10.1586/ERM.12.121 811ISSN 1473-7159© 2012 Expert Reviews Ltdwww.expert-reviews.com

Meeting Report

BRAF mutation testing in clinical practice of oncology: single gene approachPei Hui (Yale University School of Medicine, CT, USA) reported the current practice of BRAF mutation testing in cancer diagnosis, prognosis and therapeutic guidance. The high prevalence of BRAFV600E activating mutation in papillary thyroid carcinoma, cutaneous malignant mela-noma and hairy cell leukemia indicates that the mutation is an important ‘driver’ in the pathogenesis of these tumors. Cancers with a BRAF mutation are generally more aggressive than their counterparts without the mutation. Diagnostically, BRAFV600E mutation is remark-able for its positive predictive value (>99%) in the diagnosis of papillary thyroid carcinoma in a thyroid fine needle aspiration specimen, clinically helpful to further stratify patients for definitive clinical management. BRAFV600E

mutation is also a highly sensitive and specific marker of hairy cell leukemia. Therapeutically, mutant BRAF has been a highly attractive target for precision cancer therapy and recent clinical trials of BRAF inhibitors in patients with late-stage malignant melanoma are changing the treatment paradigm of this highly lethal dis-ease. BRAF mutation testing using highly sen-sitive and specific methodology in a molecular diagnostic laboratory is essential in the current clinical practice of oncology [1].

Genetically informed cancer medicine: panel gene approachCindy Vnencak-Jones (Vanderbilt University School of Medicine, TN, USA) reported her experience in target panel gene mutation testing (tumor mutation prof iling) at Vanderbilt University. Using a multidisciplinary team approach, the group has developed and

Maja H Oktay1 and Pei Hui*2

1Department of Pathology, Montefiore Medical Center/Albert Einstein College of Medicine, Bronx, NY, USA 2Department of Pathology, Yale University School of Medicine, Innovative Tissue-Based Diagnostics (ITB), Cambridge Healthtech Institute, Philadelphia, PA, USA*Author for correspondence: Tel.: +1 203 785 6498 Fax: +1 203 737 1064 pei.hui@yale.edu

Innovative Tissue-Based Diagnostics Philadelphia, PA, USA, 4–5 June 2012

This article focuses on the current and emerging molecular diagnostics relevant to clinical practice of oncology discussed in the session of Molecular Pathology as the Driving Force for Personalized Oncology, one of the three main themes of innovative tissue-based diagnostics with the other two being pathology in cancer drug development, and in vivo microscopy and intraoperative imaging. The session brought together seven topics and a keynote presentation in the area of precision cancer diagnosis and treatment: single gene molecular testing as the most popular cancer molecular diagnostics in current time, panel gene mutation analysis as an emerging theme for cancer therapy targeting at multiple signaling pathways, and the next-generation sequencing platform – an ultimate molecular analysis of cancer for future clinical practice. Novel tactics based on existing technology were emphasized including in vitro drug sensitivity testing and exploring immunohistochemistry in combination with histocytological assays for risk assessment of tumor metastasis and layered immunohistochemistry to predict tumor response to target cancer treatment. Clinical molecular assay development, verification and validation were among practical topics in molecular diagnostic operations. The conference was culminated by Marc Ladanyi’s keynote presentation of the current and future strategies for comprehensive routine clinical genotyping of lung cancers for optimal selection of targeted therapies.

Molecular pathology as the driving force for personalized oncologyExpert Rev. Mol. Diagn. 12(8), 811–813 (2012)

Keywords: molecular testing • personalized oncology

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Molecular pathology as the driving force for personalized oncology

Oktay & Hui

Expert Rev. Mol. Diagn.

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MInI FoCUs y Theranostics for Innovation in 21st Century Healthcare

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implemented tumor specif ic molecular prof iling assays (SNaPshot) to improve the clinical outcome [2,3], including 39 somatic point mutations in eight genes relevant to target therapy of lung adenocarcinoma, 43 somatic point mutations in six genes relevant to target therapy of melanoma and mutations of PI3K/AKT pathway in breast cancer. In her experience, significant numbers of double or triple mutations were found in all three cancer types. The Vanderbilt–Ingram Cancer Center has developed automated reporting in laboratory information system and comprehensive decision support services with ongoing analysis to determine the clinical utility of tumor genotype with phenotype correlation. This nation’s f irst personalized cancer decision support tool, ‘My Cancer Genome’, will enable a real-time genetically informed approach to cancer medicine [101].

next-generation sequencing in cancer: from targeted panels to whole exomeMadhuri Hegde (Emory University School of Medicine, GA, USA) focused on next-generation sequencing (NGS) technol-ogy and its impact on germline and somatic mutation testing in cancer. Advantages of mutation detection by NGS include high throughput, systemic detection of all mutation types, digital readout and drug repurposing with application of known/approved drugs to new indications/cancer types or ‘right drug at right dose to the right patient at the right time’ and, therefore, cost-effective personalized cancer treatment. Hedge introduced Ion AmpliSeq™ Ready to use Predesigned panels (Ion Torrent/Life Technology, Inc.) for potential clinical appli-cations. Ion AmpliSeq Cancer Panel (detection of 739 muta-tions in 46 genes with 190 amplicons) may accomplish the entire process to annotated variant calls in less than 10 h. The newest Ion AmpliSeq Comprehensive Cancer Panel analyzes 409 genes with scalability up to 4000 amplicons in one-tube PCR, offering highly multiplexed target selection of genes implicated in cancer therapy. While panel gene analysis by NGS will likely move into clinical practice in the next few years, testing platforms targeting the whole exome may bring lower cost and high throughput to the next level of cancer molecular diagnostics. However, signifi-cant hurdles exist, including the demand for reliable mutation detection, being able to discriminate specific genetic alterations, acceptable clinical turnaround time and daunting bioinformatics barriers.

Correlated immunohistochemical & cytological assays for the prediction of hematogenous dissemination of breast cancerMaja Hrzenjak Oktay (Montefiore Medical Center, NY, USA) described a new immunohistochemical approach for the assess-ment of metastatic risk in breast cancer patients based on the density of intravasation microenvironment sites called Tumor MicroEnvironment of Metastasis (TMEM). TMEM sites consist of macrophages in a direct apposition to endothelial cells and actin-regulatory protein Mena-expressing cancer cells. The num-ber of TMEM sites has been positively correlated with metastatic

risk [4]. In addition, Oktay described a qRT-PCR assay for the actin regulatory protein Mena isoform assessment using fine-needle aspiration samples. It has been shown that the TMEM score correlates with the expression levels of a particular isoform of Mena called MenaINV, implying that TMEM and MenaINV assays may be applied in clinical practice in a synergistic way to assess the risk of metastasis [5,6].

Layered immunohistochemistry tests to predict tumor response to targeted therapiesJohn Gillespie (20/20 GeneSystem, MD, USA) reported that layered immunohistochemistry analysis (L-IHC) can be used to quantify and correlate multiple biomarkers in a single tissue section to biologically and clinically relevant events. L-IHC is compatible with conventional formalin-fixed paraffin-embedded tissue, as well as frozen tissue, and has a capacity of multiplexing up to ten markers per tissue section. The system allows protein profiling along one signaling pathway, employing fluorescence detection, using unconventional antibodies and reliable internal controls for objective analysis. Two cancer L-IHC platforms were presented: PredicTOR™ Kidney Cancer: Prediction of Response to Torisel® and PredicTOR Breast Cancer: Prediction of Response to Herceptin®.

The COXEn principle: translating in vitro chemosensitivity signatures into tools for clinical outcome predication in cancerJae Lee (University of Virginia, VA, USA) on behalf of Dan Theodorescu (University of Colorado, CO, USA) addressed inadequacies in the current chemotherapy selection process and described a new approach called Co-eXpression ExtrapolatioN (COXEN) algorithm, which can help maximize chemotherapy benefits. COXEN approach is based on THE National Cancer Institute’s (NCI’s) Developmental Therapeutics Program’s (NCI-DTP) NCI-60 Human Tumor Cell Line Screen database which has tested 60 cancer cell lines derived from nine common cancer types tested with >110,000 compounds of which >45,000 are pub-lically available. They elaborated on how using gene-expression profiling of these 60 cell lines, coupled to the in vitro response data in the NCI-60 screen, investigators could develop signatures predictive of sensitivity to chemotherapeutics within the same cell line panel. Taken a step further, gene-expression data could be used to systematically extrapolate drug sensitivity results observed in cell culture screening to predict tumor behavior in patients [7]. They emphasized that the COXEN approach can be used for both drug discovery and personalized cancer therapy [8].

From bench to clinical laboratory, the challenges in assay development for the clinical settingStephen Hewitt (NIH, MD, USA) described numerous challenges that clinical laboratories face when trying to introduce new assays into clinical practice [9]. He methodically laid out details regarding preanalytic (patient pathophysiology, specimen handing and storage), analytic (reagents, detection systems) and interpretative phase (cut-off points for making binary clinical decisions) of assay

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development and elaborated on the most important aspects of this process; verification and validation. Hewitt’s talk was a very informative summary on why only the most robust markers make it into the clinic.

Marc Ladanyi (Memorial Sloan–Kettering Cancer Center [MSKCC], NY, USA) summarized lung cancer clinical behavior and pathological classification and outlined mutations associated with particular histological subtypes and risk factors. He empha-sized different mutational profile in adenocarcinomas compared with squamous cell carcinomas and presented molecular testing for lung cancer that is in place at MSKCC. Briefly, MSKCC rec-ommends reflex profiling for 91 somatic mutations in eight genes (EGFR, KRAS, NRAS, ERBB2, PIKCA, MEK1, BRAF, AKT ), multiplex sizing assays for deletions/insertions for EGFR, and ERBB2 as well as FISH for EML4-ALK, RET and Ros1 transloca-tion [10,11]. For squamous cell carcinoma, MSKCC recommends testing for FGFR1 amplification, loss of PTEN, as well as testing for mutations in PTEN, PIOK3CA and KRAS [12]. Ladanyi also addressed Integrated Mutation Profiling of Actionable Cancer Targets (IMPACT @MSKCC) exons and selected introns testing program of 230 cancer genes. Molecular testing of lung cancer is crucial because multiple studies showed dramatically different response to targeted therapies in patients whose tumors harbor selected mutations compared with tumors without mutations. For example, Iressa pan Asian Study showed that 74% of patients with mutation responded to gefitinib compared with only 1% patients without mutation. Since the advent of targeted therapy, the survival in patients with unresectable non-small-cell lung

cancer has dramatically improved and they now live 30 months compared with 5 months 40 years ago. With continued mutation and targeted therapy discovery, patient survival is expected to improve even further.

ConclusionThe emergence and increasing clinical practice of targeted thera-pies will add many new dimensions to molecular cancer testing in the next decade. The current single-agent approach based on one specific genetic signature is far from adequate for the treatment of many cancers, resistance often develops rapidly through complex mechanisms among various cancer types. Combinatorial therapy is likely to evolve to target multiple points along the involved signaling pathways simultaneously or in sequence, depending on how well we understand the genomic alterations in cancers and the dynamic signaling network response to various target gene inhibitors. Further studies using newer technologies, such as NGS, to elucidate the biology of resistance and related mechanisms with clinical demo-graphics may prove useful in guiding ancillary testing, as well as identifying additional drug targets for combinatorial therapies.

Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

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Website

101 ‘My Cancer Genome’ personalized cancer decision support tool. www.mycancergenome.org

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