Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts.

BACKGROUND: Myelodysplastic syndromes are a diverse and common group of chronic hematologic cancers. The identification of new genetic lesions could facilitate new diagnostic and therapeutic strategies. METHODS: We used massively parallel sequencing technology to identify somatically acquired point mutations across all protein-coding exons in the genome in 9 patients with low-grade myelodysplasia. Targeted resequencing of the gene encoding RNA splicing factor 3B, subunit 1 (SF3B1), was also performed in a cohort of 2087 patients with myeloid or other cancers. RESULTS: We identified 64 point mutations in the 9 patients. Recurrent somatically acquired mutations were identified in SF3B1. Follow-up revealed SF3B1 mutations in 72 of 354 patients (20%) with myelodysplastic syndromes, with particularly high frequency among patients whose disease was characterized by ring sideroblasts (53 of 82 [65%]). The gene was also mutated in 1 to 5% of patients with a variety of other tumor types. The observed mutations were less deleterious than was expected on the basis of chance, suggesting that the mutated protein retains structural integrity with altered function. SF3B1 mutations were associated with down-regulation of key gene networks, including core mitochondrial pathways. Clinically, patients with SF3B1 mutations had fewer cytopenias and longer event-free survival than patients without SF3B1 mutations. CONCLUSIONS: Mutations in SF3B1 implicate abnormalities of messenger RNA splicing in the pathogenesis of myelodysplastic syndromes. (Funded by the Wellcome Trust and others.).

LKB1/KRAS mutant lung cancers constitute a genetic subset of NSCLC with increased sensitivity to MAPK and mTOR signalling inhibition.

LKB1/STK11 is a multitasking tumour suppressor kinase. Germline inactivating mutations of the gene are responsible for the Peutz-Jeghers hereditary cancer syndrome. It is also somatically inactivated in approximately 30% of non-small-cell lung cancer (NSCLC). Here, we report that LKB1/KRAS mutant NSCLC cell lines are sensitive to the MEK inhibitor CI-1040 shown by a dose-dependent reduction in proliferation rate, whereas LKB1 and KRAS mutations alone do not confer similar sensitivity. We show that this subset of NSCLC is also sensitised to the mTOR inhibitor rapamycin. Importantly, the data suggest that LKB1/KRAS mutant NSCLCs are a genetically and functionally distinct subset and further suggest that this subset of lung cancers might afford an opportunity for exploitation of anti-MAPK/mTOR-targeted therapies.

The Catalogue of Somatic Mutations in Cancer (COSMIC).

COSMIC is currently the most comprehensive global resource for information on somatic mutations in human cancer, combining curation of the scientific literature with tumor resequencing data from the Cancer Genome Project at the Sanger Institute, U.K. Almost 4800 genes and 250000 tumors have been examined, resulting in over 50000 mutations available for investigation. This information can be accessed in a number of ways, the most convenient being the Web-based system which allows detailed data mining, presenting the results in easily interpretable formats. This unit describes the graphical system in detail, elaborating an example walkthrough and the many ways that the resulting information can be thoroughly investigated by combining data, respecializing the query, or viewing the results in different ways. Alternate protocols overview the available precompiled data files available for download.

AutoCSA, an algorithm for high throughput DNA sequence variant detection in cancer genomes.

UNLABELLED: The undertaking of large-scale DNA sequencing screens for somatic variants in human cancers requires accurate and rapid processing of traces for variants. Due to their often aneuploid nature and admixed normal tissue, heterozygous variants found in primary cancers are often subtle and difficult to detect. To address these issues, we have developed a mutation detection algorithm, AutoCSA, specifically optimized for the high throughput screening of cancer samples. AVAILABILITY: http://www.sanger.ac.uk/genetics/CGP/Software/AutoCSA.

Frequent activating FGFR2 mutations in endometrial carcinomas parallel germline mutations associated with craniosynostosis and skeletal dysplasia syndromes.

Endometrial carcinoma is the most common gynecological malignancy in the United States. Although most women present with early disease confined to the uterus, the majority of persistent or recurrent tumors are refractory to current chemotherapies. We have identified a total of 11 different FGFR2 mutations in 3/10 (30%) of endometrial cell lines and 19/187 (10%) of primary uterine tumors. Mutations were seen primarily in tumors of the endometrioid histologic subtype (18/115 cases investigated, 16%). The majority of the somatic mutations identified were identical to germline activating mutations in FGFR2 and FGFR3 that cause Apert Syndrome, Beare-Stevenson Syndrome, hypochondroplasia, achondroplasia and SADDAN syndrome. The two most common somatic mutations identified were S252W (in eight tumors) and N550K (in five samples). Four novel mutations were identified, three of which are also likely to result in receptor gain-of-function. Extensive functional analyses have already been performed on many of these mutations, demonstrating they result in receptor activation through a variety of mechanisms. The discovery of activating FGFR2 mutations in endometrial carcinoma raises the possibility of employing anti-FGFR molecularly targeted therapies in patients with advanced or recurrent endometrial carcinoma.

Mutations in the RSK2(RPS6KA3) gene cause Coffin-Lowry syndrome and nonsyndromic X-linked mental retardation.

We describe three families with X-linked mental retardation, two with a deletion of a single amino acid and one with a missense mutation in the proximal domain of the RSK2(RPS6KA3) (ribosomal protein S6 kinase, 90 kDa, polypeptide 3) protein similar to mutations found in Coffin-Lowry syndrome (CLS). In two families, the clinical diagnosis had been nonsyndromic X-linked mental retardation. In the third family, although CLS had been suspected, the clinical features were atypical and the degree of intellectual disability much less than expected. These families show that strict reliance on classical clinical criteria for mutation testing may result in a missed diagnosis. A less targeted screening approach to mutation testing is advocated.

COSMIC 2005.

The Catalogue Of Somatic Mutations In Cancer (COSMIC) database and web site was developed to preserve somatic mutation data and share it with the community. Over the past 25 years, approximately 350 cancer genes have been identified, of which 311 are somatically mutated. COSMIC has been expanded and now holds data previously reported in the scientific literature for 28 known cancer genes. In addition, there is data from the systematic sequencing of 518 protein kinase genes. The total gene count in COSMIC stands at 538; 25 have a mutation frequency above 5% in one or more tumour type, no mutations were found in 333 genes and 180 are rarely mutated with frequencies <5% in any tumour set. The COSMIC web site has been expanded to give more views and summaries of the data and provide faster query routes and downloads. In addition, there is a new section describing mutations found through a screen of known cancer genes in 728 cancer cell lines including the NCI-60 set of cancer cell lines.

Somatic mutations in human cancer: insights from resequencing the protein kinase gene family.

All cancers arise due to the accumulation of mutations in critical target genes that, when altered, give rise to selective advantage in the cell and its progeny that harbor them. Knowledge of these mutations is key in understanding the biology of cancer initiation and progression, as well as the development of more targeted therapeutic strategies. We have undertaken a systematic screen of all annotated protein kinases in the human genome for mutations in a series of cancers including breast, non-small-cell lung, and testicular cancer. Our results show a wide diversity in mutation prevalence within and between tumor types. We have identified a mutator phenotype in human breast previously undescribed. The results presented from sequencing the same 1.3 million base pairs through several tumor types suggest that most of the observed mutations are likely to be passenger events rather than causally implicated in oncogenesis. However, this work does provide evidence for the likely existence of multiple, infrequently mutated kinases.

The COSMIC (Catalogue of Somatic Mutations in Cancer) database and website.

The discovery of mutations in cancer genes has advanced our understanding of cancer. These results are dispersed across the scientific literature and with the availability of the human genome sequence will continue to accrue. The COSMIC (Catalogue of Somatic Mutations in Cancer) database and website have been developed to store somatic mutation data in a single location and display the data and other information related to human cancer. To populate this resource, data has currently been extracted from reports in the scientific literature for somatic mutations in four genes, BRAF, HRAS, KRAS2 and NRAS. At present, the database holds information on 66 634 samples and reports a total of 10 647 mutations. Through the web pages, these data can be queried, displayed as figures or tables and exported in a number of formats. COSMIC is an ongoing project that will continue to curate somatic mutation data and release it through the website.

Confirmation of a gene locus for medullary cystic kidney disease (MCKD2) on chromosome 16p12.

BACKGROUND: Autosomal-dominant medullary cystic kidney disease (MCKD) is an interstitial nephropathy characterized by structural renal tubular defects that result in salt wasting and a reduction in urinary concentration. The condition has clinical and morphological similarities to autosomal-recessive juvenile nephronophthisis. Two genes predisposing to MCKD have been localized. MCKD1 on chromosome 1q21 was localized in two Cypriot families, and MCKD2 on chromosome 16p12 was localized in a single Italian family. We have evaluated a large Welsh MCKD family for linkage at these two loci. METHODS: Clinical data and DNA samples were collected from affected family members. Polymorphic microsatellite markers spanning the critical regions on chromosome 1 and chromosome 16 that encompass MCKD1 and MCKD2 were analyzed. Two-point and multipoint LOD scores were calculated. RESULTS: The family fulfilled previously published criteria for the diagnosis of MCKD, but hyperuricemia and gout were not prominent features. Twenty-one affected individuals were identified. Mean age at death or end-stage renal disease was 47 years (37 to 60). Linkage and haplotype analysis generated strongly negative results at MCKD1 on chromosome 1q21 (two-point LOD score = -5.32). Strong evidence of linkage to MCKD2 was generated with a maximum multi-point LOD score of 3.75. CONCLUSION: These results provide the first independent confirmation of a gene predisposing to MCKD on chromosome 16p12 and indicate that mutation of this gene is not restricted to a single family or population. The absence of hyperuricemia and gout in our family indicates that these are not obligatory features of MCKD2 mutations.

Allele loss on chromosome 1p36 in epithelial ovarian cancers.

OBJECTIVES: Prior studies have shown that allelic loss on chromosome 1p36 occurs frequently in ovarian as well as several other types of cancer. This suggests that inactivation of gene(s) in this region may play a role in the pathogenesis of these cancers. The aim of this study was to further delineate the region of loss on chromosome 1p36 in ovarian cancers and to identify associated patient or tumor characteristics. METHODS: Paired normal/cancer DNA samples from 75 ovarian cancers (21 early stage I/II and 54 advanced stage III/IV) were analyzed using microsatellite markers. RESULTS: Forty-nine of 75 (65%) ovarian cancers had loss of at least one marker. The marker demonstrating the most frequent loss was D1S1597, which was lost in 29/57 (51%) informative cases. Allele loss on 1p36 was significantly more common in poorly differentiated ovarian cancers (73%) relative to well or moderately differentiated cases (48%) (P = 0.03). Evidence was obtained for two common regions of deletion: one flanked by D1S1646/D1S244 and another more proximally by D1S244/D1S228. CONCLUSION: These findings further delineate regions on chromosome 1p36 proposed to contain tumor suppressor gene(s) that may play a role in the development and/or progression of epithelial ovarian carcinoma. Allele loss on 1p36 is associated with poor histologic grade.

Localization of the gene for distal hereditary motor neuronopathy VII (dHMN-VII) to chromosome 2q14.

Distal hereditary motor neuronopathy type VII (dHMN-VII) is an autosomal dominant disorder characterized by distal muscular atrophy and vocal cord paralysis. We performed a genomewide linkage search in a large Welsh pedigree with dHMN-VII and established linkage to chromosome 2q14. Analyses of a second family with dHMN-VII confirmed the location of the gene and provided evidence for a founder mutation segregating in both pedigrees. The maximum three-point LOD score in the combined pedigree was 7.49 at D2S274. Expansion of a polyalanine tract in Engrailed-1, a transcription factor strongly expressed in the spinal cord, was excluded as the cause of dHMN-VII.

Cancer and genomics.

Identification of the genes that cause oncogenesis is a central aim of cancer research. We searched the proteins predicted from the draft human genome sequence for paralogues of known tumour suppressor genes, but no novel genes were identified. We then assessed whether it was possible to search directly for oncogenic sequence changes in cancer cells by comparing cancer genome sequences against the draft genome. Apparently chimaeric transcripts (from oncogenic fusion genes generated by chromosomal translocations, the ends of which mapped to different genomic locations) were detected to the same degree in both normal and neoplastic tissues, indicating a significant level of false positives. Our experiment underscores the limited amount and variable quality of DNA sequence from cancer cells that is currently available.

K-ras mutations in Müllerian inclusion cysts associated with serous borderline tumors of the ovary.

OBJECTIVE: Müllerian inclusion cysts (MIC) are small benign appearing glands that are occasionally noted in lymph nodes and peritoneal biopsies. They occur most frequently in women with serous ovarian tumors, with borderline tumors (SBOT) having a higher incidence than invasive cancers. The aim of this study was to examine whether MIC and SBOT have identical K-ras mutations, which would suggest that they are related. Methods. Six patients in whom adequate tissue was available from SBOT, MIC, and normal tissue were identified from a consecutive series of patients with SBOT who underwent lymph node sampling from 1992 to 1997 at Duke University Medical Center. DNA extraction was performed using laser capture microdissection. Exon 1 of the K-ras gene was amplified using PCR and subjected to single-strand conformation analysis to screen for mutations. Shifted bands were sequenced to confirm the presence of mutations. RESULTS: Mutations in codon 12 of K-ras were found in three of six (50%) SBOT. In two of these three cases, the identical mutation was found in the SBOT and the MIC (gly to val in both cases), but not in the corresponding normal DNA. In one case, a mutation was seen in the ovarian tumor (gly to asp), but not in the corresponding MIC. CONCLUSIONS: Mutations in codon 12 of the K-ras gene are a hallmark of serous borderline tumors. The presence of identical K-ras mutations in some SBOT and their associated MIC suggests that they are related processes. Both may arise due to a field effect, or alternatively some MIC may represent metastases from the primary ovarian tumor.

High frequency of hypermethylation at the 14-3-3 sigma locus leads to gene silencing in breast cancer.

Expression of 14-3-3 final sigma (final sigma) is induced in response to DNA damage, and causes cells to arrest in G(2). By SAGE (serial analysis of gene expression) analysis, we identified final sigma as a gene whose expression is 7-fold lower in breast carcinoma cells than in normal breast epithelium. We verified this finding by Northern blot analysis. Remarkably, final sigma mRNA was undetectable in 45 of 48 primary breast carcinomas. Genetic alterations at final sigma such as loss of heterozygosity were rare (1/20 informative cases), and no mutations were detected (0/34). On the other hand, hypermethylation of CpG islands in the final sigma gene was detected in 91% (75/82) of breast tumors and was associated with lack of gene expression. Hypermethylation of final sigma is functionally important, because treatment of final sigma-non-expressing breast cancer cell lines with the drug 5-aza-2'-deoxycytidine resulted in demethylation of the gene and synthesis of final sigma mRNA. Breast cancer cells lacking final sigma expression showed increased number of chromosomal breaks and gaps when exposed to gamma-irradiation. Therefore, it is possible that loss of final sigma expression contributes to malignant transformation by impairing the G(2) cell cycle checkpoint function, thus allowing an accumulation of genetic defects. Hypermethylation and loss of final sigma expression are the most consistent molecular alterations in breast cancer identified so far.

Testing for hereditary breast and ovarian cancer in the southeastern United States.

OBJECTIVES: To detail characterization of mutations and uncharacterized variants in the breast cancer susceptibility genes BRCA1 and BRCA2, as observed in a population of breast cancer patients from the southeastern United States, and to examine baseline characteristics of women referred for counseling and testing and provide a preliminary look at how counseling and testing affected intentions toward prophylactic surgery. BACKGROUND: Mutations in the BRCA1 and BRCA2 genes give rise to a dramatically increased risk of developing breast or ovarian cancer or both. There are many reports about special populations in which deleterious mutations are present at a high frequency. It is useful to study these genes in more heterogeneous populations, reflecting different geographic regions. Interest in preventive surgery for gene carriers is high in women and their surgeons. METHODS: Women were recruited through a prospective clinical trial of counseling and free genetic testing. BRCA1 and BRCA2 were screened for mutations using standard techniques, and results were given to participants. Baseline questionnaires determined interest in preventive surgery at the beginning of the study. Follow-up questionnaires for those who completed testing surveyed interest in prophylactic surgery after counseling and receiving test results. RESULTS: Of 213 women who completed counseling and testing, 44 (20.6%) had 29 separate mutations; there were 11 Jewish women carrying three founder mutations. Twenty-eight women (13.1%) had uncharacterized variants in BRCA1 or BRCA2; nine were not previously reported. Women overestimated their chances of possessing a deleterious gene mutation compared to a statistical estimate of carrier risk. A number of women changed their intentions toward preventive surgery after genetic counseling and testing. CONCLUSIONS: Hereditary breast cancer due to mutations in BRCA1 and BRCA2 was a heterogeneous syndrome in the southeastern United States. Most mutations were seen just once, and uncharacterized variants were common and of uncertain clinical significance. In general, positive test results tended to reinforce intentions toward prophylactic surgery. In contrast, women not interested in surgery at the time of entry tended to remain reluctant after testing and counseling.

The pathology of familial breast cancer: histological features of cancers in families not attributable to mutations in BRCA1 or BRCA2.

Breast cancers arising in carriers of mutations in the breast cancer susceptibility genes, BRCA1 and BRCA2, differ histologically from each other and from breast cancers unselected for a family history. However, a substantial proportion of families with multiple cases of breast cancer is not attributable to these two genes (non-BRCA1/2 families). We have now characterized the pathology of 82 breast cancers from non-BRCA1/2 families. Breast cancers in non-BRCA1/2 families were of lower grade (P = 0.0018), showed fewer mitoses (P < 0.0001), less nuclear pleomorphism (P = 0.0014), less lymphocytic infiltrate (P < 0.0001), a lesser extent of the tumor with a continuous pushing margin (P = 0.004), a lesser extent of the tumor composed of solid sheets of cells (P = 0.0047), less necrosis (P = 0.002), and wereparison with BRCA2 tumors, non-BRCA1/2 tumors were lower grade (P = 0.017) and exhibited less pleomorphism (P = 0.01) and more tubule formation (P = 0.05). In comparison with control breast cancers unselected for a family history of the disease, non-BRCA1/2 tumors were of significantly lower grade (P = 0.001), showed less pleomorphism (P = 0.0002), and had a lower mitotic count (P = 0.003). The results indicate that non-BRCA1/2 breast cancers differ histologically from both BRCA1 and BRCA2 breast cancers and are overall of lower grade. They also suggest that non-BRCA1/2 breast cancers differ from nonfamilial breast cancers, but these differences may be attributable to various types of bias.

Bcl10 is not a target for frequent mutation in human carcinomas.

The recently described Bcl10 gene has been suggested to be a major target gene for inactivation in a variety of human cancers. In order to further evaluate the role of this gene in human adult malignancies, we have analysed a series of carcinomas for mutations in the Bcl10 gene. We have screened a panel of 174 carcinoma samples in total, comprised of 47 breast, 36 epithelial ovarian, 36 endometrial, 12 cervical, 23 colorectal and 20 head/neck carcinomas, all unselected for grade or stage. This panel reflects, in part, tumours reported to have involvement of the 1p22 region of chromosome 1, the region harbouring the Bcl10 gene. No deleterious mutations were detected in any of the samples analysed, strongly suggesting that Bcl10 is not a common target for inactivation in adult malignancies and that BCL10 is not the gene targeted for frequent inactivation at 1p22.

Genetic susceptibility testing and prophylactic oophorectomy.

About 10% of ovarian cancer cases are thought to have a hereditary basis and family history is the strongest risk factor for the development of this disease. In the past, prophylactic oophorectomy has been advocated for women with two or more affected first-degree relatives. More recently, with the identification of the genes responsible for most hereditary ovarian cancers (BRCA1, BRCA2), oophorectomy can now be offered specifically to women who are mutation carriers. Conversely, non-carriers in these families can be reassured that their risk of ovarian cancer is not increased. The value of oophorectomy in mutation carriers has not yet been proven, however, and there are concerns that the benefit may be less than intuitively expected. First, although the lifetime risk of ovarian cancer initially was reported to be as high as 60%, more recent studies have reported risks in the range of 15-30%. A better understanding of the genetic and/or environmental basis of variable penetrance is needed to augment our ability to counsel women regarding their risk. In addition, peritoneal papillary serous carcinoma indistinguishable from ovarian cancer occurs in some women following oophorectomy. Studies that better define how often this occurs also are needed to establish more firmly the value of prophylactic oophorectomy. In view of the uncertainty regarding the efficacy of prophylactic oophorectomy, chemopreventive and early detection approaches also deserve consideration as strategies for decreasing ovarian cancer mortality in women who carry mutations in ovarian cancer susceptibility genes.

Managing hereditary ovarian cancer risk.

Ovarian cancer is the fourth leading cause of cancer deaths in American women. About 10% of cases are thought to have a hereditary basis, and family history is the strongest known risk factor. In the past, prophylactic oophorectomy has been advocated for women with two or more affected first-degree relatives. More recently, with the identification of the genes responsible for most hereditary ovarian cancers (BRCA1, BRCA2), oophorectomy can now be offered specifically to women who are mutation carriers. Conversely, noncarriers in these families can be reassured that their risk of ovarian cancer is not increased. The value of oophorectomy in mutation carriers has not yet been proven, however, and concern exists that the benefit may be less than intuitively expected. First, although the lifetime risk of ovarian cancer initially was reported to be as high as 60%, more recent studies have suggested risks in the range of 15 to 30%. A better understanding of the factors that underlie variable penetrance in mutation carriers is needed to augment our ability to counsel individual women. In addition, peritoneal papillary serous carcinoma indistinguishable from ovarian cancer occurs in some women after oophorectomy. Studies that better define the frequency with which this occurs are needed to establish the magnitude of the protective effect conferred by prophylactic oophorectomy. In view of the uncertainty regarding the efficacy of prophylactic oophorectomy, chemopreventive and early detection approaches also deserve consideration as strategies for decreasing ovarian cancer mortality in women who carry mutations in ovarian cancer susceptibility genes.