Graham R. Taylor

KRAS and BRAF Mutations in Advanced Colorectal Cancer Are Associated With Poor Prognosis but Do Not Preclude Benefit From Oxaliplatin or Irinotecan: Results From the MRC FOCUS Trial

PURPOSE Activating mutation of the KRAS oncogene is an established predictive biomarker for resistance to anti-epidermal growth factor receptor (anti-EGFR) therapies in advanced colorectal cancer (aCRC). We wanted to determine whether KRAS and/or BRAF mutation is also a predictive biomarker for other aCRC therapies. PATIENTS AND METHODS The Medical Research Council Fluorouracil, Oxaliplatin and Irinotecan: Use and Sequencing (MRC FOCUS) trial compared treatment sequences including first-line fluorouracil (FU), FU/irinotecan or FU/oxaliplatin in aCRC. Tumor blocks were obtained from 711 consenting patients. DNA was extracted and KRAS codons 12, 13, and 61 and BRAF codon 600 were assessed by pyrosequencing. Mutation (mut) status was assessed first as a prognostic factor and then as a predictive biomarker for the benefit of adding irinotecan or oxaliplatin to FU. The association of BRAF-mut with loss of MLH1 was assessed by immunohistochemistry. RESULTS Three hundred eight (43.3%) of 711 patients had KRAS-mut and 56 (7.9%) of 711 had BRAF-mut. Mutation of KRAS, BRAF, or both was present in 360 (50.6%) of 711 patients. Mutation in either KRAS or BRAF was a poor prognostic factor for overall survival (OS; hazard ratio [HR], 1.40; 95% CI, 1.20 to 1.65; P < .0001) but had minimal impact on progression-free survival (PFS; HR, 1.16; 95% CI, 1.00 to 1.36; P = .05). Mutation status did not affect the impact of irinotecan or oxaliplatin on PFS or OS. BRAF-mut was weakly associated with loss of MLH1 staining (P = .012). CONCLUSION KRAS/BRAF mutation is associated with poor prognosis but is not a predictive biomarker for irinotecan or oxaliplatin. There is no evidence that patients with KRAS/BRAF mutated tumors are less likely to benefit from these standard chemotherapy agents.

Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction.

Several DNA extraction techniques were quantitatively and qualitatively compared using both fresh and paraffin wax embedded tissue and their suitability investigated for providing DNA and RNA for the polymerase chain reaction (PCR). A one hour incubation with proteinase K was the most efficient DNA extraction procedure for fresh tissue. For paraffin wax embedded tissue a five day incubation with proteinase K was required to produce good yields of DNA. Incubation with sodium dodecyl sulphate produced very poor yields, while boiling produced 20% as much DNA as long enzyme digestion. DNA extracted by these methods was suitable for the PCR amplification of a single copy gene. Proteinase K digestion also produced considerable amounts of RNA which has previously been shown to be suitable for PCR analysis. A delay before fixation had no effect on the amount of DNA obtained while fixation in Carnoys reagent results in a much better preservation of DNA than formalin fixation, allowing greater yields to be extracted.

Meta-Analysis on the Use of Zidovudine and Interferon-Alfa in Adult T-Cell Leukemia/Lymphoma Showing Improved Survival in the Leukemic Subtypes

PURPOSE Human T-cell lymphotropic virus type-I-associated adult T-cell leukemia/lymphoma (ATL) is an aggressive, chemotherapy-resistant malignancy. Multiple small studies using zidovudine (AZT) and interferon-alfa (IFN-α) have shown response in patients with ATL. However, the impact of this innovative antiviral treatment strategy on long-term survival remains undetermined. PATIENTS AND METHODS We report a meta-analysis of antiviral therapy of ATL. Medical records of 254 patients with ATL who were treated in the United States, the United Kingdom, Martinique, and continental France were individually reviewed. RESULTS According to Shimoyama classification, there were 116 patients with acute ATL, 18 patients with chronic ATL, 11 patients with smoldering ATL, and 100 patients with ATL lymphoma. In 231 patients with available survival data, first-line therapy was recorded in 207 patients. Five-year overall survival rates were 46% for 75 patients who received first-line antiviral therapy (P = .004), 20% for 77 patients who received first-line chemotherapy, and 12% for 55 patients who received first-line chemotherapy followed by antiviral therapy. Patients with acute, chronic, and smoldering ATL significantly benefited from first-line antiviral therapy, whereas patients with ATL lymphoma experienced a better outcome with chemotherapy. In acute ATL, achievement of complete remission with antiviral therapy resulted in 82% 5-year survival. Antiviral therapy in chronic and smoldering ATL resulted in 100% 5-year survival. Multivariate analysis confirmed that first-line antiviral therapy significantly improves overall survival of patients with ATL (hazard ratio, 0.47; 95% CI, 0.27 to 0.83; P = .021). CONCLUSION These results confirm the high efficacy of AZT and IFN, which should now be considered the gold standard first-line therapy in leukemic subtypes of ATL.

Prognostic value of p53 overexpression and c-Ki-ras gene mutations in colorectal cancer

BACKGROUND Mutations in Ki-ras codon 12 and the p53 gene are common abnormalities in colorectal cancer. The occurrence of p53 overexpression and/or Ki-ras codon 12 mutations were analyzed in 100 colorectal adenomas to determine if they were related to patient survival. METHODS p53 overexpression was identified by immunohistochemistry, and Ki-ras codon 12 mutations were detected using the polymerase chain reaction and a restriction enzyme digestion method. RESULTS p53 overexpression was identified in 45% of tumors, with a higher frequency identified in DNA aneuploid and left-sided tumors than in DNA diploid and right-sided tumors. Mutations in Ki-ras codon 12 were identified in 24% of carcinomas. Individually, mutations in Ki-ras codon 12 or p53 overexpression were not prognostic indicators of survival. However, a statistically significant difference in survival was identified when these two oncogenic abnormalities were analyzed together. The median survival of patients whose tumors contained both oncogenic abnormalities was less than half of that of patients with either alteration alone or without either abnormality. CONCLUSIONS Screening for multiple genetic abnormalities in colorectal cancers excised at surgery may prove to be a useful tool in determining prognosis.

Rapid detection of allele loss in colorectal tumours using microsatellites and fluorescent DNA technology.

In order to investigate allele loss in colorectal tumours we have developed a rapid technique which overcomes most of the problems associated with radioactive Restriction Fragment Length Polymorphism (RFLP) analysis of allele loss. We utilise microsatellite length polymorphisms which are highly informative and are closely linked to loci of interest. Sequences containing microsatellites can be amplified from normal and tumour DNA pairs by a polymerase chain reaction (PCR) in which one of the primers is fluorescently labelled. This enables us to detect the products on polyacrylamide gels run on an automated DNA sequencer using dedicated software, by which results are automatically quantitated in terms of peak size, height, and area. Using this technique we have analysed 26 normal tissue: cancer pairs for allele loss at two loci linked to the adenomatous polyposis coli (APC) gene on chromosome 5q. Repeated assays yielded identical results for each pair. Allele loss was found in 10 out of 25 informative samples (40%).

A common founder for the 35delG GJB2 gene mutation in connexin 26 hearing impairment

Fifty to eighty percent of autosomal recessive congenital severe to profound hearing impairment result from mutations in a single gene, GJB2, that encodes the protein connexin 26. One mutation of this gene, the 35delG allele, is particularly common in white populations. We report evidence that the high frequency of this allelic variant is the result of a founder effect rather than a mutational hot spot inGJB2, which was the prevailing hypothesis. Patients homozygous for the 35delG mutation and normal hearing controls originating from Belgium, the UK, and the USA were genotyped for different single nucleotide polymorphisms (SNPs). Four SNPs mapped in the immediate vicinity of GJB2, while two were positioned up to 76 kb from it. Significant differences between the genotypes of patients and controls for the five SNPs closest toGJB2 were found, with nearly complete association of one SNP allele with the 35delG mutation. For the most remote SNP, we could not detect any association. We conclude that the 35delG mutation is derived from a common, albeit ancient founder.

Mutations Causing Familial Biparental Hydatidiform Mole Implicate C6orf221 as a Possible Regulator of Genomic Imprinting in the Human Oocyte

Familial biparental hydatidiform mole (FBHM) is the only known pure maternal-effect recessive inherited disorder in humans. Affected women, although developmentally normal themselves, suffer repeated pregnancy loss because of the development of the conceptus into a complete hydatidiform mole in which extraembryonic trophoblastic tissue develops but the embryo itself suffers early demise. This developmental phenotype results from a genome-wide failure to correctly specify or maintain a maternal epigenotype at imprinted loci. Most cases of FBHM result from mutations of NLRP7, but genetic heterogeneity has been demonstrated. Here, we report biallelic mutations of C6orf221 in three families with FBHM. The previously described biological properties of their respective gene families suggest that NLRP7 and C6orf221 may interact as components of an oocyte complex that is directly or indirectly required for determination of epigenetic status on the oocyte genome.

Mutation of the Variant α-Tubulin TUBA8 Results in Polymicrogyria with Optic Nerve Hypoplasia

The critical importance of cytoskeletal function for correct neuronal migration during development of the cerebral cortex has been underscored by the identities of germline mutations underlying a number of human neurodevelopmental disorders. The proteins affected include TUBA1A, a major alpha-tubulin isoform, and microtubule-associated components such as doublecortin, and LIS1. Mutations in these genes are associated with the anatomical abnormality lissencephaly, which is believed to reflect failure of neuronal migration. An important recent observation has been the dependence of cortical neuronal migration upon acetylation of alpha-tubulin at lysine 40 by the histone acetyltransferase Elongator complex. Here, we describe a recognizable autosomal recessive syndrome, characterized by generalized polymicrogyria in association with optic nerve hypoplasia (PMGOH). By autozygosity mapping, we show that the molecular basis for this condition is mutation of the TUBA8 gene, encoding a variant alpha-tubulin of unknown function that is not susceptible to the lysine 40 acetylation that regulates microtubule function during cortical neuron migration. Together with the unique expression pattern of TUBA8 within the developing cerebral cortex, these observations suggest a role for this atypical microtubule component in regulating mammalian brain development.

Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens

The use of next-generation sequencing technologies to produce genomic copy number data has recently been described. Most approaches, however, reply on optimal starting DNA, and are therefore unsuitable for the analysis of formalin-fixed paraffin-embedded (FFPE) samples, which largely precludes the analysis of many tumour series. We have sought to challenge the limits of this technique with regards to quality and quantity of starting material and the depth of sequencing required. We confirm that the technique can be used to interrogate DNA from cell lines, fresh frozen material and FFPE samples to assess copy number variation. We show that as little as 5 ng of DNA is needed to generate a copy number karyogram, and follow this up with data from a series of FFPE biopsies and surgical samples. We have used various levels of sample multiplexing to demonstrate the adjustable resolution of the methodology, depending on the number of samples and available resources. We also demonstrate reproducibility by use of replicate samples and comparison with microarray-based comparative genomic hybridization (aCGH) and digital PCR. This technique can be valuable in both the analysis of routine diagnostic samples and in examining large repositories of fixed archival material.

Mutations in NMNAT1 cause Leber congenital amaurosis and identify a new disease pathway for retinal degeneration

Leber congenital amaurosis (LCA) is a blinding retinal disease that presents within the first year after birth. Using exome sequencing, we identified mutations in the nicotinamide adenine dinucleotide (NAD) synthase gene NMNAT1 encoding nicotinamide mononucleotide adenylyltransferase 1 in eight families with LCA, including the family in which LCA was originally linked to the LCA9 locus. Notably, all individuals with NMNAT1 mutations also have macular colobomas, which are severe degenerative entities of the central retina (fovea) devoid of tissue and photoreceptors. Functional assays of the proteins encoded by the mutant alleles identified in our study showed that the mutations reduce the enzymatic activity of NMNAT1 in NAD biosynthesis and affect protein folding. Of note, recent characterization of the slow Wallerian degeneration (Wlds) mouse model, in which prolonged axonal survival after injury is observed, identified NMNAT1 as a neuroprotective protein when ectopically expressed. Our findings identify a new disease mechanism underlying LCA and provide the first link between endogenous NMNAT1 dysfunction and a human nervous system disorder.