Iain B. Tan

Paradoxical Relationship between Chromosomal Instability and Survival Outcome in Cancer

Chromosomal instability (CIN) is associated with poor prognosis in human cancer. However, in certain animal tumor models elevated CIN negatively impacts upon organism fitness, and is poorly tolerated by cancer cells. To better understand this seemingly contradictory relationship between CIN and cancer cell biological fitness and its relationship with clinical outcome, we applied the CIN70 expression signature, which correlates with DNA-based measures of structural chromosomal complexity and numerical CIN in vivo, to gene expression profiles of 2,125 breast tumors from 13 published cohorts. Tumors with extreme CIN, defined as the highest quartile CIN70 score, were predominantly of the estrogen receptor negative (ER(-)), basal-like phenotype and displayed the highest chromosomal structural complexity and chromosomal numerical instability. We found that the extreme CIN/ER(-) tumors were associated with improved prognosis relative to tumors with intermediate CIN70 scores in the third quartile. We also observed this paradoxical relationship between CIN and prognosis in ovarian, gastric, and non-small cell lung cancer, with poorest outcome in tumors with intermediate, rather than extreme, CIN70 scores. These results suggest a nonmonotonic relationship between gene signature expression and HR for survival outcome, which may explain the difficulties encountered in the identification of prognostic expression signatures in ER(-) breast cancer. Furthermore, the data are consistent with the intolerance of excessive CIN in carcinomas and provide a plausible strategy to define distinct prognostic patient cohorts with ER(-) breast cancer. Inclusion of a surrogate measurement of CIN may improve cancer risk stratification and future therapeutic approaches.

Genetics: an 18-gene signature (ColoPrint®) for colon cancer prognosis.

ColoPrint® is an 18-gene expression signature designed to predict disease relapse in patients with early-stage colorectal cancer (CRC). We discuss the potential impact of ColoPrint® on clinical practice, and its contribution to our knowledge of CRC molecular heterogeneity.

Prognostic impact of bleomycin-induced pneumonitis on the outcome of Hodgkin’s lymphoma

Bleomycin-induced pneumonitis (BIP) has been well described in Hodgkin’s lymphoma (HL) patients. The impact of BIP on patients uniformly treated with adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD) is not clear; previous studies have included patients treated with both ABVD and hybrid regimens. We reviewed our experience with BIP in HL to better understand the impact of BIP on overall survival. One hundred and eighty four consecutive patients who were treated with ABVD for newly diagnosed HL were eligible for retrospective review. BIP was defined by the presence of pulmonary symptoms, bilateral interstitial infiltrates on chest X-ray, computed tomography or presence of lung fibrosis on transbronchial lung biopsy, and the absence of infection. Patients were required to meet all three criteria to be included in the BIP group. BIP was observed in 28 patients (15%). A low albumin level and the use of colony granulocyte stimulating factor were associated with a higher risk of developing BIP. Age, smoking history, and underlying lung function were not predictive of BIP. Importantly, patients with BIP had similar rates of 5-year overall survival compared to unaffected patients. There were no deaths from BIP. Omission of bleomycin from subsequent treatment did not adversely affect the outcomes.

Technical Reproducibility of Single-Nucleotide and Size-Based DNA Biomarker Assessment Using DNA Extracted from Formalin-Fixed, Paraffin-Embedded Tissues

DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues has been used in the past to analyze genetic polymorphisms. We evaluated the technical reproducibility of different types of assays for gene polymorphisms using DNA extracted from FFPE material. By using the MassARRAY iPLEX system, we investigated polymorphisms in DPYD (rs1801159 and rs3918290), UMPS (rs1801019), ERCC1 (rs11615), ERCC1 (rs3212986), and ERCC2 (rs13181) in 56 FFPE DNA samples. By using PCR, followed by size-based gel electrophoresis, we also examined TYMS 5 untranslated region 2R/3R repeats and GSTT1 deletions in 50 FFPE DNA samples and 34 DNAs extracted from fresh-frozen tissues and cell lines. Each polymorphism was analyzed by two independent runs. We found that iPLEX biomarker assays measuring single-nucleotide polymorphisms provided consistent concordant results. However, by using FFPE DNA, size-based PCR biomarkers (GSTT1 and TYMS 5 untranslated region) were discrepant in 32.7% (16/49, with exact 95% CI, 19.9%-47.5%; exact binomial confidence limit test) and 4.2% (2/48, with exact 95% CI, 0.5%-14.3%) of cases, respectively, whereas no discrepancies were observed using intact genomic DNA. Our findings suggest that DNA from FFPE material can be used to reliably test single-nucleotide polymorphisms. However, results based on size-based PCR biomarkers, and particularly GSTT1 deletions, using FFPE DNA need to be interpreted with caution. Independent repeated assays should be performed on all cases to assess potential discrepancies.

Technical Validation of a Next-Generation Sequencing Assay for Detecting Actionable Mutations in Patients with Gastrointestinal Cancer.

Targeted next-generation sequencing is becoming increasingly common as a clinical diagnostic and prognostic test for patient- and tumor-specific genetic profiles as well as to optimally select targeted therapies. Here, we describe a custom-developed, next-generation sequencing test for detecting single-nucleotide variants (SNVs) and short insertions and deletions (indels) in 93 genes related to gastrointestinal cancer from routine formalin-fixed, paraffin-embedded clinical specimens. We implemented a validation strategy, based on the College of American Pathologists requirements, using reference DNA mixtures from cell lines with known genetic variants, which model a broad range of allele frequencies. Test sensitivity achieved >99% for both SNVs and indels, with allele frequencies >10%, with high specificity (97.4% for SNVs and 93.6% for indels). We further confirmed test accuracies using primary formalin-fixed, paraffin-embedded colorectal cancer specimens characterized by alternative and conventional clinical diagnostic technologies. Robust performance was observed on the formalin-fixed, paraffin-embedded specimens: sensitivity was 97.2% and specificity was 99.2%. We also observed high intrarun and inter-run reproducibility, as well as a low cross-contamination rate. Overall assessment using cell line samples and formalin-fixed, paraffin-embedded samples showed that our custom next-generation sequencing assay has consistent detection sensitivity down to 10% variant frequency.

A formalin-fixed paraffin-embedded (FFPE)-based prognostic signature to predict metastasis in clinically low risk stage I/II microsatellite stable colorectal cancer

Approximately 20% early-stage (I/II) colorectal cancer (CRC) patients develop metastases despite curative surgery. We aim to develop a formalin-fixed and paraffin-embedded (FFPE)-based predictor of metastases in early-stage, clinically-defined low risk, microsatellite-stable (MSS) CRC patients. We considered genome-wide mRNA and miRNA expression and mutation status of 20 genes assayed in 150 fresh-frozen tumours with known metastasis status. We selected 193 genes for further analysis using NanoString nCounter arrays on corresponding FFPE tumours. Neither mutation status nor miRNA expression improved the estimated prediction. The final predictor, ColoMet19, based on the top 19 genes mRNA levels trained by Random Forest machine-learning strategy, had an estimated positive-predictive-value (PPV) of 0.66. We tested ColoMet19 on an independent test-set of 131 tumours and obtained a population-adjusted PPV of 0.67 indicating that early-stage CRC patients who tested positive have a 67% risk of developing metastases, substantially higher than the metastasis risk of 40% for node-positive (Stage III) patients who are generally treated with chemotherapy. Predicted-positive patients also had poorer metastasis-free survival (hazard ratios [HR]xa0=xa01.92, design-set; HRxa0=xa02.05, test-set). Thus, early-stage CRC patients who test positive may be considered for adjuvant therapy after surgery.

Genetics: An 18-gene signature (ColoPrint|[reg]|) for colon cancer prognosis

ColoPrint® is an 18-gene expression signature designed to predict disease relapse in patients with early-stage colorectal cancer (CRC). We discuss the potential impact of ColoPrint® on clinical practice, and its contribution to our knowledge of CRC molecular heterogeneity.

An 18-gene signature (ColoPrint®) for colon cancer prognosis: Genetics

ColoPrint® is an 18-gene expression signature designed to predict disease relapse in patients with early-stage colorectal cancer (CRC). We discuss the potential impact of ColoPrint® on clinical practice, and its contribution to our knowledge of CRC molecular heterogeneity.

Abstract 2167: Unbiased genomic approaches identify 2 major subclasses of Gastric Cancer with prognostic and predictive value superior to Lauren's and also reveals subtype-specific treatment opportunities

Introduction: The Lauren9s classification of gastric cancer (GC) is widely used with 2 subtypes, intestinal and diffuses having distinct histo-pathological and epidemiological characteristics. Beyond diagnosis, Lauren9s classes have limited prognostic and no predictive value. Methods: We performed gene expression profiling on 37 GC cell lines (GCCL) and 2 independent cohorts of 200 (SG) and 68 (AU) primary GC tumors. We used integrative molecular profiling to discover and characterise molecular subtypes of GC. Results: Unsupervised hierarchal clustering in GCCLs identified 2 distinct major molecular subtypes termed “G1”and “G2”. Linear models for microarray data (LIMMA) identified 171 distinguishing genes. These were mapped into 2 independent datasets of primary tumors where G1 and G2 subclasses were again identified. G1/G2 subtypes, discovered by unbiased techniques, were found to be related to intestinal and diffuse histology respectively (p Conclusion: We describe an unbiased genomic approach that improves upon Lauren9s to identify the two major subclasses of gastric cancer. G1/G2 classes have superior prognostic and predictive value and subclass specific therapeutic opportunities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2167.