blohmer, Abrecht Stenzinger, Manfred Dietel, Wilko Weichert, Carsten Denkert. Detection of copy number variations in breast cancer using targeted sequencing without normal tissue controls. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 630. doi:10.1158/1538-7445.AM2015-630

Status of estrogen receptor 1 (ESR1) gene in mastopathy predicts subsequent development of breast cancer.

Mastopathy is a common disease of the breast likely associated with elevated estrogen levels and a putative risk factor for breast cancer. The role of estrogen receptor alpha (ESR1) in mastopathy has not been investigated previously. Here, we investigated the prevalence of ESR1 gene amplification in mastopathy and its prediction for breast cancer. Paraffin-embedded tissues from 58 women with invasive breast cancer were analyzed. For all women, tissues with mastopathy taken at least 1.5years before first diagnosis of breast cancer were available. Tissue from 46 women with mastopathy without a diagnosis of breast carcinoma in the observed time frame (12-18years) was used as control. Fluorescence in situ hybridization analysis revealed that ESR1 was amplified in nine of 58 (15.5%) breast cancers. All ESR1-amplified breast cancers were strongly positive for estrogen receptor with ER immunohistochemistry. Interestingly, in women with ESR1 amplification in breast cancer, the amplification was detectable in mastopathic tissues prior to the first diagnosis of breast cancer but was absent in tissues from women with mastopathy who did not develop breast cancer. Our study suggests that ESR1 gene amplification is an early event in breast pathology and might be a helpful predictive marker to identify patients at high risk of developing breast cancer.

Comparison of the 2007 and 2013 ASCO/CAP evaluation systems for HER2 amplification in breast cancer

It has been proven that chromosome 17 centromere (CEP17) amplification causes misleading human epidermal growth factor receptor 2 (HER2) gene fluorescence in situ hybridization (FISH) results, precluding anti-HER2-based therapy in some patients with breast carcinoma. We used the 2013 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) scoring criteria to evaluate HER2 amplification status in 175 cases of breast carcinoma with chromosome 17 polysomy. We used immunohistochemistry (IHC) to determine the HER2 amplification status, and 2-color FISH to detect CEP17, and reviewed the results of initial evaluation using the 2007 ASCO/CAP criteria. Of the 175 cases, 17, 95, and 63 were IHC 0/1+, 2+, and 3+, respectively. Evaluation of IHC HER2 status according to the 2013 ASCO/CAP criteria identified significantly more HER2-positive cases compared to cases evaluated using the 2007 criteria (p<0.05). When the FISH results were evaluated in parallel with the 2013 criteria, we found that 22 cases were not HER2-negative despite the presence of polysomy 17, which, according to the 2013 criteria, indicates HER2-positive status. Our findings indicate that in breast carcinoma, HER2 status in the presence of polysomy 17 may vary with the scoring criteria used. In turn, performing FISH and evaluating samples using the 2013 ASCO/CAP criteria means that more patients with breast cancer may be appropriate for targeted treatment with trastuzumab, potentially improving their outcome.

Abstract 1829: Regulation of Mixed Lineage Kinase-3 activity by Her2 and its implication in death or survival

Abstract Human Epidermal Growth Factor Receptor 2 (Her2) is a receptor tyrosine kinase that regulates cell growth and differentiation signaling pathways. The protein Her2 is significantly overexpressed in 20-30% of breast cancer. The available clinical data proves that Her2 amplification in breast cancer is associated with worst overall survival. Therefore, Her2 has become a very important candidate for drug targeting and currently Her2 positive patients are treated with humanized monoclonal antibody, Trastuzumab (Herceptin) and a small molecule, Lapatinib that blocks Her2 tyrosine kinase activity. It is reported that Her2 amplification leads to activation of multiple survival pathways, including PI3K-Akt. However, it is not clearly known how Trastuzumab promotes cell death and what pro-apoptotic pathways are activated downstream of Her2 blockage? It is also reported that patients on Trastuzumab develop subsequent resistance to the therapy. Previously we published that in breast cancer tumors, a pro-apoptotic kinase, Mixed Lineage Kinase-3 (MLK-3) was inhibited, specifically in estrogen receptor (ER) positive tumors. The kinase activity of MLK-3 was down regulated by estrogen and this prevented MLK-3-mediated cell death. Since Her2 serves as a drug target in breast cancer, therefore, we asked whether Her2 plays any role in regulating MLK-3 kinase activation and whether MLK-3 plays any role in Trastuzumab resistance. Our results showed that the endogenous MLK-3 kinase activity was up-regulated in SkBr3 cells upon treatment with Trastuzumab and Lapatinib. Whether the activation of MLK-3 upon Her2 blockage leads to cell death or survival is still not known and experiments are underway to investigate these aspects. We also observed that Her3 was the dimerization partner of Her2 in Trastuzumab- or Lapatinib- mediated MLK-3 activation. Not knowing any role of MLK-3 in Trastuzumab resistance, we used Trastuzumab sensitive and resistant BT474 cell model to investigate the role of MLK-3 in Trastuzumab resistance. Our data clearly showed that knockdown of MLK-3 caused spontaneous cell death in resistant cells. The Trastuzumab treatment further amplified cell death in MLK-3 knockdown resistant cells. The Akt1 activation in these cells was also diminished upon MLK-3 knockdown. Taken together, our data demonstrate that like ER, the Her2 also down regulates MLK-3 kinase activity and perhaps promote cell survival. Furthermore, paradoxically, the MLK-3 knockdown promoted cell death in Trastuzumab resistance BT474 cells, suggesting that MLK-3 could also serve as survival factor in resistant cells. Whether MLK-3 serves as a pro-apoptotic kinase or survival kinase in Trastuzumab mediated pathway needs to be extensively examined in near future. Citation Format: Subhasis Das, Gautam Sondarva, Navin Viswakarma, Rakesh Sathish Nair, Clodia Osipo, Basabi Rana, Ajay Rana. Regulation of Mixed Lineage Kinase-3 activity by Her2 and its implication in death or survival. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1829. doi:10.1158/1538-7445.AM2014-1829

Abstract 5561: Clinical drug response can be predicted using baseline gene expression levels and in vitro drug sensitivity in cell lines

Abstract Robust prediction of in vivo chemotherapeutic response, using baseline gene expression and drug sensitivity data gathered on cancer cell lines, has been a profoundly important, long standing and controversial problem in pharmacogenomics. Here, we present for the first time, a solution to this problem. Currently, personalizing cancer chemotherapy relies on pathology and more recently molecular biomarker-based approaches (e.g. ERBB2 amplification in breast cancer). However, as the driving biology are normally not fully understood, the majority of existing biomarkers do not capture a substantial proportion of variability in drug response. This partly explains the commonly observed lack of reproducibility of findings (e.g. from many conventional gene expression signatures) when these markers are applied to new datasets. In this study, we developed an approach to predict in vivo drug sensitivity that leverages whole-genome gene expression microarray data and allows the expression of every gene to influence the prediction by a small amount. The method works by fitting a ridge regression model of baseline genome-wide gene expression levels against in vitro drug sensitivity in a very large panel of approximately 700 cancer cell lines. Then, after a (crucial) data homogenization step, these models are applied to baseline expression levels from primary tumor biopsies. Our method successfully predicted patient response to different chemotherapeutic agents in three (of four total suitable) independent, publicly available clinical trials, each investigating different drugs and different types of cancer. In each of these cases, we predicted drug response at least as accurately as previously published models that had been derived from the clinical data itself. Interestingly, our approach could also predict clinical response in the absence of any known drug sensitivity biomarker. We effectively enriched for drug responders in breast, myeloma and lung cancers, treated with docetaxel, bortezomib and erlotinib respectively, thus identifying responders to both cytotoxic and targeted agents. Many previous clinical trials and in vitro assays have attempted to discover biomarkers of drug sensitivity, but found that the genes/aberrations which they had identified, performed poorly as predictors, once applied to out-of-batch sets of samples. Our models, on the other hand, are trained on an independent set of cancer cell lines and performed well on three completely separate and independent clinical trial datasets (all assessed using different microarray platforms). These results have far-reaching implications for personalized medicine and drug development (e.g. for the development of companion diagnostics). All datasets and bioinformatics tools to reproduce our results are publicly available. Citation Format: Paul Geeleher, Nancy Cox, R. Stephanie Huang. Clinical drug response can be predicted using baseline gene expression levels and in vitro drug sensitivity in cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5561. doi:10.1158/1538-7445.AM2014-5561

乳癌におけるDual Color in situ Hybridization法によるHER2遺伝子増幅の検出―FISH法との比較―

乳癌におけるDual Color in situ Hybridization法によるHER2遺伝子増幅の検出―FISH法との比較―

Comparison of Dako fluorescence in situ hybridization assays (FISH and IQFISH) in the assessment of HER2 amplification in breast cancer

Determining human epidermal growth factor receptor 2 (HER2) status in breast cancer patients can be attained by fluorescent in situ hybridization (FISH) analysis, which prompts targeted treatment options. This study was performed to compare the staining results of Dako’s manual overnight HER2 FISH pharmDx™ assay with the manual (4 hours) HER2 IQFISH pharmDx™ assay and the Dako Omnis automated (4 hours) HER2 IQFISH assay, for assessment of HER2 amplification.Thirty-four formalin-fixed paraffin-embedded (FFPE) tissue blocks from patients diagnosed with invasive breast cancer were used to compare three FISH assays in three experiments. All assays included Texas Red-labeled HER2 and fluorescein-labeled centromere 17 (CEN-17) probes. First, 20 blocks with known HER2 immunohistochemistry (IHC) and FISH results from a reference laboratory were used in the validation of HER2 FISH pharmDx™ assay. Secondly, 19 of the same blocks were used in a comparison of HER2 FISH pharmDx™ assay and HER2 Instant Quality FISH pharmDx (IQFISH pharmDx™) assays. The third experiment, which included 23 blocks, compared the HER2 FISH pharmDx™ assay with the Dako Omnis IQFISH assay (stained on a Beta version of Dako’s new Omnis platform). Stained slides were counted following American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines (2007 version), with three categories of labeling: <1·8 (negative), 1·8–2·2 (equivocal), and >2·2 (positive) calculated HER2/CEN-17 ratio. The results showed high agreement between all assays employed. Initial validation of HER2 FISH pharmDx™ assay to the results provided by a reference laboratory had a concordance of 95%. The concordance between HER2 FISH pharmDx™ assay and HER2 IQFISH pharmDx™ assay was 100%. There was also 100% concordance between manual and Omnis IQFISH staining. A high level of concordance was obtained in all three experiments, indicating that manual and automated IQFISH are comparable to HER2 FISH pharmDx™. IQFISH has a faster turn-around-time (TAT) and showed comparable results in quality and quantitation.

Absence of estrogen receptor alpha (ESR1) gene amplification in a series of breast cancers in Taiwan.

Immunohistochemical expression of ERα, encoded by the ESR1 (estrogen receptor 1) gene located at 6q25.1, is the most important determinant of responsiveness to endocrine therapy in breast cancer. The prevalence and significance of ESR1 amplification in breast cancer remain controversial. We set out to assess ESR1 status and its relevance in breast cancer in Taiwan. We tested tissue samples from 311 invasive carcinomas in a tissue microarray for ESR1 status by fluorescent in situ hybridization (FISH) and chromogenic in situ hybridization (CISH). In order to examine its association with ERα and ESR1 status, HER2 status was determined by FISH. Of the carcinomas, 58.8% (183/311) was ERα positive. None of the carcinomas showed amplification of ESR1 by either method, whereas 24.1% (75/311) of the carcinomas harbored HER2 amplification. Of the carcinomas, 9.6% (26/301) showed ESR1 gain (1.3 ≤ ratio ESR1/chromosome 6 < 2) by FISH and 10% (24/299) by CISH. FISH and CISH results showed a good correlation (κ-coefficient = 0.786). ESR1 gain by FISH and CISH was significantly associated with high-grade (P = 0.0294 and 0.0417, respectively) but not with ERα expression, HER2 status, or overall survival. ERα positivity was significantly associated with better overall survival (P = 0.039). HER2 amplification was significantly related with poor overall survival (P = 0.002). Our data confirm that in breast cancer, HER2 amplification is a frequent genetic aberration and a negative prognostic factor, and show that ESR1 amplification is not a key genetic abnormality in the tumorigenesis of breast cancer in Taiwan.

Down-Regulation of UNC5D in Bladder Cancer: UNC5D as a Possible Mediator of Cisplatin Induced Apoptosis in Bladder Cancer Cells

Identifying potential targets would improve therapeutic planning anddisease management. Therefore, we investigated whether the novel identified dependence receptor UNC5D acts as a tumor suppressor in bladder malignancies. We assessed the UNC5D level in a panel of 15 primary bladder carcinomas and 6 cell lines using real-time reverse transcriptase-polymerase chain reaction and Western blot. MTT assay, TUNEL staining, colony formation assay and Western blot were done in cells untransfected and transfected with UNC5D vector, siUNC5D or siDAPK. UNC5D was dramatically down-regulated in bladder cancer tissue samples and malignant cell lines. Restoration of UNC5D expression in bladder cancer cells lacking endogenous UNC5D expression suppressed cell proliferation and survival. Cisplatin treatment significantly induced UNC5D expression and DAPK dephosphorylation while UNC5D knockdown decreased bladder cancer cell sensitivity to cisplatin. DAPK silencing significantly inhibited the effect of UNC5D on apoptosis induced by cisplatin. Our study suggests that UNC5D may have important roles as a novel suppressor in bladder cancer via the UNC5D/DAPK pathway.

Correction: ESR1 Amplification in Breast Cancer by Optimized RNase FISH: Frequent but Low-Level and Heterogeneous

The Competing Interests statement is incorrect. The correct Competing Interests statement is: The University Medical Centre Hamburg-Eppendorf has filed patent US8101352B2 “Detection of ESR1 Amplification in Breast Cancer” and accordant patent applications in EU and CA for certain technology described in this paper.

Molecular testing for targeted therapy – sequencing based assay

Significant advances have been made in molecular and genetic studies, and our knowledge of genomic alterations in various cancers has increased. It is generally accepted that oncogenesis is initiated by genetic alteration of drivers. Detection of mutations of these driver genes can lead to targeted therapy agents, referred as personalized medicine. Examples include HER2 amplification in breast cancer, EGFR mutations in lung cancer and BRAF mutations in melanoma. Single gene assays for selected driver genes were the method of choice until more robust gene panel testing by next generation sequencing came to the horizon. Several different methodologies are available to detect mutations and each assay has its own pros and cons, which will be discussed in this session. Sanger chain termination sequencing has been considered the gold standard for long time, but it is time consuming and has lower sensitivity. Pyrosequencing, sequencing by synthesis, is more sensitive and quick, but is limited to short read lengths. Allele-specific real-time PCR has been utilized for clinical testing for oncogene mutations as FDA-cleared assays are available. It is very sensitive, but is limited to hot spot mutation detection. Real-time PCR melt curve analysis is also sensitive, but cannot identify the exact mutation.