Publications by authors named "Paul Waring"

92 Publications

Retrospective analysis of Schlafen11 (SLFN11) to predict the outcomes to therapies affecting the DNA damage response.

Br J Cancer 2021 Oct 18. Epub 2021 Oct 18.

Translational Medicine, Oncology R&D, AstraZeneca, Cambridge, UK.

Background: The absence of the putative DNA/RNA helicase Schlafen11 (SLFN11) is thought to cause resistance to DNA-damaging agents (DDAs) and PARP inhibitors.

Methods: We developed and validated a clinically applicable SLFN11 immunohistochemistry assay and retrospectively correlated SLFN11 tumour levels to patient outcome to the standard of care therapies and olaparib maintenance.

Results: High SLFN11 associated with improved prognosis to the first-line treatment with DDAs platinum-plus-etoposide in SCLC patients, but was not strongly linked to paclitaxel-platinum response in ovarian cancer patients. Multivariate analysis of patients with relapsed platinum-sensitive ovarian cancer from the randomised, placebo-controlled Phase II olaparib maintenance Study19 showed SLFN11 tumour levels associated with sensitivity to olaparib. Study19 patients with high SLFN11 had a lower progression-free survival (PFS) hazard ratio compared to patients with low SLFN11, although both groups had the benefit of olaparib over placebo. Whilst caveated by small sample size, this trend was maintained for PFS, but not overall survival, when adjusting for BRCA status across the olaparib and placebo treatment groups, a key driver of PARP inhibitor sensitivity.

Conclusion: We provide clinical evidence supporting the role of SLFN11 as a DDA therapy selection biomarker in SCLC and highlight the need for further clinical investigation into SLFN11 as a PARP inhibitor predictive biomarker.
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http://dx.doi.org/10.1038/s41416-021-01560-1DOI Listing
October 2021

Synergistic targeting of BRCA1 mutated breast cancers with PARP and CDK2 inhibition.

NPJ Breast Cancer 2021 Aug 31;7(1):111. Epub 2021 Aug 31.

The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, NSW, Australia.

Basal-like breast cancers (BLBC) are aggressive breast cancers that respond poorly to targeted therapies and chemotherapies. In order to define therapeutically targetable subsets of BLBC we examined two markers: cyclin E1 and BRCA1 loss. In high grade serous ovarian cancer (HGSOC) these markers are mutually exclusive, and define therapeutic subsets. We tested the same hypothesis for BLBC. Using a BLBC cohort enriched for BRCA1 loss, we identified convergence between BRCA1 loss and high cyclin E1 protein expression, in contrast to HGSOC in which CCNE1 amplification drives increased cyclin E1. In cell lines, BRCA1 loss was associated with stabilized cyclin E1 during the cell cycle, and BRCA1 siRNA led to increased cyclin E1 in association with reduced phospho-cyclin E1 T62. Mutation of cyclin E1 T62 to alanine increased cyclin E1 stability. We showed that tumors with high cyclin E1/BRCA1 mutation in the BLBC cohort also had decreased phospho-T62, supporting this hypothesis. Since cyclin E1/CDK2 protects cells from DNA damage and cyclin E1 is elevated in BRCA1 mutant cancers, we hypothesized that CDK2 inhibition would sensitize these cancers to PARP inhibition. CDK2 inhibition induced DNA damage and synergized with PARP inhibitors to reduce cell viability in cell lines with homologous recombination deficiency, including BRCA1 mutated cell lines. Treatment of BRCA1 mutant BLBC patient-derived xenograft models with combination PARP and CDK2 inhibition led to tumor regression and increased survival. We conclude that BRCA1 status and high cyclin E1 have potential as predictive biomarkers to dictate the therapeutic use of combination CDK inhibitors/PARP inhibitors in BLBC.
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http://dx.doi.org/10.1038/s41523-021-00312-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8408175PMC
August 2021

BCL-XL exerts a protective role against anemia caused by radiation-induced kidney damage.

EMBO J 2020 12 25;39(24):e105561. Epub 2020 Nov 25.

The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia.

Studies of gene-targeted mice identified the roles of the different pro-survival BCL-2 proteins during embryogenesis. However, little is known about the role(s) of these proteins in adults in response to cytotoxic stresses, such as treatment with anti-cancer agents. We investigated the role of BCL-XL in adult mice using a strategy where prior bone marrow transplantation allowed for loss of BCL-XL exclusively in non-hematopoietic tissues to prevent anemia caused by BCL-XL deficiency in erythroid cells. Unexpectedly, the combination of total body γ-irradiation (TBI) and genetic loss of Bcl-x caused secondary anemia resulting from chronic renal failure due to apoptosis of renal tubular epithelium with secondary obstructive nephropathy. These findings identify a critical protective role of BCL-XL in the adult kidney and inform on the use of BCL-XL inhibitors in combination with DNA damage-inducing drugs for cancer therapy. Encouragingly, the combination of DNA damage-inducing anti-cancer therapy plus a BCL-XL inhibitor could be tolerated in mice, at least when applied sequentially.
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http://dx.doi.org/10.15252/embj.2020105561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7737616PMC
December 2020

Expanded Low Allele Frequency and V600E Testing in Metastatic Colorectal Cancer as Predictive Biomarkers for Cetuximab in the Randomized CO.17 Trial.

Clin Cancer Res 2021 01 21;27(1):52-59. Epub 2020 Oct 21.

University of Texas, MD Anderson Cancer Center, Houston, Texas.

Purpose: Expanded mutations have not been assessed as predictive for single-agent cetuximab in metastatic colorectal cancer (mCRC), and low mutant allele frequency (MAF) mutations are of unclear significance. We aimed to establish cetuximab efficacy in optimally selected patients using highly sensitive beads, emulsion, amplification, and magnetics (BEAMing) analysis, capable of detecting alterations below standard clinical assays.

Patients And Methods: CO.17 trial compared cetuximab versus best supportive care (BSC) in -unselected mCRC. We performed analysis on microdissected tissue of 242 patients in CO.17 trial using BEAMing for (codons 12/13/59/61/117/146) and V600E. Patients without BEAMing but with previous Sanger sequencing-detected mutations were included.

Results: , and mutations were present in 53%, 4%, and 3% of tumors, respectively. Cetuximab improved overall survival [OS; HR, 0.51; 95% confidence interval (CI), 0.32-0.81; = 0.004] and progression-free survival (PFS; HR, 0.25; 95% CI, 0.15-0.41; < 0.0001) compared with BSC in wild-type patients. Cetuximab did not improve OS/PFS for , or mutated tumors, and tests of interaction confirmed expanded ( = 0.0002) and ( = 0.006) as predictive, while mutations were not ( = 0.089). BEAMing identified 14% more tumors as mutant than Sanger sequencing, and cetuximab lacked activity in these patients. Mutations at MAF < 5% were noted in 6 of 242 patients (2%). One patient with a A59T mutation (MAF = 2%) responded to cetuximab. More than mutations were low MAF (OR, 20.50; 95% CI, 3.88-96.85; = 0.0038).

Conclusions: We establish single-agent cetuximab efficacy in optimally selected patients and show that subclonal alterations are uncommon and remain of indeterminate significance.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-2710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7785657PMC
January 2021

Intratumoral Transcriptome Heterogeneity Is Associated With Patient Prognosis and Sidedness in Patients With Colorectal Cancer Treated With Anti-EGFR Therapy From the CO.20 Trial.

JCO Precis Oncol 2020 29;4. Epub 2020 Sep 29.

Division of Molecular Pathology, The Institute of Cancer Research, London, United Kingdom.

Purpose: Metastatic colorectal cancers (mCRCs) assigned to the transit-amplifying (TA) CRCAssigner subtype are more sensitive to anti-epidermal growth factor receptor (EGFR) therapy. We evaluated the association between the intratumoral presence of TA signature (TA-high/TA-low, dubbed as TA-ness classification) and outcomes in CRCs treated with anti-EGFR therapy.

Patients And Methods: The TA-ness classes were defined in a discovery cohort (n = 84) and independently validated in a clinical trial (CO.20; cetuximab monotherapy arm; n = 121) and other samples using an established NanoString-based gene expression assay. Progression-free survival (PFS), overall survival (OS), and disease control rate (DCR) according to TA-ness classification were assessed by univariate and multivariate analyses.

Results: The TA-ness was measured in 772 samples from 712 patients. Patients (treated with anti-EGFR therapy) with TA-high tumors had significantly longer PFS (discovery hazard ratio [HR], 0.40; 95% CI, 0.25 to 0.64; < .001; validation HR, 0.65; 95% CI, 0.45 to 0.93; = .018), longer OS (discovery HR, 0.48; 95% CI, 0.29 to 0.78; = .003; validation HR, 0.67; 95% CI, 0.46 to 0.98; = .04), and higher DCR (discovery odds ratio [OR]; 14.8; 95% CI, 4.30 to 59.54; < .001; validation OR, 4.35; 95% CI, 2.00 to 9.09; < .001). TA-ness classification and its association with anti-EGFR therapy outcomes were further confirmed using publicly available data (n = 80) from metastatic samples (PFS < .001) and patient-derived xenografts ( = .042). In an exploratory analysis of 55 patients with wild-type and left-sided tumors, TA-high class was significantly associated with longer PFS and trend toward higher response rate (PFS HR, 0.53; 95% CI, 0.28 to 1.00; = .049; OR, 5.88; 95% CI, 0.71 to 4.55; = .09; response rate 33% in TA-high and 7.7% in TA-low).

Conclusion: TA-ness classification is associated with prognosis in patients with mCRC treated with anti-EGFR therapy and may further help understanding the value of sidedness in patients with wild-type tumors.
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http://dx.doi.org/10.1200/PO.20.00050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529528PMC
September 2020

Cetuximab Alone or With Irinotecan for Resistant KRAS-, NRAS-, BRAF- and PIK3CA-wild-type Metastatic Colorectal Cancer: The AGITG Randomized Phase II ICECREAM Study.

Clin Colorectal Cancer 2018 12 8;17(4):313-319. Epub 2018 Jun 8.

Department of Oncology, Monash Health and Monash University, Clayton, Victoria, Australia.

Background: The Irinotecan Cetuximab Evaluation and Cetuximab Response Evaluation (ICECREAM) study assessed the efficacy of cetuximab monotherapy compared with cetuximab combined with chemotherapy for quadruple wild-type (KRAS, NRAS, BRAF, or P13KCA exon 20) metastatic colorectal cancer.

Patients And Methods: Patients were enrolled in an open-label, multicenter, phase II trial and randomly assigned to cetuximab 400 mg/m, then 250 mg/m cetuximab weekly, with or without irinotecan 180 mg/m every 2 weeks. The primary endpoint was 6-month progression-free survival; secondary endpoints were response rate, overall survival, toxicity, and quality of life.

Results: From 2012 to 2016, 48 patients were recruited. Two were ineligible, and 2 were not evaluable for response. Characteristics were balanced, except gender (male, 62% vs. 72%) and primary sidedness (left, 95% vs. 68%). For cetuximab compared with cetuximab-irinotecan, progression-free survival was 14% versus 41% (hazard ratio, 0.39; 95% confidence interval, 0.20-0.78; P = .008); response rate was 10% (2 partial responses) versus 38% (1 complete, 8 partial); P = .04. Grade 3 to 4 toxicities were less with cetuximab monotherapy (23% vs. 50%); global and specific quality of life scores did not differ.

Conclusion: In comparison with cetuximab alone, cetuximab plus irinotecan increases the response rate and delays progression in irinotecan-resistant RAS wild-type colorectal cancer. This echoes data from molecularly unselected patients.
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http://dx.doi.org/10.1016/j.clcc.2018.06.002DOI Listing
December 2018

19q12 amplified and non-amplified subsets of high grade serous ovarian cancer with overexpression of cyclin E1 differ in their molecular drivers and clinical outcomes.

Gynecol Oncol 2018 11 9;151(2):327-336. Epub 2018 Sep 9.

Centre for Translational Pathology, Department of Pathology, University of Melbourne, Parkville, Victoria 3010, Australia; Department of Surgery, University of Melbourne, Parkville, Victoria 3010, Australia. Electronic address:

Objectives: Readily apparent cyclin E1 expression occurs in 50% of HGSOC, but only half are linked to 19q12 locus amplification. The amplified/cyclin E1 subset has intact BRCA1/2, unfavorable outcome, and is potentially therapeutically targetable. We studied whether non-amplified/cyclin E1 HGSOC has similar characteristics. We also assessed the expression of cyclin E1 degradation-associated proteins, FBXW7 and USP28, as potential drivers of high cyclin E1 expression in both subsets.

Methods: 262 HGSOC cases were analyzed by in situ hybridization for 19q12 locus amplification and immunohistochemistry for cyclin E1, URI1 (another protein encoded by the 19q12 locus), FBXW7 and USP28 expression. Tumors were classified by 19q12 amplification status and correlated to cyclin E1 and URI1 expression, BRCA1/2 germline mutation, FBXW7 and USP28 expression, and clinical outcomes. Additionally, we assessed the relative genomic instability of amplified/cyclin E1 and non-amplified/cyclin E1 groups of HGSOC datasets from The Cancer Genome Atlas.

Results: Of the 82 cyclin E1 cases, 43 (52%) were amplified and 39 (48%) were non-amplified. Unlike amplified tumors, non-amplified/cyclin E1 tumor status was not mutually exclusive with gBRCA1/2 mutation. The non-amplified/cyclin E1 group had significantly increased USP28, while the amplified/cyclin E1 cancers had significantly lower FBXW7 expression consistent with a role for both in stabilizing cyclin E1. Notably, only the amplified/cyclin E1 subset was associated with genomic instability and had a worse outcome than non-amplified/cyclin E1 group.

Conclusions: Amplified/cyclin E1 and non-amplified/cyclin E1 tumors have different pathological and biological characteristics and clinical outcomes indicating that they are separate subsets of cyclin E1 HGSOC.
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http://dx.doi.org/10.1016/j.ygyno.2018.08.039DOI Listing
November 2018

PI3K activation in neural stem cells drives tumorigenesis which can be ameliorated by targeting the cAMP response element binding protein.

Neuro Oncol 2018 09;20(10):1344-1355

Department of Pathology, The University of Melbourne, Parkville, Victoria, Australia.

Background: Hyperactivation of phosphoinositide 3-kinase (PI3K) signaling is common in cancers, but the precise role of the pathway in glioma biology remains to be determined. Some understanding of PI3K signaling mechanisms in brain cancer comes from studies on neural stem/progenitor cells (NSPCs), where signals transmitted via the PI3K pathway cooperate with other intracellular pathways and downstream transcription factors to regulate critical cell functions.

Methods: To investigate the role of the PI3K pathway in glioma initiation and development, we generated a mouse model targeting the inducible expression of a PIK3CAH1047A oncogenic mutant and deletion of the PI3K negative regulator, phosphatase and tensin homolog (PTEN), to NSPCs.

Results: Expression of a Pik3caH1047A was sufficient to generate tumors with oligodendroglial features, but simultaneous loss of PTEN was required for the development of invasive, high-grade glioma. Pik3caH1047A-PTEN mutant NSPCs exhibited enhanced neurosphere formation which correlated with increased Wnt signaling, while loss of cAMP response element binding protein (CREB) in Pik3caH1047A-Pten mutant tumors led to longer symptom-free survival in mice.

Conclusion: Taken together, our findings present a novel mouse model for glioma demonstrating that the PI3K pathway is important for initiation of tumorigenesis and that disruption of downstream CREB signaling attenuates tumor expansion.
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http://dx.doi.org/10.1093/neuonc/noy068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6140786PMC
September 2018

Exploring the feasibility and utility of exome-scale tumour sequencing in a clinical setting.

Intern Med J 2018 07;48(7):786-794

Department of Medical Oncology, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.

Background: Technology has progressed from single gene panel to large-scale genomic sequencing. This is raising expectations from clinicians and patients alike. The utility and performance of this technology in a clinical setting needs to be evaluated.

Aim: This pilot study investigated the feasibility of using exome-scale sequencing (ESS) to identify molecular drivers within cancers in real-time for Precision Oncology in the clinic.

Methods: Between March 2014 and March 2015, the Victorian Comprehensive Cancer Centre Alliance explored the feasibility and utility of ESS in a pilot study. DNA extracted from the tumour specimens underwent both ESS and targeted 'hotspot' sequencing (TS). Blood was taken for germline analysis. A multi-disciplinary molecular tumour board determined the clinical relevance of identified mutations; in particular, whether they were 'actionable' and/or 'druggable'.

Results: Of 23 patients screened, 15 (65%) met the tissue requirements for genomic analysis. TS and ESS were successful in all cases. ESS identified pathogenic somatic variants in 73% (11/15 cases) versus 53% (8/15 cases) using TS. Clinically focused ESS identified 63 variants, consisting of 30 somatic variants (including all 13 identified by TS) and 33 germline variants. Overall, there were 48 unique variants. ESS had a clinical impact in 53% (8/15 cases); 47% (7/15 cases) were referred to the familial cancer clinic, and 'druggable' targets were identified in 53% (8/15 cases).

Conclusion: ESS of tumour DNA impacted clinical decision-making in 53%, with 20% more pathogenic variants identified through ESS than TS. The identification of germline variants in 47% was an unexpected finding.
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http://dx.doi.org/10.1111/imj.13806DOI Listing
July 2018

Identification of Mutation as a Genetic Driver of Prostate Cancer That Cooperates with Loss to Accelerate Progression and Castration-Resistant Growth.

Cancer Discov 2018 06 26;8(6):764-779. Epub 2018 Mar 26.

Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.

Genetic alterations that potentiate PI3K signaling are frequent in prostate cancer, yet how different genetic drivers of the PI3K cascade contribute to prostate cancer is unclear. Here, we report mutation/amplification correlates with poor survival of patients with prostate cancer. To interrogate the requirement of different PI3K genetic drivers in prostate cancer, we employed a genetic approach to mutate in mouse prostate epithelium. We show mutation causes p110α-dependent invasive prostate carcinoma Furthermore, we report that mutation and loss coexist in patients with prostate cancer and can cooperate to accelerate disease progression via AKT-mTORC1/2 hyperactivation. Contrasting single mutants that slowly acquire castration-resistant prostate cancer (CRPC), concomitant mutation and loss caused CRPC. Thus, mutation and deletion are not functionally redundant. Our findings indicate that mutation is an attractive prognostic indicator for prostate cancer that may cooperate with loss to facilitate CRPC in patients. We show mutation correlates with poor prostate cancer prognosis and causes prostate cancer in mice. Moreover, mutation and loss coexist in prostate cancer and can cooperate to accelerate tumorigenesis and facilitate CRPC. Delineating this synergistic relationship may present new therapeutic/prognostic approaches to overcome castration/PI3K-AKT-mTORC1/2 inhibitor resistance. .
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http://dx.doi.org/10.1158/2159-8290.CD-17-0867DOI Listing
June 2018

Loss of NF-κB1 Causes Gastric Cancer with Aberrant Inflammation and Expression of Immune Checkpoint Regulators in a STAT-1-Dependent Manner.

Immunity 2018 03;48(3):570-583.e8

The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia. Electronic address:

Polymorphisms in NFKB1 that diminish its expression have been linked to human inflammatory diseases and increased risk for epithelial cancers. The underlying mechanisms are unknown, and the link is perplexing given that NF-κB signaling reportedly typically exerts pro-tumorigenic activity. Here we have shown that NF-κB1 deficiency, even loss of a single allele, resulted in spontaneous invasive gastric cancer (GC) in mice that mirrored the histopathological progression of human intestinal-type gastric adenocarcinoma. Bone marrow chimeras revealed that NF-κB1 exerted tumor suppressive functions in both epithelial and hematopoietic cells. RNA-seq analysis showed that NF-κB1 deficiency resulted in aberrant JAK-STAT signaling, which dysregulated expression of effectors of inflammation, antigen presentation, and immune checkpoints. Concomitant loss of STAT1 prevented these immune abnormalities and GC development. These findings provide mechanistic insight into how polymorphisms that attenuate NFKB1 expression predispose humans to epithelial cancers, highlighting the pro-tumorigenic activity of STAT1 and identifying targetable vulnerabilities in GC.
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http://dx.doi.org/10.1016/j.immuni.2018.03.003DOI Listing
March 2018

An evaluation of the challenges to developing tumor BRCA1 and BRCA2 testing methodologies for clinical practice.

Hum Mutat 2018 03 28;39(3):394-405. Epub 2017 Dec 28.

Translational Science, Oncology, IMED Biotech Unit, AstraZeneca, Waltham, Massachusetts.

Ovarian cancer patients with germline or somatic pathogenic variants benefit from treatment with poly ADP ribose polymerase (PARP) inhibitors. Tumor BRCA1/2 testing is more challenging than germline testing as the majority of samples are formalin-fixed paraffin embedded (FFPE), the tumor genome is complex, and the allelic fraction of somatic variants can be low. We collaborated with 10 laboratories testing BRCA1/2 in tumors to compare different approaches to identify clinically important variants within FFPE tumor DNA samples. This was not a proficiency study but an inter-laboratory comparison to identify common issues. Each laboratory received the same tumor DNA samples ranging in genotype, quantity, quality, and variant allele frequency (VAF). Each laboratory performed their preferred next-generation sequencing method to report on the variants. No false positive results were reported in this small study and the majority of methods detected the low VAF variants. A number of variants were not detected due to the bioinformatics analysis, variant classification, or insufficient DNA. The use of hybridization capture or short amplicon methods are recommended based on a bioinformatic assessment of the data. The study highlights the importance of establishing standards and standardization for tBRCA testing particularly when the test results dictate clinical decisions regarding life extending therapies.
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http://dx.doi.org/10.1002/humu.23375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5838520PMC
March 2018

Intratumorous heterogeneity for RAS mutations in a treatment-naïve colorectal tumour.

J Clin Pathol 2017 Aug 17;70(8):720-723. Epub 2017 Mar 17.

Department of Pathology, University of Melbourne, Melbourne, Victoria, Australia.

Activating mutations in and genes in patients with colorectal cancer (CRC) are associated with a lack of response to treatment with anti-epidermal growth factor receptor (EGFR) therapies. Mutations in these genes are thought to be mutually exclusive, however reports have described CRCs with two activating rat sarcoma (RAS) mutations. This has fuelled discussion about whether these mutations are the result of intratumorous heterogeneity, or if they are co-occurring in the same cancer cell clone. We present a case of a colorectal tumour with three RAS mutations detected during routine diagnostic testing. Further detailed analysis with laser capture microdissection and next generation sequencing excluded the possibility of all three mutations being present in the same clone, presenting the highest resolution evidence of intratumorous heterogeneity of RAS mutations to date.
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http://dx.doi.org/10.1136/jclinpath-2017-204327DOI Listing
August 2017

Examining the impact of regular aspirin use and PIK3CA mutations on survival in stage 2 colon cancer.

Intern Med J 2017 Jan;47(1):88-98

Department of Medical Oncology, Western Health, Melbourne, Victoria, Australia.

Background/aim: Data suggest aspirin improves survival in colorectal cancer (CRC) harbouring PIK3CA mutations. The impact of aspirin is thought predominantly to be through an anti-inflammatory effect. The aim of this study is to explore the effect of aspirin use on survival in a real-world cohort of stage 2 colon cancer (CC) patients.

Methods: A prospective CRC database identified patients diagnosed with stage 2 CC between 2000 and 2011. PIK3CA mutation status was determined by next generation sequencing. Neutrophil-lymphocyte ratio greater than 5 at diagnosis represented systemic inflammation. Chart review was used to record regular aspirin use at diagnosis. Clinico-pathological features and survival data were available. Survival analyses used the Cox proportional hazards method.

Results: Of 488 patients with stage 2 CC, 95 patients were aspirin users and 70 patients had PIK3CA mutations. Aspirin users were more likely to be older (median: 76.4 years vs 68.3 years, P < 0.001), to be less fit (American Society of Anaesthetists Score 3-4: 58% vs 31%, P < 0.001) and to have systemic inflammation (neutrophil-lymphocyte ratio > 5: 39% vs 27%, P = 0.027). Regular aspirin use did not significantly improve recurrence-free survival. In the PIK3CA mutated group, there was a trend towards improved recurrence-free survival (hazard ratio: 0.45, P = 0.42).

Conclusions: Our study did not demonstrate a significant survival advantage from aspirin use in stage 2 PIK3CA mutated CC. The 'real-world' nature of our cohort and the subsequent uncontrolled differences in age and fitness in aspirin users are likely to have contributed to this result. Defining the true impact of aspirin in CRC requires prospective randomised clinical trials.
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http://dx.doi.org/10.1111/imj.13312DOI Listing
January 2017

Immunomodulation by MYB is associated with tumor relapse in patients with early stage colorectal cancer.

Oncoimmunology 2016 Jul 29;5(7):e1149667. Epub 2016 Jun 29.

Peter MacCallum Cancer Center and The Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne ,Victoria, Australia; The Department of Pathology, University of Melbourne, Melbourne Victoria, Australia.

The presence of tumor immune infiltrating cells (TILs), particularly CD8(+) T-cells, is a robust predictor of outcome in patients with colorectal cancer (CRC). We revisited TIL abundance specifically in patients with microsatellite stable (MSS) CRC without evidence of lymph node or metastatic spread. Examination of the density of CD8(+) T-cells in primary tumors in the context of other pro-oncogenic markers was performed to investigate potential regulators of TILs. Two independent cohorts of patients with MSS T2-4N0M0 CRC, enriched for cases with atypical relapse, were investigated. We quantified CD8(+) and CD45RO(+) -TILs, inflammatory markers, NFkBp65, pStat3, Cyclo-oxygenase-2 (COX2) and GRP78 as well as transcription factors (TF), β-catenin and MYB. High CD8(+) TILs correlated with a better relapse-free survival in both cohorts (p = 0.002) with MYB and its target gene, GRP78 being higher in the relapse group (p = 0.001); no difference in pSTAT3 and p65 was observed. A mouse CRC (CT26) model was employed to evaluate the effect of MYB on GRP78 expression as well as T-cell infiltration. MYB over-expressing in CT26 cells increased GRP78 expression and the analysis of tumor-draining lymph nodes adjacent to tumors showed reduced T-cell activation. Furthermore, MYB over-expression reduced the efficacy of anti-PD-1 to modulate CT26 tumor growth. This high MYB and GRP78 show a reciprocal relationship with CD8(+) TILs which may be useful refining the prediction of patient outcome. These data reveal a new immunomodulatory function for MYB suggesting a basis for further development of anti-GRP78 and/or anti-MYB therapies.
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http://dx.doi.org/10.1080/2162402X.2016.1149667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5006930PMC
July 2016

Clustered somatic mutations are frequent in transcription factor binding motifs within proximal promoter regions in melanoma and other cutaneous malignancies.

Oncotarget 2016 Oct;7(41):66569-66585

Research Division, Peter MacCallum Cancer Centre, Victorian Comprehensive Cancer Centre, Victoria, Australia.

Most cancer DNA sequencing studies have prioritized recurrent non-synonymous coding mutations in order to identify novel cancer-related mutations. Although attention is increasingly being paid to mutations in non-coding regions, standard approaches to identifying significant mutations may not be appropriate and there has been limited analysis of mutational clusters in functionally annotated non-coding regions. We sought to identify clustered somatic mutations (hotspot regions across samples) in functionally annotated regions in melanoma and other cutaneous malignancies (cutaneous squamous cell carcinoma, basal cell carcinoma and Merkel cell carcinoma). Sliding window analyses revealed numerous recurrent clustered hotspot mutations in proximal promoters, with some specific clusters present in up to 25% of cases. Mutations in melanoma were clustered within ETS and Sp1 transcription factor binding motifs, had a UV signature and were identified in other cutaneous malignancies. Clinicopathologic correlation and mutation analysis support a causal role for chronic UV irradiation generating somatic mutations in transcription factor binding motifs of proximal promoters.
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http://dx.doi.org/10.18632/oncotarget.11892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341821PMC
October 2016

ICECREAM: randomised phase II study of cetuximab alone or in combination with irinotecan in patients with metastatic colorectal cancer with either KRAS, NRAS, BRAF and PI3KCA wild type, or G13D mutated tumours.

BMC Cancer 2016 05 31;16:339. Epub 2016 May 31.

Cabrini Hospital, Melbourne, Australia.

Background: Patients with metastatic colorectal cancer whose disease has progressed on oxaliplatin- and irinotecan-containing regimens may benefit from EGFR-inhibiting monoclonal antibodies if they do not contain mutations in the KRAS gene (are "wild type"). It is unknown whether these antibodies, such as cetuximab, are more efficacious in refractory metastatic colorectal cancer as monotherapy, or in combination with irinotecan. Lack of mutation in KRAS, BRAF and PIK3CA predicts response to EFGR-inhibitors. The ICECREAM trial examines the question of monotherapy versus combination with chemotherapy in two groups of patients: those with a "quadruple wild type" tumour genotype (no mutations in KRAS, NRAS, PI3KCA or BRAF genes) and those with the specific KRAS mutation in codon G13D, for whom possibly EGFR-inhibitor efficacy may be equivalent.

Methods And Design: ICECREAM is a randomised, phase II, open-label, controlled trial comparing the efficacy of cetuximab alone or with irinotecan in patients with "quadruple wild type" or G13D-mutated metastatic colorectal cancer, whose disease has progressed on, or who are intolerant of oxaliplatin- and fluoropyrimidine-based chemotherapy. The primary endpoint is the 6-month progression-free survival benefit of the treatment regimen. Secondary endpoints are response rate, overall survival, and quality of life. The tertiary endpoint is prediction of outcome with further biological markers. International collaboration has facilitated recruitment in this prospective trial of treatment in these infrequently found molecular subsets of colorectal cancer.

Discussion: This unique trial will yield prospective information on the efficacy of cetuximab and whether this is further enhanced with chemotherapy in two distinct populations of patients with metastatic colorectal cancer: the "quadruple wild type", which may 'superselect' for tumours sensitive to EGFR-inhibition, and the rare KRAS G13D mutated tumours, which are also postulated to be sensitive to the drug. The focus on establishing both positive and negative predictive factors for the response to targeted therapy is an attempt to improve outcomes, reduce toxicity and contain treatment costs. Tissue and blood will yield a resource for molecular studies. Recruitment, particularly of patients with the rare G13D mutation, will demonstrate the ability for international collaboration to run prospective trials in small colorectal cancer molecular subgroups.

Trial Registration: Australian and New Zealand Clinical Trials Registry: ACTRN12612000901808 , registered 16 August 2012.
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http://dx.doi.org/10.1186/s12885-016-2389-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4886432PMC
May 2016

Response to Cetuximab With or Without Irinotecan in Patients With Refractory Metastatic Colorectal Cancer Harboring the KRAS G13D Mutation: Australasian Gastro-Intestinal Trials Group ICECREAM Study.

J Clin Oncol 2016 07 25;34(19):2258-64. Epub 2016 Apr 25.

Eva Segelov, St Vincent's Clinical School, University of New South Wales; Subotheni Thavaneswaran, Kristy P. Robledo, Val J. Gebski, Mustafa Khasraw, Kate Wilson, and John Simes, National Health and Medical Research Council Clinical Trials Centre (NHMRC), University of Sydney; Lorraine A. Chantrill, Macarthur Cancer Therapy Centre, Campbelltown Hospital and Kinghorn Cancer Centre; Nick Pavlakis and Mustafa Khasraw, Royal North Shore Hospital University of Sydney, Sydney; Paul M. Waring and Sebastian Lunke, Centre for Translational Pathology, University of Melbourne; Jayesh Desai, Royal Melbourne Hospital; Jayesh Desai and Michael Jefford, Peter MacCallum Cancer Centre; Andrew Haydon, Alfred Hospital; Jeremy D. Shapiro, Cabrini Hospital, Melbourne; Louise M. Nott, Royal Hobart Hospital, Hobart; Christos S. Karapetis, Flinders University and Flinders Medical Centre; Timothy J. Price, Queen Elizabeth Hospital and Lyell McEwin Hospital, Adelaide; Craig Underhill, Border Medical Oncology, Albury-Wodonga; Guy van Hazel, Sir Charles Gairdner Hospital, Perth, Australia; Elena Elez, Vall d'Hebron University Hospital, Barcelona, Spain; Fortunato Ciardiello, Seconda Università degli Studi di Napoli, Naples, Italy; and Harpreet Wasan, Hammersmith Hospital, London, United Kingdom.

Purpose: RAS mutations predict lack of response to epidermal growth factor receptor monoclonal antibody therapy in patients with metastatic colorectal cancer (mCRC), but preclinical studies and retrospective clinical data suggest that patients with tumors harboring the exon 2 KRAS G13D mutation may benefit from cetuximab. We aimed to assess cetuximab monotherapy and cetuximab plus irinotecan in patients with molecularly selected (G13D mutation) chemotherapy-refractory mCRC in a randomized phase II trial of this rare molecular subtype.

Patients And Methods: Patients with chemotherapy-refractory KRAS G13D mutation-positive mCRC who had progressed within 6 months of irinotecan therapy were randomly assigned to cetuximab 400 mg/m(2) loading dose and then 250 mg/m(2) once per week with or without irinotecan 180 mg/m(2) once every 2 weeks. The primary end point was 6-month progression-free survival; secondary end points were response rate, overall survival, quality of life, and toxicity.

Results: Fifty-one of 53 patients recruited over 2 years were eligible. The 6-month progression-free survival rate was 10% (95% CI, 2% to 26%) for cetuximab versus 23% (95% CI, 9% to 40%) for cetuximab plus irinotecan with a hazard ratio of 0.74 (95% CI, 0.42 to 1.32). Response and stable disease rates were 0% and 58% for monotherapy versus 9% and 70% for combination treatment, respectively. Overall survival and quality of life were similar; toxicities were higher with combination therapy.

Conclusion: In patients with G13D-mutated chemotherapy-refractory mCRC, there was no statistically significant improvement in disease control at 6 months with either cetuximab monotherapy or cetuximab plus irinotecan. No responses were seen with single-agent cetuximab. The responses observed with the combination of cetuximab and irinotecan may reflect true drug synergy or persistent irinotecan sensitivity. The ICECREAM (Irinotecan Cetuximab Evaluation and Cetuximab Response Evaluation Among Patients with a G13D Mutation) study demonstrates the need to prospectively evaluate hypotheses that were previously supported by retrospective analyses and exemplifies the value of international collaboration in trials of rare molecular subtypes.
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http://dx.doi.org/10.1200/JCO.2015.65.6843DOI Listing
July 2016

Girdin (GIV) Expression as a Prognostic Marker of Recurrence in Mismatch Repair-Proficient Stage II Colon Cancer.

Clin Cancer Res 2016 07 30;22(14):3488-98. Epub 2016 Mar 30.

Walter and Eliza Hall Institute, Melbourne, Australia. The Royal Melbourne Hospital, Melbourne, Australia. Western Hospital, Melbourne, Australia.

Purpose: Prognostic markers that identify patients with stage II colon cancers who are at the risk of recurrence are essential to personalize therapy. We evaluated the potential of GIV/Girdin as a predictor of recurrence risk in such patients.

Experimental Design: Expression of full-length GIV was evaluated by IHC using a newly developed mAb together with a mismatch repair (MMR)-specific antibody panel in three stage II colon cancer patient cohorts, that is, a training (n = 192), test (n = 317), and validation (n = 181) cohort, with clinical follow-up data. Recurrence risk stratification models were established in the training cohort of T3, proficient MMR (pMMR) patients without chemotherapy and subsequently validated.

Results: For T3 pMMR tumors, GIV expression and the presence of lymphovascular invasion (LVI) were the only factors predicting recurrence in both training (GIV: HR, 2.78, P = 0.013; LVI: HR, 2.54, P = 0.025) and combined test and validation (pooled) cohorts (GIV: HR, 1.85, P = 0.019; LVI: HR, 2.52, P = 0.0004). A risk model based on GIV expression and LVI status classified patients into high- or low-risk groups; 3-year recurrence-free survival was significantly lower in the high-risk versus low-risk group across all cohorts [Training: 52.3% vs. 84.8%; HR, 3.74, 95% confidence interval (CI), 1.50-9.32; Test: 85.9% vs. 97.9%, HR, 7.83, 95% CI, 1.03-59.54; validation: 59.4% vs. 84.4%, HR, 3.71, 95% CI, 1.24-11.12].

Conclusions: GIV expression status predicts recurrence risk in patients with T3 pMMR stage II colon cancer. A risk model combining GIV expression and LVI status information further enhances prediction of recurrence. Further validation studies are warranted before GIV status can be routinely included in patient management algorithms. Clin Cancer Res; 22(14); 3488-98. ©2016 AACR.
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http://dx.doi.org/10.1158/1078-0432.CCR-15-2290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4947424PMC
July 2016

RAS Mutations as Predictive Biomarkers in Clinical Management of Metastatic Colorectal Cancer.

Clin Colorectal Cancer 2016 06 11;15(2):95-103. Epub 2015 Nov 11.

Flinders University, Flinders Centre for Innovation in Cancer, Flinders Medical Centre, Adelaide, South Australia, Australia. Electronic address:

The use of anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapies in patients with metastatic colorectal cancer is guided by the presence of activating point mutations in codons 12, 13, 59, 61, 117, and 146 of the KRAS and NRAS genes in the primary tumor. Although these mutations have been incorporated into the prescribing information for both cetuximab and panitumumab, highlighted in the National Comprehensive Cancer Network Guidelines, and routinely tested, a number of controversial issues and unanswered questions related to these mutations and their clinical significance remain. In the present review, we explored the contradictory data related to the prognostic value of KRAS mutations, the reported frequent discordance of KRAS mutations, and the reported nonequivalence of some of these mutations. We also considered the issues related to incorporating additional mutations into the already accredited and approved assays and the challenges created by changing an assay's analytical and clinical limits of detection. We also discuss the lack of biologic data supporting the pathogenicity of newly described clinically actionable mutations and explore the uncertainty regarding the clinical significance of low-frequency mutations, highlighting the importance of correcting allele frequencies for tumor purity. We also considered the importance of distinguishing the significance of low-frequency RAS mutations in tumors previously not treated or treated with anti-EGFR therapies and explore new technologies capable of detecting emerging polyclonal RAS mutations that appear to confer drug resistance.
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http://dx.doi.org/10.1016/j.clcc.2015.10.006DOI Listing
June 2016

Stomach-Specific Activation of Oncogenic KRAS and STAT3-Dependent Inflammation Cooperatively Promote Gastric Tumorigenesis in a Preclinical Model.

Cancer Res 2016 04 2;76(8):2277-87. Epub 2016 Feb 2.

Cancer and Inflammation, Olivia Newton-John Cancer Research Institute and School of Cancer Medicine La Trobe University, Heidelberg, Australia.

About 5% to 10% of human gastric tumors harbor oncogenic mutations in the KRAS pathway, but their presence alone is often insufficient for inducing gastric tumorigenesis, suggesting a requirement for additional mutagenic events or microenvironmental stimuli, including inflammation. Assessing the contribution of such events in preclinical mouse models requires Cre recombinase-mediated conditional gene expression in stem or progenitor cells of normal and transformed gastric epithelium. We therefore constructed a bacterial artificial chromosome containing transgene (Tg), comprising the regulatory elements of the trefoil factor 1 (Tff1) gene and the tamoxifen-inducible Cre recombinase (CreERT2)-coding sequence. The resulting Tg(Tff1-CreERT2) mice were crossed with mice harboring conditional oncogenic mutations in Kras or Braf The administration of tamoxifen to the resulting adult Tg(Tff1-CreERT2);Kras(LSL-G12D/+) and Tg(Tff1-CreERT2);Braf(LSL-V600E/+) mice resulted in gastric metaplasia, inflammation, and adenoma development, characterized by excessive STAT3 activity. To assess the contribution of STAT3 to the spontaneously developing gastric adenomas in gp130(F/F) mice, which carry a knockin mutation in the Il6 signal transducer (Il6st), we generated Tg(Tff1-CreERT2);Stat3(fl/fl);gp130(F/F) mice that also harbor a conditional Stat3 knockout allele and found that tamoxifen administration conferred a significant reduction in their tumor burden. Conversely, excessive Kras activity in Tg(Tff1-CreERT2);Kras(LSL-G12D/+);gp130(F/F) mice promoted more extensive gastric inflammation, metaplastic transformation, and tumorigenesis than observed in Tg(Tff1-CreERT2);Kras(LSL-G12D/+) mice. Collectively, our findings demonstrate that advanced gastric tumorigenesis requires oncogenic KRAS or BRAF in concert with aberrant STAT3 activation in epithelial precursor cells of the glandular stomach, providing a new conditional model of gastric cancer in which to investigate candidate therapeutic targets and treatment strategies. Cancer Res; 76(8); 2277-87. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-3089DOI Listing
April 2016

High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq.

PLoS One 2015 16;10(11):e0143006. Epub 2015 Nov 16.

Department of Pathology, University of Melbourne, Melbourne, VIC, Australia.

Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143006PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646639PMC
June 2016

Incorporating traditional and emerging biomarkers in the clinical management of metastatic colorectal cancer.

Expert Rev Mol Diagn 2015 12;15(8):1033-48. Epub 2015 Jul 12.

Flinders Medical Centre, Sturt Road, Bedford Park, South Australia 5042, Australia.

The role of biomarker assessment in determining the best therapeutic options for patients with metastatic colorectal cancer has become increasingly complex and important. Biomarkers that predict the efficacy and/or toxicity of such treatments can affect medical decision making, leading to decreased harm and/or costs associated with treatment and improvements in therapeutic outcomes for patients. This review discusses traditional and emerging biomarkers of potential clinical utility for patients with metastatic colorectal cancer, current assays and methods used in clinical practice, technologies that have allowed the identification of new biomarkers and key considerations for oncologists and pathologists when determining appropriate biomarker evaluations to be undertaken for their patients.
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http://dx.doi.org/10.1586/14737159.2015.1052797DOI Listing
April 2016

Whole-genome characterization of chemoresistant ovarian cancer.

Nature 2015 May;521(7553):489-94

Victorian Institute of Forensic Medicine, Southbank, Victoria 3006, Australia.

Patients with high-grade serous ovarian cancer (HGSC) have experienced little improvement in overall survival, and standard treatment has not advanced beyond platinum-based combination chemotherapy, during the past 30 years. To understand the drivers of clinical phenotypes better, here we use whole-genome sequencing of tumour and germline DNA samples from 92 patients with primary refractory, resistant, sensitive and matched acquired resistant disease. We show that gene breakage commonly inactivates the tumour suppressors RB1, NF1, RAD51B and PTEN in HGSC, and contributes to acquired chemotherapy resistance. CCNE1 amplification was common in primary resistant and refractory disease. We observed several molecular events associated with acquired resistance, including multiple independent reversions of germline BRCA1 or BRCA2 mutations in individual patients, loss of BRCA1 promoter methylation, an alteration in molecular subtype, and recurrent promoter fusion associated with overexpression of the drug efflux pump MDR1.
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http://dx.doi.org/10.1038/nature14410DOI Listing
May 2015

Temporal patterns in Saturnidae (silk moth) and Sphingidae (hawk moth) assemblages in protected forests of central Uganda.

Ecol Evol 2015 Apr 25;5(8):1746-57. Epub 2015 Mar 25.

Department of Biology, University of Bergen Bergen, Norway.

Forest-dependent biodiversity is threatened throughout the tropics by habitat loss and land-use intensification of the matrix habitats. We resampled historic data on two moth families, known to play central roles in many ecosystem processes, to evaluate temporal changes in species richness and community structure in three protected forests in central Uganda in a rapidly changing matrix. Our results show some significant declines in the moth species richness and the relative abundance and richness of forest-dependent species over the last 20-40 years. The observed changes in species richness and composition among different forests, ecological types, and moth groups highlight the need to repeatedly monitor biodiversity even within protected and relatively intact forests.
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http://dx.doi.org/10.1002/ece3.1477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4409421PMC
April 2015

Equivocal ALK fluorescence in-situ hybridization (FISH) cases may benefit from ancillary ALK FISH probe testing.

Histopathology 2015 Nov 24;67(5):654-63. Epub 2015 May 24.

Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.

Aims: Accurate assessment of anaplastic lymphoma kinase (ALK) gene rearrangement in non-small-cell lung cancers (NSCLCs) is critical to identify patients who are likely to respond to crizotinib. The aim of this study was to evaluate the ALK/EML4 TriCheck FISH probe in a series of NSCLCs enriched for tumours with equivocal ALK status.

Methods And Results: ALK FISH was prospectively performed on 45 NSCLCs with the ALK/EML4 TriCheck probe (ZytoVision) and the Vysis ALK break-apart probe (Abbott Molecular). ALK immunohistochemistry was performed with 5A4 and D5F3 antibodies. Fourteen cases had equivocal ALK status, based on borderline or focal FISH positivity, an atypical FISH pattern, or discrepancy between ALK FISH and immunohistochemistry. Four of the 14 equivocal cases showed discordance between the two FISH probes. All other cases were concordant. The TriCheck probe showed that, of 31 unequivocal cases, 15 were ALK-rearranged, and 60% of these had EML4 as the translocation partner. Within the group of 14 equivocal cases, 12 showed rearrangement with the Tricheck probe; only one of these showed EML4 rearrangement. Of the six equivocal cases that received crizotinib, four showed clinical benefit.

Conclusions: The ALK/EML4 TriCheck FISH probe may be useful for the detection of ALK rearrangements, especially in borderline or atypical cases, where an additional unique ALK FISH probe may provide further confirmation of rearrangement.
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http://dx.doi.org/10.1111/his.12708DOI Listing
November 2015

Applicability of next generation sequencing technology in microsatellite instability testing.

Genes (Basel) 2015 Feb 12;6(1):46-59. Epub 2015 Feb 12.

Department of Pathology, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Victoria 3052, Australia.

Microsatellite instability (MSI) is a useful marker for risk assessment, prediction of chemotherapy responsiveness and prognosis in patients with colorectal cancer. Here, we describe a next generation sequencing approach for MSI testing using the MiSeq platform. Different from other MSI capturing strategies that are based on targeted gene capture, we utilize "deep resequencing", where we focus the sequencing on only the microsatellite regions of interest. We sequenced a series of 44 colorectal tumours with normal controls for five MSI loci (BAT25, BAT26, BAT34c4, D18S55, D5S346) and a second series of six colorectal tumours (no control) with two mononucleotide loci (BAT25, BAT26). In the first series, we were able to determine 17 MSI-High, 1 MSI-Low and 26 microsatellite stable (MSS) tumours. In the second series, there were three MSI-High and three MSS tumours. Although there was some variation within individual markers, this NGS method produced the same overall MSI status for each tumour, as obtained with the traditional multiplex PCR-based method.
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http://dx.doi.org/10.3390/genes6010046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4377833PMC
February 2015
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