Prognostic gene expression signature for high-grade serous ovarian cancer.

BACKGROUND: Median overall survival (OS) for women with high-grade serous ovarian cancer (HGSOC) is ∼4 years, yet survival varies widely between patients. There are no well-established, gene expression signatures associated with prognosis. The aim of this study was to develop a robust prognostic signature for OS in patients with HGSOC. PATIENTS AND METHODS: Expression of 513 genes, selected from a meta-analysis of 1455 tumours and other candidates, was measured using NanoString technology from formalin-fixed paraffin-embedded tumour tissue collected from 3769 women with HGSOC from multiple studies. Elastic net regularization for survival analysis was applied to develop a prognostic model for 5-year OS, trained on 2702 tumours from 15 studies and evaluated on an independent set of 1067 tumours from six studies. RESULTS: Expression levels of 276 genes were associated with OS (false discovery rate < 0.05) in covariate-adjusted single-gene analyses. The top five genes were TAP1, ZFHX4, CXCL9, FBN1 and PTGER3 (P < 0.001). The best performing prognostic signature included 101 genes enriched in pathways with treatment implications. Each gain of one standard deviation in the gene expression score conferred a greater than twofold increase in risk of death [hazard ratio (HR) 2.35, 95% confidence interval (CI) 2.02-2.71; P < 0.001]. Median survival [HR (95% CI)] by gene expression score quintile was 9.5 (8.3 to -), 5.4 (4.6-7.0), 3.8 (3.3-4.6), 3.2 (2.9-3.7) and 2.3 (2.1-2.6) years. CONCLUSION: The OTTA-SPOT (Ovarian Tumor Tissue Analysis consortium - Stratified Prognosis of Ovarian Tumours) gene expression signature may improve risk stratification in clinical trials by identifying patients who are least likely to achieve 5-year survival. The identified novel genes associated with the outcome may also yield opportunities for the development of targeted therapeutic approaches.

A clinically applicable molecular-based classification for endometrial cancers.

BACKGROUND: Classification of endometrial carcinomas (ECs) by morphologic features is inconsistent, and yields limited prognostic and predictive information. A new system for classification based on the molecular categories identified in The Cancer Genome Atlas is proposed. METHODS: Genomic data from the Cancer Genome Atlas (TCGA) support classification of endometrial carcinomas into four prognostically significant subgroups; we used the TCGA data set to develop surrogate assays that could replicate the TCGA classification, but without the need for the labor-intensive and cost-prohibitive genomic methodology. Combinations of the most relevant assays were carried forward and tested on a new independent cohort of 152 endometrial carcinoma cases, and molecular vs clinical risk group stratification was compared. RESULTS: Replication of TCGA survival curves was achieved with statistical significance using multiple different molecular classification models (16 total tested). Internal validation supported carrying forward a classifier based on the following components: mismatch repair protein immunohistochemistry, POLE mutational analysis and p53 immunohistochemistry as a surrogate for 'copy-number' status. The proposed molecular classifier was associated with clinical outcomes, as was stage, grade, lymph-vascular space invasion, nodal involvement and adjuvant treatment. In multivariable analysis both molecular classification and clinical risk groups were associated with outcomes, but differed greatly in composition of cases within each category, with half of POLE and mismatch repair loss subgroups residing within the clinically defined 'high-risk' group. Combining the molecular classifier with clinicopathologic features or risk groups provided the highest C-index for discrimination of outcome survival curves. CONCLUSIONS: Molecular classification of ECs can be achieved using clinically applicable methods on formalin-fixed paraffin-embedded samples, and provides independent prognostic information beyond established risk factors. This pragmatic molecular classification tool has potential to be used routinely in guiding treatment for individuals with endometrial carcinoma and in stratifying cases in future clinical trials.

Cancer-associated somatic DICER1 hotspot mutations cause defective miRNA processing and reverse-strand expression bias to predominantly mature 3p strands through loss of 5p strand cleavage.

Our group recently described recurrent somatic mutations of the miRNA processing gene DICER1 in non-epithelial ovarian cancer. Mutations appeared to be clustered around each of four critical metal-binding residues in the RNase IIIB domain of DICER1. This domain is responsible for cleavage of the 3' end of the 5p miRNA strand of a pre-mRNA hairpin. To investigate the effects of these cancer-associated 'hotspot' mutations, we engineered mouse DICER1-deficient ES cells to express wild-type and an allelic series of the mutant DICER1 variants. Global miRNA and mRNA profiles from cells carrying the metal-binding site mutations were compared to each other and to wild-type DICER1. The miRNA and mRNA profiles generated through the expression of the hotspot mutations were virtually identical, and the DICER1 hotspot mutation-carrying cells were distinct from both wild-type and DICER1-deficient cells. Further, miRNA profiles showed that mutant DICER1 results in a dramatic loss in processing of mature 5p miRNA strands but were still able to create 3p strand miRNAs. Messenger RNA (mRNA) profile changes were consistent with the loss of 5p strand miRNAs and showed enriched expression for predicted targets of the lost 5p-derived miRNAs. We therefore conclude that cancer-associated somatic hotspot mutations of DICER1, affecting any one of four metal-binding residues in the RNase IIIB domain, are functionally equivalent with respect to miRNA processing and are hypomorphic alleles, yielding a global loss in processing of mature 5p strand miRNA. We further propose that this resulting 3p strand bias in mature miRNA expression likely underpins the oncogenic potential of these hotspot mutations.

Germline mutations in CDH1 are infrequent in women with early-onset or familial lobular breast cancers.

BACKGROUND: Germline mutations in CDH1 are associated with hereditary diffuse gastric cancer; lobular breast cancer also occurs excessively in families with such condition. METHOD: To determine if CDH1 is a susceptibility gene for lobular breast cancer in women without a family history of diffuse gastric cancer, germline DNA was analysed for the presence of CDH1 mutations in 318 women with lobular breast cancer who were diagnosed before the age of 45 years or had a family history of breast cancer and were not known, or known not, to be carriers of germline mutations in BRCA1 or BRCA2. Cases were ascertained through breast cancer registries and high-risk cancer genetic clinics (Breast Cancer Family Registry, the kConFab and a consortium of breast cancer genetics clinics in the United States and Spain). Additionally, Multiplex Ligation-dependent Probe Amplification was performed for 134 cases to detect large deletions. RESULTS: No truncating mutations and no large deletions were detected. Six non-synonymous variants were found in seven families. Four (4/318 or 1.3%) are considered to be potentially pathogenic through in vitro and in silico analysis. CONCLUSION: Potentially pathogenic germline CDH1 mutations in women with early-onset or familial lobular breast cancer are at most infrequent.

The NMD mRNA surveillance pathway downregulates aberrant E-cadherin transcripts in gastric cancer cells and in CDH1 mutation carriers.

Germline mutations in the gene encoding the tumour suppressor E-cadherin (CDH1) are the underlying genetic defect responsible for hereditary diffuse gastric cancer (HDGC). A remarkably high percentage ( approximately 80%) of CDH1 mutations in HDGC patients and carriers generate premature termination codons (PTCs). Here, we examined whether CDH1 transcripts harbouring PTCs are downregulated by nonsense-mediated decay (NMD), an RNA surveillance pathway that degrades PTC-bearing transcripts. Using an allele-specific expression (ASE) assay to differentiate between mutated and wild-type CDH1 alleles, we found that PTC-bearing CDH1 mRNAs are strongly downregulated in normal gastric tissue from several CDH1 mutation carriers. We show that NMD is responsible for this robust downregulation, as CDH1 transcripts harbouring PTCs in the KATO-III gastric tumour cell line were upregulated in response to protein synthesis inhibitors or depletion of the NMD factors UPF1 and eIF4AIII. Analysis of HDGC patients harbouring CDH1 alleles with PTCs at a wide variety of different positions indicates an association of their predicted ability to induce NMD and an earlier age of onset of gastric cancer. This suggests that NMD may be detrimental for HDGC patients and therefore NMD is a potentially useful therapeutic target for CDH1 mutation carriers.

Germline E-cadherin mutations in familial lobular breast cancer.

BACKGROUND: The cell surface glycoprotein E-cadherin (CDH1) is a key regulator of adhesive properties in epithelial cells. Germline mutations in CDH1 are well established as the defects underlying hereditary diffuse gastric cancer (HDGC) syndrome, and an increased risk of lobular breast cancer (LBC) has been described in HDGC kindreds. However, germline CDH1 mutations have not been described in patients with LBC in non-HDGC families. This study aimed to investigate the frequency of germline CDH1 mutations in patients with LBC with early onset disease or family histories of breast cancer without DGC. METHODS: Germline DNA was analysed in 23 women with invasive lobular or mixed ductal and lobular breast cancers who had at least one close relative with breast cancer or had themselves been diagnosed before the age of 45 years, had tested negative for a germline BRCA1 or BRCA2 mutation, and reported no personal or family history of diffuse gastric cancer. The full coding sequence of CDH1 including splice junctions was amplified using PCR and screened for mutations using DHPLC and sequencing. RESULTS: A novel germline CDH1 truncating mutation in the extracellular portion of the protein (517insA) was identified in one woman who had LBC at the age of 42 years and a first degree relative with invasive LBC. CONCLUSIONS: Germline CDH1 mutations can be associated with invasive LBC in the absence of diffuse gastric cancer. The finding, if confirmed, may have implications for management of individuals at risk for this breast cancer subtype. Clarification of the cancer risks in the syndrome is essential.

CDH1/E-cadherin germline mutations in early-onset gastric cancer.

BACKGROUND: Gastric cancer remains a leading cause of cancer deaths worldwide. Genetic factors, including germline mutations in E-cadherin (CDH1, MIM#192090) in hereditary diffuse gastric cancer (HDGC, MIM#137215), are implicated in this disease. Family studies have reported CDH1 germline mutations in HDGC but the role of CDH1 germline mutations in the general population remains unclear. AIMS: To examine the frequency of CDH1 germline mutations in a population-based series of early-onset gastric cancer (EOGC <50 years old). METHODS: 211 cases of EOGC were identified in Central-East Ontario region from 1989 to 1993, with archival material and histological confirmation of non-intestinal type gastric cancer available for 81 subjects. Eligible cases were analysed for CDH1 germline mutations by single-strand conformation polymorphism, variants were sequenced, and tumours from cases with functional mutations were stained for E-cadherin (HECD-1) using immunohistochemistry. RESULTS: 1155 (89%) of 1296 polymerase chain reactions amplified successfully. One new germline deletion (nt41delT) was identified in a 30-year-old patient with isolated cell gastric cancer. The overall frequency of germline CDH1 mutations was 1.3% (1/81) for EOGC and 2.8% (1/36) for early-onset isolated cell gastric cancer. CONCLUSION: This is the first population-based study, in a low-incidence region, of genetic predisposition to gastric cancer. Combined with our previous report of germline hMLH1 mutations in two other subjects from this series, it is suggested that 2-3% of EOCG cases in North Americans may be owing to high-risk genetic mutations. These data should inform cancer geneticists on the utility of searching for specific genetic mutations in EOGC.

Germline E-cadherin mutations in hereditary diffuse gastric cancer: assessment of 42 new families and review of genetic screening criteria.

BACKGROUND: Mutations in the E-cadherin (CDH1) gene are a well documented cause of hereditary diffuse gastric cancer (HDGC). Development of evidence based guidelines for CDH1 screening for HDGC have been complicated by its rarity, variable penetrance, and lack of founder mutations. METHODS: Forty three new gastric cancer (GC) families were ascertained from multiple sources. In 42 of these families at least one gastric cancer was pathologically confirmed to be a diffuse gastric cancer (DGC); the other family had intestinal type gastric cancers. Screening of the entire coding region of the CDH1 gene and all intron/exon boundaries was performed by bi-directional sequencing. RESULTS: Novel mutations were found in 13 of the 42 DGC families (31% overall). Twelve of these mutations occur among the 25 families with multiple cases of gastric cancer and with pathologic confirmation of diffuse gastric cancer phenotype in at least one individual under the age of 50 years. The mutations found include small insertions and deletions, splice site mutations, and three non-conservative amino acid substitutions (A298T, W409R, and R732Q). All three missense mutations conferred loss of E-cadherin function in in vitro assays. Multiple cases of breast cancers including pathologically confirmed lobular breast cancers were observed both in mutation positive and negative families. CONCLUSION: Germline truncating CDH1 mutations are found in 48% of families with multiple cases of gastric cancer and at least one documented case of DGC in an individual under 50 years of age. We recommend that these criteria be used for selecting families for CDH1 mutational analysis.

Hereditary coproporphyria: exon screening by heteroduplex analysis detects three novel mutations in the coproporphyrinogen oxidase gene.

Hereditary coproporphyria is a dominantly inherited disorder of porphyrin metabolism caused by a partial deficiency of coproporphyrinogen oxidase, the sixth enzyme in the heme synthetic pathway. We investigated the molecular basis of hereditary coproporphyria in three unrelated patients, amplifying each exon of the coproporphyrinogen oxidase gene and performing heteroduplex analysis to look for mutations. Unique heteroduplex patterns were noted in exons 2, 3, and 6. Sequencing revealed different mutations in each patient: a G-->A point mutation encoding a glutamic acid to lysine substitution at codon 101 (E101K), a C-->T point mutation encoding a proline to serine substitution at codon 149 (P149S), and a one base-pair insertion in exon 6 (968insT). No other mutations were found on sequencing the remaining exons and their intron-exon junctions. The two point mutations affect amino acids that are conserved in all species studied to date. The one base-pair insertion in exon 6 is the first frameshift mutation to be described in the coproporphyrinogen oxidase gene. This study adds three new mutations to those that have been previously reported, and all have been restricted to single families. These results indicate that hereditary coproporphyria is a genetically heterogeneous disease.

Substrate and positional specificities of human and mouse lecithin-cholesterol acyltransferases. Studies with wild type recombinant and chimeric enzymes expressed in vitro.

Human lecithin-cholesterol acyltransferase (LCAT) preferentially attacks sn-1 position of 16:0-20:4 phosphatidylcholine (PC), producing more 16:0 cholesteryl ester (CE) than 20:4 CE. In contrast, rat and mouse LCATs produce mostly 20:4 CE from the same PC. To understand the structural basis for this difference in positional specificity, we studied the specificities of recombinant mouse and human LCATs and several chimeric constructs of the two. The rLCATs retained the substrate and positional specificities of the plasma enzymes when expressed in COS-1 cells. Human and mouse LCAT cDNAs were each cleaved into three fragments, recombined in various combinations, and the chimeric products were analyzed for their specificities. When the N-terminal, or (and) C-terminal segments of human LCAT were replaced by the corresponding mouse LCAT segments, the chimeric products exhibited the specificity of intact human enzyme. However, when the middle segment, containing the residues 130-306 was replaced by the corresponding mouse LCAT segment, the enzyme exhibited the specificity of mouse LCAT. Similarly, the mouse rLCAT exhibited the specificity of human enzyme when its central segment, but not its N-terminal or C-terminal segment was replaced by the corresponding segment from human LCAT. These results show that the substrate and positional specificities of LCAT are controlled by the central domain of LCAT protein, corresponding to the amino acid residues 130-306.