Preferences for return of incidental findings from genome sequencing among women diagnosed with breast cancer at a young age.

While experts have made recommendations, information is needed regarding what genome sequencing results patients would want returned. We investigated what results women diagnosed with breast cancer at a young age would want returned and why. We conducted 60 semi-structured, in-person individual interviews with women diagnosed with breast cancer at age 40 or younger. We examined interest in six types of incidental findings and reasons for interest or disinterest in each type. Two coders independently coded interview transcripts; analysis was conducted using NVivo 10. Most participants were at least somewhat interested in all six result types, but strongest interest was in actionable results (i.e. variants affecting risk of a preventable or treatable disease and treatment response). Reasons for interest varied between different result types. Some participants were not interested or ambivalent about results not seen as currently actionable. Participants wanted to be able to choose what results are returned. Participants distinguished between types of individual genome sequencing results, with different reasons for wanting different types of information. The findings suggest that a focus on actionable results can be a common ground for all stakeholders in developing a policy for returning individual genome sequencing results.

The mutational spectrum of FOXA2 in endometrioid endometrial cancer points to a tumor suppressor role.

BACKGROUND: Forkhead box protein A2 (FOXA2) plays an important in development, cellular metabolism and tumorigenesis. The Cancer Genome Atlas (TCGA) identified a modest frequency of FOXA2 mutations in endometrioid endometrial cancers (EEC). The current study sought to determine the relationship between FOXA2 mutation and clinicopathologic features in EEC and FOXA2 expression. METHODS: Polymerase chain reaction (PCR) amplification and sequencing were used to identify mutations in 542 EEC. Western blot, quantitative reverse transcriptase PCR (qRT-PCR) and immunohistochemistry (IHC) were used to assess expression. Methylation analysis was performed using combined bisulfite restriction analysis (COBRA) and sequencing. Chi-squared, Fisher's exact, Student's t- and log-rank tests were performed. RESULTS: Fifty-one mutations were identified in 49 tumors (9.4% mutation rate). The majority of mutations were novel, loss of function (LOF) (78.4%) mutations, and most disrupted the DNA-binding domain (58.8%). Six recurrent mutations were identified. Only two tumors had two mutations and there was no evidence for FOXA2 allelic loss. Mutation status was associated with tumor grade and not associated with survival outcomes. Methylation of the FOXA2 promoter region was highly variable. Most tumors expressed FOXA2 at both the mRNA and protein level. In those tumors with mutations, the majority of cases expressed both alleles. CONCLUSION: FOXA2 is frequently mutated in EEC. The pattern of FOXA2 mutations and expression in tumors suggests complex regulation and a haploinsufficient or dominant-negative tumor suppressor function. In vitro studies may shed light on how mutations in FOXA2 affect FOXA2 pioneer and/or transcription factor functions in EEC.

The T-box transcription factor gene TBX22 is mutated in X-linked cleft palate and ankyloglossia.

Formation of the secondary palate is a complex step during craniofacial development. Disturbance of the events affecting palatogenesis results in a failure of the palate to close. As a consequence of deformity, an affected child will have problems with feeding, speech, hearing, dentition and psychological development. Cleft palate occurs frequently, affecting approximately 1 in 1,500 births; it is usually considered a sporadic occurrence resulting from an interaction between genetic and environmental factors. Although several susceptibility loci have been implicated, attempts to link genetic variation to functional effects have met with little success. Cleft palate with ankyloglossia (CPX; MIM 303400) is inherited as a semidominant X-linked disorder previously described in several large families of different ethnic origins and has been the subject of several studies that localized the causative gene to Xq21 (refs. 10-13). Here we show that CPX is caused by mutations in the gene encoding the recently described T-box transcription factor TBX22 (ref. 14). Members of the T-box gene family are known to play essential roles in early vertebrate development, especially in mesoderm specification. We demonstrate that TBX22 is a major gene determinant crucial to human palatogenesis. The spectrum of nonsense, splice-site, frameshift and missense mutations we have identified in this study indicates that the cleft phenotype results from a complete loss of TBX22 function.

A pseudoautosomal gene in man.

The X/Y homologous gene MIC2 was shown to exchange between the sex chromosomes, thus demonstrating that it is a pseudoautosomal gene in man. MIC2 recombines with the sex-determining gene(s) TDF at a frequency of 2 to 3 percent. It is the most proximal pseudoautosomal locus thus far described and as such is an important marker for use in studies directed towards the isolation of TDF.

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.

Inherited cancers associated with the RET proto-oncogene.

Mutations in the RET proto-oncogene have been identified in the constitutional DNA of patients with the inherited disorders multiple endocrine neoplasia type 2A and 2B and familial medullary thyroid carcinoma. This review focuses on the discoveries over the past year that pointed to RET as a candidate gene, and on the nature and spectrum of what appear to be dominant mutations associated with an inherited predisposition to tumor development.

RET gene and its implications for cancer.

The RET proto-oncogene encodes a protein receptor tyrosine kinase. RET mutations are associated with the dominantly inherited cancer syndromes multiple endocrine neoplasia (MEN) types 2A and 2B and familial medullary thyroid carcinoma (FMTC). In MEN 2A, MEN 2B, and FMTC, direct detection of RET mutations can be used to identify disease allele carriers prior to the development of clinically evident neoplasms. RET mutations are also associated with sporadic thyroid carcinomas. The effects of RET mutation on protein function have been investigated both in vivo and in vitro, and the study of RET has served to provide insights into the mechanisms of tumorigenesis in general.

Allelotype analysis of esophageal adenocarcinomas: evidence for the involvement of sequences on the long arm of chromosome 4.

The incidence of esophageal adenocarcinoma has increased dramatically over the past 20 years. The causes for this change in incidence and the genetic defects that underlie tumorigenesis are unknown. We performed loss of heterozygosity (LOH) studies in esophageal adenocarcinomas in an effort to map the location of tumor suppressor genes involved in the initiation or progression of this cancer. A genome-wide search for LOH was undertaken using microsatellite repeat polymorphisms and a panel of 27 tumor and matched normal DNAs. This is the first report of an allelotype analysis of esophageal adenocarcinomas. We observed frequent loss of sequences on the short arm of chromosome 17 in the region of the TP53 gene. We also identified a region on 4q lost in more than half of the tumors investigated. The high rate of LOH for 4q sequences speaks to the involvement of an as yet unidentified tumor suppressor gene in esophageal adenocarcinoma tumorigenesis.

Allelic loss of sequences from the long arm of chromosome 10 and replication errors in endometrial cancers.

Thirty-seven endometrial cancers were subjected to an allelotype analysis in an attempt to identify chromosomal regions that are lost in a significant portion of tumors and to identify tumors characterized by replication errors. Thirty-nine highly polymorphic microsatellite markers representing all chromosomal arms, excluding the X and the short arms of the acrocentrics, were examined. An average of 20 informative cases were evaluated for each marker. Genetic alterations were detected in 30 of the 37 tumors. Replication errors were identified in 8 tumor specimens. Loss of heterozygosity was observed for loci on all chromosomes examined with the exception of chromosomes 4 and 20. The two most frequent sites of loss were at the marker loci examined on 10q (40%) and 17p (29%). Six additional simple sequence repeat markers from 10q were genotyped in an effort to refine the region of 10q loss. The chromosome 10 markers used in these studies were physically mapped with the use of a panel of somatic hybrids that retain defined portions of chromosome 10. The observed patterns of loss of sequences on 10q suggest a role for a tumor suppressor gene in the 10q23-26 region in the development or progression of endometrial cancers.

Mlh1 deficiency accelerates myeloid leukemogenesis in neurofibromatosis 1 (Nf1) heterozygous mice.

Defects in DNA mismatch repair (MMR) have been implicated in the genesis of a diverse set of human cancers. Recent studies have suggested that one of the targets of MMR is the neurofibromatosis 1 (NF1) gene. To evaluate the contribution of Mlh1 MMR deficiency to Nf1 tumorigenesis, Mlh1-/-;Nf1+/- mice were generated. All Mlh1-/-;Nf1+/- mice (n=21) were dead by 260 days compared to none of the Nf1+/- mice. In all, 50% of the Mlh1-/-;Nf1+/- mice were dead at 150 days compared to 252 days for Mlh1-/- mice. Nine of the Mlh1-/-;Nf1+/- mice were found to harbor intrathoracic NOS2-immunoreactive myeloid leukemias similar to the hematopoietic malignancies observed in older Nf1+/- mice. As expected, significant microsatellite instability was observed in six of six tumors and neurofibromin expression was lost in all tumors analysed. These results suggest that MMR deficiency can accelerate myeloid leukemogenesis in Nf1+/- mice, presumably by inactivating Nf1 gene expression.

Novel germline RET proto-oncogene mutations associated with medullary thyroid carcinoma (MTC): mutation analysis in Japanese patients with MTC.

Germ-like and somatic mutations in the RET proto-oncogene are associated with inherited and sporadic medullary thyroid carcinoma (MTC). The majority of patients with multiple endocrine neoplasia type 2A (MEN2A) and familial medullary thyroid carcinoma (FMTC) carry germ-line point mutations that result in the substitution of one of five cysteine residues. We investigated exons 10, 11, 13, 14 and 16 of the RET proto-oncogene in 33 unrelated Japanese patients with MTC. Eleven of the 33 cases (33%) were found to have germ-line mutations. Three previously unreported mutations in exon 10 and 11 were identified: one in codon 620, (TGC-->GGC), resulting in a cysteine to glycine substitution, and two in codon 630, (TGC-->TCC) and (TGC-->TAC), resulting in cysteine to serine and cysteine to tyrosine changes, respectively. The new mutations were present in the germ-line DNA of four unrelated patients for whom a family history of MTC had not been documented. Because the new RET alleles described here involve cysteine residues in a region of protein previously associated with FMTC and MEN2A, it is very likely that they represent mutations that predispose to the development of MTC.

Confirmation that chromosome 18q allelic loss in colon cancer is a prognostic indicator.

PURPOSE: Recent studies suggest that allelic loss of sequences from the long arm of chromosome 18 may be a useful prognostic indicator in colorectal cancer. The aim of the present study was to confirm whether 18q loss of heterozygosity (LOH) is of prognostic value in patients with colon cancer. METHODS: Genomic DNA was prepared from archival tumor and corresponding normal tissue specimens from 151 patients who had undergone potentially curative surgery for adenocarcinoma of the colon. Polymerase chain reaction (PCR) was used to assess allelic loss of five chromosome 18q microsatellite markers in the tumors. The relationship between allelic loss and disease-free and disease-specific survival was investigated. RESULTS: LOH was detected in 67 of 126 tumors. Chromosome 18q allelic loss was a negative prognostic indicator of both disease-free (relative risk [RR], 1.65; P = .01) and disease-specific survival (RR, 2.0; P = .003). 18q loss was also associated with significantly reduced disease-free and disease-specific survival in patients with stage II (P = .05 and P = .0156) and III (P = .038 and P = .032) disease. CONCLUSION: Chromosome 18q allelic loss is a prognostic marker in colorectal cancers. Chromosome 18 LOH studies may be useful in identifying patients with stage II disease who are at high risk for recurrence, and as such might benefit from adjuvant chemotherapy.

Hereditary motor and sensory neuropathy IIB: clinical and electrodiagnostic characteristics.

Axonal forms of autosomal dominant hereditary motor and sensory neuropathies (HMSNs) represent a heterogeneous group of disorders based on genetic linkage studies. We recently identified one large family with axonal HMSN exhibiting linkage to chromosome 3q, designated HMSN IIB, and report here the clinical and electrodiagnostic features. We clinically evaluated 10 individuals with HMSN IIB and performed detailed electrophysiologic studies in 5 of these patients. HMSN IIB is characterized clinically by the presence of distal symmetric motor weakness and prominent sensory loss affecting the lower extremities with preserved ankle reflexes. Symptomatic age at onset is in the second or early third decade of life. Six patients with HMSN IIB had distal trophic ulcerations in the feet, leading to eventual toe amputations in four cases. Electrodiagnostic studies confirmed a distal sensorimotor axonopathy involving the lower limbs with normal motor conduction velocities. Tibial H-reflexes were preserved in HMSN IIB, despite the uniform loss of sural nerve potentials. Overall, individuals with HMSN IIB demonstrated a consistent clinical and electrodiagnostic phenotype that had no overlap with genetically unaffected family members. The identification of specific clinical and electrodiagnostic features of HMSN IIB may prove useful in the diagnosis and differentiation between various subtypes of HMSN II.

Mxi1 tumor suppressor gene is not mutated in primary pancreatic adenocarcinoma.

Amplification of the c-myc oncogene occurs in a variety of solid tumors, including pancreatic adenocarcinomas. The MXI1 gene, located at 10q24-q25, may serve to negatively regulate c-myc oncogene activity, and potentially has tumor suppressor function. As such, altered MXI1 function might contribute to tumorigenesis. We examined 40 primary human pancreatic adenocarcinomas for MXI1 mutations. Single-strand conformation variant analysis and direct sequencing of the variants revealed a MXI1 polymorphism in 1 of 40 tumors. No MXI1 mutations were identified. Southern blot analyses did not reveal any gross rearrangements of MXI1. These results suggest that MXI1 is unlikely to play a role in human pancreatic adenocarcinoma tumorigenesis.

"Cold" single-strand conformational variants for mutation analysis of the RET protooncogene.

BACKGROUND: RET protooncogene mutation analysis is a routinely performed predictive DNA test in kindreds affected by multiple endocrine neoplasia (MEN) types 2A and 2B and familial medullary thyroid carcinoma (FMTC), and is a valuable diagnostic tool in newly diagnosed cases of medullary thyroid carcinoma (MTC). METHODS: We tested the suitability of the recently introduced "cold" single-strand conformational variant (SSCV) technique, which promises rapid, simple, nonradioactive detection of sequence variants in the identification of germline and somatic RET mutations. A total of 11 different mutations in exon 10 (codons 609, 611, 618, and 620) and 6 mutations in exon 11 (codon 634) were studied. RESULTS: Conditions were optimized so that conformational variants were demonstrated for all mutations examined in a single setting for exons 10 and 11. A novel six base pair (bp) inframe deletion between cysteines 630 and 634 was detected in a sporadic MTC. This adds to the evidence that not only cysteine deletions and substitutions but also changes in the spacing between cysteine residues have a pathogenic effect. CONCLUSIONS: Our results indicate that the cold SSCV method offers the advantages of simplicity, time savings, and nonradioactive detection for screening for RET sequence variants in hereditary and sporadic MTCs.

Linkage analysis of multiple endocrine neoplasia type 2A (MEN-2A) and three DNA markers on chromosome 20: evidence against synteny.

Members of four families in which multiple endocrine neoplasia type 2A (MEN-2A) is segregating were genotyped for three chromosome #20 DNA markers. Close linkage is excluded for the genetic locus of MEN-2A (MEN2A) with each marker locus tested. Using multipoint analysis, the MEN2A locus was excluded from a region 32.5 cM in length encompassing D20S5 and D20S6, two loci that previously had been mapped to the chromosome 20p12.2 region containing the putative deletion associated with MEN-2. In total, we excluded approximately 90.5 cM of chromosome #20 as a possible location of the MEN2A locus. The data from this study do not support increased recombination on the short arms of chromosome #20 and were insufficient to support heterogeneity of MEN-2A as possible explanations for failure to detect linkage between MEN2A and D20S5 or D20S6.

Analysis of MSH3 in endometrial cancers with defective DNA mismatch repair.

OBJECTIVE: To clarify the origin of defective mismatch repair (MMR) in sporadic endometrial cancers with microsatellite instability (MSI), a thorough mutation analysis was performed on the human mismatch repair gene MSH3. METHODS: Twenty-eight MSI-positive endometrial cancers were investigated for mutations in the human mismatch repair gene MSH3 using single-strand conformation variant (SSCV) analysis of all 24 exons. All variants were sequenced. Loss of heterozygosity was investigated at all MSH3 polymorphisms discovered. A subset of tumors were investigated for methylation of the 5' promoter region of MSH3 using Southern blot hybridization. RESULTS: An identical single-base deletion (delta A) predicted to result in a truncated proteins was discovered in six tumors (21.4%). This deletion occurs in a string of eight consecutive adenosine residues (A8). Because simple repeat sequences are unstable in cells with defective MMR, the observed mutation may be an effect, rather than a cause, of MSI. Evidence of inactivation of the second MSH3 allele in tumors with the delta A mutation would strongly support a causal role for these MSH3 mutations. However, there was no evidence of a second mutation, loss of sequences, or methylation of the promoter region in any of the tumors with the delta A mutation. CONCLUSION: Although the delta A mutation is a frequent event in sporadic MSI-positive endometrial cancers, it may not be causally associated with defective DNA MMR.

16q loss of heterozygosity and microsatellite instability in Wilms' tumor.

BACKGROUND/PURPOSE: Wilms' tumor is the most common renal malignancy of childhood. Loss of heterozygosity (LOH) at 16q is seen in about 17% of cases and has been associated with a poor prognosis. To more precisely define the pattern of 16q deletion exhibited by Wilms' tumor, the authors performed a detailed LOH analysis of 96 specimens using polymorphic microsatellite repeat markers. The authors also evaluated the neoplasms for the presence of microsatellite instability (MSI). METHODS: A total of 96 DNA samples were studied using polymerase chain reaction-based LOH analyses amplifying polymorphic microsatellite repeat markers. Screening for MSI using 2 additional genetic markers also was carried out. RESULTS: The authors found 16q LOH in 14 of the specimens evaluated. Comprehensive analysis of these LOH-positive specimens showed a region of loss spanning 16p11.2-q22.1 and a separate distal region of LOH at 16q23.2-24.2. The distal region of deletion is very small, estimated to be approximately 2.4 megabases. In addition to the observed LOH, 2 specimens were found to consistently exhibit MSI, which has not been reported previously in Wilms' tumor. CONCLUSIONS: The smallest consensus region of deletion in our analysis of Wilms' tumor 16q LOH measures 2.4 megabases at 16q23.2-q24.2. Additionally, MSI was present in a subset of tumor specimens suggesting that defects in DNA mismatch repair may contribute to the pathogenesis of Wilms' tumor.