A MXI1-NUTM1 fusion protein with MYC-like activity suggests a novel oncogenic mechanism in a subset of NUTM1-rearranged tumors.

Most NUTM1-rearranged neoplasms (NRNs) have fusions between NUTM1 and BRD (bromodomain-containing) family members and are termed NUT carcinomas (NCs) because they show some squamous differentiation. However, some NRNs are associated with fusions between NUTM1 and members of the MAD (MAX dimerization) gene family of MYC antagonists. Here we describe a small round cell malignancy from the gastro-esophageal junction with a previously unreported fusion between NUTM1 and the MAD family member MXI1. In contrast to NCs, the MXI1-NUTM1 tumor did not show squamous differentiation and did not express MYC, TP63 or SOX2, genes known to be targets of BRD-NUTM1 proteins and critical for NC oncogenesis. Transcriptome analysis showed paradoxical enrichment of MYC target genes in the MXI1-NUTM1 tumor despite the lack of MYC expression. When expressed in vitro MXI1-NUTM1 partially phenocopied MYC, enhancing cell proliferation and cooperating with oncogenic HRAS to produce anchorage-independent cell growth. These data provide evidence that MAD family members, which are normally repressors of MYC activity, can be converted into MYC-like mimics by fusion to NUTM1. The pathological features and novel oncogenic mechanism of the MXI1-NUTM1 tumor show that identification of NUTM1 fusion partners can be important for accurate diagnostic classification of some NRN subtypes, and potentially may guide therapeutic options.

Copy number analysis of ductal carcinoma in situ with and without recurrence.

Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer and a frequent mammographic finding requiring treatment. Up to 25% of DCIS can recur and half of recurrences are invasive, but there are no reliable biomarkers for recurrence. We hypothesised that copy number aberrations could predict likelihood of recurrence. We analysed a cohort of pure DCIS cases treated only with wide local excision for genome-wide copy number and loss of heterozygosity using Affymetrix OncoScan MIP arrays. Cases included those without recurrence within 7 years (n = 25) and with recurrence between 1 and 5 years after diagnosis (n = 15). Pure DCIS were broadly similar in copy number changes compared with invasive breast cancer, with the consistent exception of a greater frequency of ERBB2 amplification in DCIS. There were no significant differences in age or ER status between the cases with a recurrence vs those without. Overall, the DCIS cases with recurrence had more copy number events than the DCIS without recurrence. The increased copy number appeared non-random with several genomic regions showing an increase in frequency in recurrent cases, including 20 q gain, ERBB2 amplification and 15q loss. Copy number changes may provide prognostic information for DCIS recurrence, but validation in additional cohorts is required.

Exome sequencing identifies rare deleterious mutations in DNA repair genes FANCC and BLM as potential breast cancer susceptibility alleles.

Despite intensive efforts using linkage and candidate gene approaches, the genetic etiology for the majority of families with a multi-generational breast cancer predisposition is unknown. In this study, we used whole-exome sequencing of thirty-three individuals from 15 breast cancer families to identify potential predisposing genes. Our analysis identified families with heterozygous, deleterious mutations in the DNA repair genes FANCC and BLM, which are responsible for the autosomal recessive disorders Fanconi Anemia and Bloom syndrome. In total, screening of all exons in these genes in 438 breast cancer families identified three with truncating mutations in FANCC and two with truncating mutations in BLM. Additional screening of FANCC mutation hotspot exons identified one pathogenic mutation among an additional 957 breast cancer families. Importantly, none of the deleterious mutations were identified among 464 healthy controls and are not reported in the 1,000 Genomes data. Given the rarity of Fanconi Anemia and Bloom syndrome disorders among Caucasian populations, the finding of multiple deleterious mutations in these critical DNA repair genes among high-risk breast cancer families is intriguing and suggestive of a predisposing role. Our data demonstrate the utility of intra-family exome-sequencing approaches to uncover cancer predisposition genes, but highlight the major challenge of definitively validating candidates where the incidence of sporadic disease is high, germline mutations are not fully penetrant, and individual predisposition genes may only account for a tiny proportion of breast cancer families.

Analysis of RAD51C germline mutations in high-risk breast and ovarian cancer families and ovarian cancer patients.

There is strong evidence that overtly inactivating mutations in RAD51C predispose to hereditary breast and ovarian cancer but the prevalence of such mutations, and whether they are associated with a particular clinical phenotype, remains unclear. Resolving these questions has important implications for the implementation of RAD51C into routine clinical genetic testing. Consequently, we have performed a large RAD51C mutation screen of hereditary breast and ovarian cancer families, and the first study of unselected patients diagnosed with ovarian cancer. Our data confirm a consistent but low frequency (2/335 families) of inactivating RAD51C mutations among families with a history of both breast and ovarian cancer and an absence of mutations among breast cancer only families (0/1,053 families). Our data also provide support for the designation of the missense variant p.Gly264Ser as a moderate penetrance allele.

Analysis of KLLN as a high-penetrance breast cancer predisposition gene.

KLLN is a p53 target gene with DNA binding function and represents a highly plausible candidate breast cancer predisposition gene. We screened for predisposing variants in 860 high-risk breast cancer families using high resolution melt analysis. A germline c.339_340delAG variant predicted to cause premature termination of the protein after 57 alternative amino acid residues was identified in 3/860 families who tested negative for BRCA1 and BRCA2 mutations and in 1/84 sporadic breast cancer cases. However, the variant was also detected in 2/182 families with known BRCA1 or BRCA2 mutations and in 2/464 non-cancer controls. Furthermore, loss of the mutant allele was detected in 2/2 breast tumors. Our data suggest that pathogenic mutations in KLLN are rare in breast cancer families and the c.339_340delAG variant does not represent a high-penetrance breast cancer risk allele.

Analysis of the mitogen-activated protein kinase kinase 4 (MAP2K4) tumor suppressor gene in ovarian cancer.

BACKGROUND: MAP2K4 is a putative tumor and metastasis suppressor gene frequently found to be deleted in various cancer types. We aimed to conduct a comprehensive analysis of this gene to assess its involvement in ovarian cancer. METHODS: We screened for mutations in MAP2K4 using High Resolution Melt analysis of 149 primary ovarian tumors and methylation at the promoter using Methylation-Specific Single-Stranded Conformation Polymorphism analysis of 39 tumors. We also considered the clinical impact of changes in MAP2K4 using publicly available expression and copy number array data. Finally, we used siRNA to measure the effect of reducing MAP2K4 expression in cell lines. RESULTS: In addition to 4 previously detected homozygous deletions, we identified a homozygous 16 bp truncating deletion and a heterozygous 4 bp deletion, each in one ovarian tumor. No promoter methylation was detected. The frequency of MAP2K4 homozygous inactivation was 5.6% overall, and 9.8% in high-grade serous cases. Hemizygous deletion of MAP2K4 was observed in 38% of samples. There were significant correlations of copy number and expression in three microarray data sets. There was a significant correlation between MAP2K4 expression and overall survival in one expression array data set, but this was not confirmed in an independent set. Treatment of JAM and HOSE6.3 cell lines with MAP2K4 siRNA showed some reduction in proliferation. CONCLUSIONS: MAP2K4 is targeted by genetic inactivation in ovarian cancer and restricted to high grade serous and endometrioid carcinomas in our cohort.

Frequent somatic mutations of GATA3 in non-BRCA1/BRCA2 familial breast tumors, but not in BRCA1-, BRCA2- or sporadic breast tumors.

Heterozygous somatic mutations of the transcription factor, GATA-3, have recently been reported in approximately 5% breast of tumors unselected for family history. We sequenced the GATA-3 gene in 55 breast tumors from women with familial breast cancer, and found seven heterozygous somatic mutations, all in non-BRCA1/2 cases in which the frequency was 22%. In contrast, we found mutations of GATA-3 in only 4% of 81 sporadic tumors analysed. It is possible that GATA3 mutations occur earlier in the evolution of BRCAx tumors, compared to BRCA1, BRCA2 or sporadic tumors, and are therefore easier to detect by direct sequencing in the presence of some stromal contamination.

Improved next-generation sequencing pre-capture library yields and sequencing parameters using on-bead PCR.

Tumor DNA sequencing results can have important clinical implications. However, its use is often limited by low DNA input, owing to small tumor biopsy size. To help overcome this limitation we have developed a simple improvement to a commonly used next-generation sequencing (NGS) capture-based library preparation method using formalin-fixed paraffin-embedded-derived tumor DNA. By using on-bead PCR for pre-capture library generation we show that library yields are dramatically increased, resulting in decreased sample failure rates. Improved yields allowed for a reduction in PCR cycles, which translated into improved sequencing parameters without affecting variant calling. This methodology should be applicable to any NGS system in which input DNA is a limiting factor.

Genetic analysis of cancer-implicated MicroRNA in ovarian cancer.

PURPOSE: There is accumulating evidence that microRNAs may function like classic tumor suppressor genes but little is known about their mechanism of inactivation in cancer cells. We investigated whether somatic mutations are a common mechanism of inactivation of microRNA genes in ovarian cancer. EXPERIMENTAL DESIGN: Ten cancer-implicated microRNA genes were analyzed for somatic mutations in 90 ovarian epithelial cancers and matching normal DNA. High-resolution melt analysis and bidirectional sequencing was used to detect sequence variations. RESULTS: High-resolution melt analysis and direct sequencing did not identify any somatic mutations but did reveal numerous novel and previously reported germ line base substitutions, deletions, and insertions surrounding the mature microRNA sequences. The majority of variants were detected in the same proportion of non-cancer control individuals suggesting that they do not represent ovarian cancer-predisposing alleles. CONCLUSION: The absence of somatic mutations in any of the 10 cancer-implicated microRNAs in our large cohort of ovarian tumors suggests that this may be an uncommon mechanism of inactivation of microRNAs in ovarian cancer.

Profound MEK inhibitor response in a cutaneous melanoma harboring a GOLGA4-RAF1 fusion.

BRAF and CRAF are critical components of the MAPK signaling pathway which is activated in many cancer types. In approximately 1% of melanomas, BRAF or CRAF are activated through structural arrangements. We describe here a metastatic melanoma with a GOLGA4-RAF1 fusion and pathogenic variants in CTNNB1 and CDKN2A. Anti-CTLA4/anti-PD1 combination immunotherapy failed to control tumor progression. In the absence of other actionable variants the patient was administered MEK inhibitor therapy on the basis of its potential action against RAF1 fusions. This resulted in a profound and clinically significant response. We demonstrated that GOLGA4-RAF1 expression was associated with ERK activation, elevated expression of the RAS/RAF downstream co-effector ETV5, and a high Ki67 index. These findings provide a rationale for the dramatic response to targeted therapy. This study shows that thorough molecular characterization of treatment-resistant cancers can identify therapeutic targets and personalize management, leading to improved patient outcomes.

Breast cancer risk and the BRCA1 interacting protein CTIP.

Mutations in BRCA1 predispose to breast cancer. CTIP interacts with BRCA1 and so could also be associated with increased risk. We screened CTIP for germline mutations in 210 probands of breast cancer families including 129 families with no mutations in BRCA1 or BRCA2. No coding variants were detected in CTIP, therefore, it is unlikely to be involved in breast cancer risk.

High-resolution single nucleotide polymorphism array analysis of epithelial ovarian cancer reveals numerous microdeletions and amplifications.

PURPOSE: Genetic changes in sporadic ovarian cancer are relatively poorly characterized compared with other tumor types. We have evaluated the use of high-resolution whole genome arrays for the genetic profiling of epithelial ovarian cancer. EXPERIMENTAL DESIGN: We have evaluated 31 primary ovarian cancers and matched normal DNA for loss of heterozygosity and copy number alterations using 500 K single nucleotide polymorphism arrays. RESULTS: In addition to identifying the expected large-scale genomic copy number changes, >380 small regions of copy number gain or loss (<500 kb) were identified among the 31 tumors, including 33 regions of high-level gain (>5 copies) and 27 homozygous deletions. The existence of such a high frequency of small regions exhibiting copy number alterations had not been previously suspected because earlier genomic array platforms lacked comparable resolution. Interestingly, many of these regions harbor known cancer genes. For example, one tumor harbored a 350-kb high-level amplification centered on FGFR1 and three tumors showed regions of homozygous loss 109 to 216 kb in size involving the RB1 tumor suppressor gene only. CONCLUSIONS: These data suggest that novel cancer genes may be located within the other identified small regions of copy number alteration. Analysis of the number of copy number breakpoints and the distribution of the small regions of copy number change indicate high levels of structural chromosomal genetic instability in ovarian cancer.

SDH-deficient renal cell carcinoma associated with biallelic mutation in succinate dehydrogenase A: comprehensive genetic profiling and its relation to therapy response.

Succinate dehydrogenase (SDH)-deficient renal cell carcinoma (RCC) is a rare RCC subtype that is caused by biallelic mutation of one of the four subunits of the SDH complex (SDHA, B, C, and D) and results in inactivation of the SDH enzyme. Here we describe a case of genetically characterized SDH-deficient RCC caused by biallelic (germline plus somatic) SDHA mutations. SDHA pathogenic variants were detected using comprehensive genomic profiling and SDH absence was subsequently confirmed by immunohistochemistry. Very little is known regarding the genomic context of SDH-deficient RCC. Interestingly we found genomic amplifications commonly observed in RCC but there was an absence of additional variants in common cancer driver genes. Prior to genetic testing a PD-1 inhibitor treatment was administered. However, following the genetic results a succession of tyrosine kinase inhibitors were administered as targeted treatment options and we highlight how the genetic results provide a rationale for their effectiveness. We also describe how the genetic results benefited the patient by empowering him to adopt dietary and lifestyle changes in accordance with knowledge of the mechanisms of SDH-related tumorigenesis.

Genetic and epigenetic analysis of the TIMP-3 gene in ovarian cancer.

Chromosome 22q shows a high frequency of loss of heterozygosity (LOH) in ovarian cancers suggesting the existence of one or more important tumor suppressor genes (TSGs). The tissue inhibitor of metalloproteinase-3 (TIMP-3) is a plausible TSG candidate since it is often encompassed within these regions of LOH. TIMP-3 has not previously been investigated for somatic mutations or promoter hypermethylation in ovarian cancer. We analyzed 65 ovarian cancers for both somatic genetic mutations and TIMP-3 promoter hypermethylation. Screening of all coding exons of TIMP-3 did not reveal any somatic genetic mutations and only 1/65 showed TIMP-3 methylation. Our data indicate that inactivation of TIMP-3 by somatic mutation or promoter hypermethylation is rare in ovarian cancer.

Genetic and epigenetic analysis of the putative tumor suppressor km23 in primary ovarian, breast, and colorectal cancers.

PURPOSE: A very high frequency of somatic mutations in the transforming growth factor-beta signaling component km23 has been reported in a small series of ovarian cancers (8 of 19, 42%). Functional studies showed that some mutations disrupt km23 function, resulting in aberrant transforming growth factor-beta signaling and presumably enhanced tumorigenicity. If verified, this would elevate mutation of km23 as the single most frequent somatic event in ovarian cancer. EXPERIMENTAL DESIGN: We sought to verify the frequency of silencing of km23 among 104 primary ovarian cancers (49 serous, 18 mucinous, 29 endometrioid/clear cell, and 8 undifferentiated) as well as 72 breast and 61 colorectal cancers by undertaking both somatic mutation and promoter methylation analyses. All four exons of km23 were individually amplified from genomic DNA with primers complementary to surrounding intronic sequences and analyzed by single-stranded conformational polymorphism analysis. RESULTS: Two germ line polymorphisms were identified, but none of the 237 tumors analyzed harbored somatic km23 mutations. In addition, promoter methylation analysis showed that in all cases, the 5' CpG island was unmethylated. CONCLUSIONS: Our data suggest that silencing of km23, either through somatic genetic mutation or promoter hypermethylation, is rare in ovarian, breast, and colorectal cancers.

No association of the MDM2 SNP309 polymorphism with risk of breast or ovarian cancer.

A functional T to G germline polymorphism in the promoter region of MDM2 (SNP309) has been reported to profoundly accelerate tumor formation suggesting that it may also represent a powerful cancer predisposing allele. To investigate the role of SNP309 in cancer predisposition we undertook a case-control study of this polymorphism among 351 women diagnosed with breast cancer, 302 women diagnosed with ovarian and 258 female controls from a British population. The GG genotype was not associated with either breast cancer (OR 1.04, 95% CI 0.67-1.60) or ovarian cancer (OR 0.86, 95% CI 0.53-1.37). This study has found no evidence that the GG genotype of the MDM2 SNP309 polymorphism is associated with early onset or familial breast cancer or with ovarian cancer.

Mutation and expression analysis of LZTS1 in ovarian cancer.

LZTS1 has been shown to have tumour suppressor activities against prostate and breast cancer and is located within a region of frequent loss of heterozygosity (LOH) at 8p22 in ovarian cancer. We have analysed the expression of LZTS1 in ovarian cancer and found no evidence of loss of expression relative to normal ovarian surface epithelial cells. We have also analysed the coding region of the LZTS1 gene in 87 primary ovarian adenocarcinomas by DHPLC and detected a single silent somatic mutation. These data indicate that LZTS1 is not the target of LOH at 8p22 in ovarian cancer.

Mutation of the PIK3CA gene in ovarian and breast cancer.

Phosphatidylinositol 3'-kinases are lipid kinases with important roles in neoplasia. Recently, a very high frequency of somatic mutations in PIK3CA has been reported among a large series of colorectal cancers. However, the relevance of PIK3CA mutation in other cancer types remains unclear because of the limited number of tumors investigated. We have screened a total of 284 primary human tumors for mutations in all coding exons of PIK3CA using a combination of single stranded conformational polymorphism and denaturing high-performance liquid chromatography analysis. Among 70 primary breast cancers, 40% (28 of 70) harbored mutations in PIK3CA, making it the most common mutation described to date in this cancer type. Mutations were not associated with histologic subtype, estrogen receptor status, grade or presence of tumor in lymph nodes. Among the primary epithelial ovarian cancers only 11 of 167 (6.6%) contain somatic mutations, but there was a clear histologic subtype bias in their distribution. Only 2 of 88 (2.3%) of serous carcinomas had PIK3CA mutations compared with 8 of 40 (20.0%) endometrioid and clear cell cancers, which was highly significant (P = 0.001). In contrast, PIK3CA gene amplification (>7-fold) was common among all histologic subtypes (24.5%) and was inversely associated with the presence of mutations. Overall, PIK3CA mutation or gene amplification was detected in 30.5% of all ovarian cancers and 45% of the endometrioid and clear cell subtypes. Our study is the first direct evidence that PIK3CA is an oncogene in ovarian cancer and greatly extends recent findings in breast cancer.

Differential hypermethylation of SOCS genes in ovarian and breast carcinomas.

Suppressor of cytokine signaling (SOCS) proteins have emerged as critical attenuators of cytokine-mediated processes, suggesting a role in the suppression of tumorigenesis. In the ovary and mammary gland, cytokines such as prolactin and IL-6 are important regulators of growth and differentiation. We have investigated whether silencing or inactivation of SOCS genes occurs in ovarian and breast carcinomas. The SOCS1 and SOCS2 CpG islands were found to be hypermethylated in 23 and 14% of primary ovarian cancers, respectively, whereas only SOCS1 was methylated in breast cancers (9%). Methylation of these genes did not occur in normal tissues. No correlation was apparent between methylation and loss of heterozygosity, and no somatic mutations were found in a large panel of carcinomas. Aberrant methylation of these SOCS genes correlated with transcriptional silencing in ovarian and breast cancer cell lines, since expression was induced by the demethylating agent 5-azadeoxycytidine. SOCS3 was not hypermethylated in either cancer type. Consistent with this data, SOCS1 and SOCS2 but not SOCS3 suppressed the growth of ovarian and breast cancer cells. Hypermethylation and silencing of specific SOCS genes in the ovary, and to a lesser extent in breast, may augment cytokine responsiveness in these tissues, thereby contributing to oncogenesis.

EDD, the human orthologue of the hyperplastic discs tumour suppressor gene, is amplified and overexpressed in cancer.

EDD (E3 isolated by differential display), located at chromosome 8q22.3, is the human orthologue of the Drosophila melanogaster tumour suppressor gene 'hyperplastic discs' and encodes a HECT domain E3 ubiquitin protein-ligase. To investigate the possible involvement of EDD in human cancer, several cancers from diverse tissue sites were analysed for allelic gain or loss (allelic imbalance, AI) at the EDD locus using an EDD-specific microsatellite, CEDD, and other polymorphic microsatellites mapped in the vicinity of the 8q22.3 locus. Of 143 cancers studied, 38 had AI at CEDD (42% of 90 informative cases). In 14 of these cases, discrete regions of imbalance encompassing 8q22.3 were present, while the remainder had more extensive 8q aberrations. AI of CEDD was most frequent in ovarian cancer (22/47 informative cases, 47%), particularly in the serous subtype (16/22, 73%), but was rare in benign and borderline ovarian tumours. AI was also common in breast cancer (31%), hepatocellular carcinoma (46%), squamous cell carcinoma of the tongue (50%) and metastatic melanoma (18%). AI is likely to represent amplification of the EDD gene locus rather than loss of heterozygosity, as quantitative RT-PCR and immunohistochemistry showed that EDD mRNA and protein are frequently overexpressed in breast and ovarian cancers, while among breast cancer cell lines EDD overexpression and increased gene copy number were correlated. These results demonstrate that AI at the EDD locus is common in a diversity of carcinomas and that the EDD gene is frequently overexpressed in breast and ovarian cancer, implying a potential role in cancer progression.