Frequency of mutations and polymorphisms in borderline ovarian tumors of known cancer genes.

Borderline ovarian tumors represent an understudied subset of ovarian tumors. Most studies investigating aberrations in borderline tumors have focused on KRAS/BRAF mutations. In this study, we conducted an extensive analysis of mutations and single-nucleotide polymorphisms (SNPs) in borderline ovarian tumors. Using the Sequenom MassArray platform, we investigated 160 mutations/polymorphisms in 33 genes involved in cell signaling, apoptosis, angiogenesis, cell cycle regulation and cellular senescence. Of 52 tumors analyzed, 33 were serous, 18 mucinous and 1 endometrioid. KRAS c.35G>A p.Gly12Asp mutations were detected in eight tumors (six serous and two mucinous), BRAF V600E mutations in two serous tumors, and PIK3CA H1047Y and PIK3CA E542K mutations in a serous and an endometrioid BOT, respectively. CTNNB1 mutation was detected in a serous tumor. Potentially functional polymorphisms were found in vascular endothelial growth factor (VEGF), ABCB1, FGFR2 and PHLPP2. VEGF polymorphisms were the most common and detected at four loci. PHLPP2 polymorphisms were more frequent in mucinous as compared with serous tumors (P=0.04), with allelic imbalance in one case. This study represents the largest and most comprehensive analysis of mutations and functional SNPs in borderline ovarian tumors to date. At least 25% of borderline ovarian tumors harbor somatic mutations associated with potential response to targeted therapeutics.

Biallelic ATM inactivation significantly reduces survival in patients treated on the United Kingdom Leukemia Research Fund Chronic Lymphocytic Leukemia 4 trial.

PURPOSE: The prognostic significance of ATM mutations in chronic lymphocytic leukemia (CLL) is unclear. We assessed their impact in the context of a prospective randomized trial. PATIENTS AND METHODS: We analyzed the ATM gene in 224 patients treated on the Leukemia Research Fund Chronic Lymphocytic Leukemia 4 (LRF-CLL4) trial with chlorambucil or fludarabine with and without cyclophosphamide. ATM status was analyzed by denaturing high-performance liquid chromatography and was related to treatment response, survival, and the impact of TP53 alterations for the same patient cohort. RESULTS: We identified 36 ATM mutations in 33 tumors, 16 with and 17 without 11q deletion. Mutations were associated with advanced disease stage and involvement of multiple lymphoid sites. Patients with both ATM mutation and 11q deletion showed significantly reduced progression-free survival (median, 7.4 months) compared with those with ATM wild type (28.6 months), 11q deletion alone (17.1 months), or ATM mutation alone (30.8 months), but survival was similar to that in patients with monoallelic (6.7 months) or biallelic (3.4 months) TP53 alterations. This effect was independent of treatment, immunoglobulin heavy chain variable gene (IGHV) status, age, sex, or disease stage. Overall survival for patients with biallelic ATM alterations was also significantly reduced compared with those with ATM wild type or ATM mutation alone (median, 42.2 v 85.5 v 77.6 months, respectively). CONCLUSION: The combination of 11q deletion and ATM mutation in CLL is associated with significantly shorter progression-free and overall survival following first-line treatment with alkylating agents and purine analogs. Assessment of ATM mutation status in patients with 11q deletion may influence the choice of subsequent therapy.

A comparison of three methods for detecting KRAS mutations in formalin-fixed colorectal cancer specimens.

BACKGROUND: KRAS mutation testing is required to select patients with metastatic colorectal cancer (CRC) to receive anti-epidermal growth factor receptor antibodies, but the optimal KRAS mutation test method is uncertain. METHODS: We conducted a two-site comparison of two commercial KRAS mutation kits - the cobas KRAS Mutation Test and the Qiagen therascreen KRAS Kit - and Sanger sequencing. A panel of 120 CRC specimens was tested with all three methods. The agreement between the cobas test and each of the other methods was assessed. Specimens with discordant results were subjected to quantitative massively parallel pyrosequencing (MPP). DNA blends were tested to determine detection rates at 5% mutant alleles. RESULTS: Reproducibility of the cobas test between sites was 98%. Six mutations were detected by cobas that were not detected by Sanger, and five were confirmed by MPP. The cobas test detected eight mutations which were not detected by the therascreen test, and seven were confirmed by MPP. Detection rates with 5% mutant DNA blends were 100% for the cobas and therascreen tests and 19% for Sanger. CONCLUSION: The cobas test was reproducible between sites, and detected several mutations that were not detected by the therascreen test or Sanger. Sanger sequencing had poor sensitivity for low levels of mutation.

Tyrosine kinase inhibitor insensitivity of non-cycling CD34+ human acute myeloid leukaemia cells with FMS-like tyrosine kinase 3 mutations.

The efficacy of tyrosine kinase (TK) inhibitors on non-cycling acute myeloid leukaemia (AML) cells, previously shown to have potent tumourigenic potential, is unknown. This pilot study describes the first attempt to characterize non-cycling cells from a small series of human FMS-like tyrosine kinase 3 (FLT3) mutation positive samples. CD34+ AML cells from patients with FLT3 mutation positive AML were cultured on murine stroma. In expansion cultures, non-cycling cells were found to retain CD34+ expression in contrast to dividing cells. Leukaemic gene rearrangements could be detected in non-cycling cells, indicating their leukaemic origin. Significantly, the FLT3-internal tandem duplication (ITD) mutation was found in the non-cycling fraction of four out of five cases. Exposure to the FLT3-directed inhibitor TKI258 clearly inhibited the growth of AML CD34+ cells in short-term cultures and colony-forming unit assays. Crucially, non-cycling cells were not eradicated, with the exception of one case, which exhibited exquisite sensitivity to the compound. Moreover, in longer-term cultures, TKI258-treated non-cycling cells showed no growth impairment compared to treatment-naive non-cycling cells. These findings suggest that non-cycling cells in AML may constitute a disease reservoir that is resistant to TK inhibition. Further studies with a larger sample size and other inhibitors are warranted.

Characterising the TP53-deleted subgroup of chronic lymphocytic leukemia: an analysis of additional cytogenetic abnormalities detected by interphase fluorescence in situ hybridisation and array-based comparative genomic hybridisation.

Deletion of the TP53 gene on chromosome 17p13.1 is the prognostic factor associated with the shortest survival in CLL. We used array-based comparative genomic hybridisation (arrayCGH) to identify additional DNA copy number changes in peripheral blood samples from 74 LRF CLL4 trial patients, 37 with >or=5% and 37 without TP53-deleted cells. ArrayCGH reliably detected deletions on 17p, including the TP53 locus, in cases with >or=50%TP53-deleted cells detected by fluorescence in situ hybridisation, plus seven additional cases with deleted regions on 17p excluding TP53. Losses on chromosomal regions 18p and/or 20p were found exclusively in cases with >or=5%TP53-deleted cells (por=5%TP53-deleted cases (p=0.02). In particular, amplification of 2p and deletion of 6q were both more frequent. Cases with >20%TP53-deleted cells had the worst prognosis in the LRF CLL4 trial.

Mutations in CTC1, Encoding Conserved Telomere Maintenance Component 1, Cause Coats Plus

Coats plus is a highly pleiotropic disorder particularly affecting the eye, brain, bone and gastrointestinal tract. Here, we show that Coats plus results from mutations in CTC1, encoding conserved telomere maintenance component 1, a member of the mammalian homolog of the yeast heterotrimeric CST telomeric capping complex. Consistent with the observation of shortened telomeres in an Arabidopsis CTC1 mutant and the phenotypic overlap of Coats plus with the telomeric maintenance disorders comprising dyskeratosis congenita, we observed shortened telomeres in three individuals with Coats plus and an increase in spontaneous γH2AX-positive cells in cell lines derived from two affected individuals. CTC1 is also a subunit of the α-accessory factor (AAF) complex, stimulating the activity of DNA polymerase-α primase, the only enzyme known to initiate DNA replication in eukaryotic cells. Thus, CTC1 may have a function in DNA metabolism that is necessary for but not specific to telomeric integrity.

NOD2 mutations and colorectal cancer - Where do we stand?

Due to the overwhelming burden of colorectal cancer (CRC), great effort has been placed on identifying genetic mutations that contribute to disease development and progression. One of the most studied polymorphisms that could potentially increase susceptibility to CRC involves the nucleotide-binding and oligomerization-domain containing 2 (NOD2) gene. There is growing evidence that the biological activity of NOD2 is far greater than previously thought and a link with intestinal microbiota and mucosal immunity is increasingly sought after. In fact, microbial composition may be an important contributor not only to inflammatory bowel diseases (IBD) but also to CRC. Recent studies have showed that deficient NOD2 function confers a communicable risk of colitis and CRC. Despite the evidence from experimental models, population-based studies that tried to link certain NOD2 polymorphisms and an increase in CRC risk have been described as conflicting. Significant geographic discrepancies in the frequency of such polymorphisms and different interpretations of the results may have limited the conclusions of those studies. Since being first associated to IBD and CRC, our understanding of the role of this gene has come a long way, and it is tempting to postulate that it may contribute to identify individuals with susceptible genetic background that may benefit from early CRC screening programs or in predicting response to current therapeutic tools. The aim of this review is to clarify the status quo of NOD2 mutations as genetic risk factors to chronic inflammation and ultimately to CRC. The use of NOD2 as a predictor of certain phenotypic characteristics of the disease will be analyzed as well.

Ocular manifestations of microcephaly with or without chorioretinopathy, lymphedema or intellectual disability (MCLID) syndrome associated with mutations in KIF11

Purpose Microcephaly with or without chorioretinopathy, lymphedema or intellectual disability (MCLID) is an autosomal dominant condition. Mutations in KIF11 have been found to be causative in approximately 75% of cases. This study describes the ocular phenotype in patients with confirmed KIF11 mutations. Methods Standard ophthalmic examination and investigation including visual acuity, refraction and fundus examination was carried out in all patients. Fundus autofluorescence imaging (FAF) was performed in three patients, and four patients underwent spectral domain optical coherence tomography (OCT). Flash electroretinography (ERG) was performed in seven patients, and five underwent additional pattern electroretinography (PERG). Results The patients ranged in age from 2 to 10 years. Most presented with visual acuity loss. Fundus examination revealed lacunae of chorioretinal atrophy. Pigmentary macular changes and optic disc pallor were present in three of seven patients. Fundus autofluorescence demonstrated hypoautofluorescence at the macula in two of three patients. The lacunae of chorioretinal atrophy were hypoautofluorescent. The OCT showed atrophic maculae in three of four patients. Follow-up in one patient showed no deterioration of the vision over a 9-year period. The lesions appear not to be progressive on the follow-up imaging. Electrophysiology showed generalized rod and cone dysfunction and severe macular dysfunction. Inner retinal dysfunction was evident in three of seven patients. Conclusions Patients with KIF11 mutations show a specific ocular phenotype with variable expressivity and intrafamilial variability. Macular atrophy and dysfunction have not been consistently documented before. The fundus lesions appear non-progressive. The findings assist in providing an accurate diagnosis and thus improving the management and follow-up of patients with this syndrome.