Heterozygous germ line hCHK2 mutations in Li-Fraumeni syndrome.

The hCHK2 gene encodes the human homolog of the yeast Cds1 and Rad53 G2 checkpoint kinases, whose activation in response to DNA damage prevents cellular entry into mitosis. Here, it is shown that heterozygous germ line mutations in hCHK2 occur in Li-Fraumeni syndrome, a highly penetrant familial cancer phenotype usually associated with inherited mutations in the TP53 gene. These observations suggest that hCHK2 is a tumor suppressor gene conferring predisposition to sarcoma, breast cancer, and brain tumors, and they also provide a link between the central role of p53 inactivation in human cancer and the well-defined G2 checkpoint in yeast.

MELPREDICT: a logistic regression model to estimate CDKN2A carrier probability.

BACKGROUND: Heritable alterations in CDKN2A account for a subset of familial melanoma cases although no robust method exists to identify those at risk of being a mutation carrier. METHODS: We set out to construct a model for estimating CDKN2A mutation carrier probability using a cohort of 116 consecutive familial cutaneous melanoma patients evaluated at Massachusetts General Hospital Pigmented Lesion Center between April 2001 and September 2004. Germline CDKN2A and CDK4 status on the familial melanoma cases and clinical features associated with mutational status were then used to build a multiple logistic regression model to predict carrier probability and performance of model on external validation. RESULTS: From the 116 kindreds prone to melanoma in the Boston area, 13 CDKN2A mutation carriers were identified and 12 were subsequently used in the modeling. Proband age at diagnosis, number of proband primaries, and number of additional family primaries were most closely associated with germline mutations. The estimated probability of the proband being a mutation carrier based on the logistic regression model (MELPREDICT) is given by e(L)/(1 + e(L) where L = 1.99+[0.92x(no. of proband primaries)]+[0.74x(no. of additional family primaries)]-[2.11xln(age)]. The mean estimated probabilities for subjects in the Boston dataset were 55.4% and 5.1% for the mutation carriers and non-carriers respectively. In a receiver operator characteristic analysis, the area under the curve was 0.881 (95% confidence interval 0.739 to 1.000) for the Boston model set (n = 116) and 0.803 (0.729 to 0.877) for an external Toronto hereditary melanoma cohort (n = 143). CONCLUSIONS: These results represent the first-iteration logistic regression model to approximate CDKN2A carrier probability. Validation of this model with an external dataset revealed relatively robust performance.

Loss of imprinting of insulin-like growth factor-II (IGF2) gene in distinguishing specific biologic subtypes of Wilms tumor.

BACKGROUND: Loss of imprinting (LOI) of the insulin-like growth factor-II (IGF2) gene, an epigenetic alteration associated with expression of the normally silent maternal allele, was observed first in Wilms tumor. Although LOI has subsequently been detected in most adult tumors, the biologic role of LOI in cancer remains obscure. We analyzed the imprinting status of Wilms tumors with respect to pathologic subtype, stage, and patient's age at diagnosis and examined the expression of genes potentially affected by LOI. METHODS: Of 60 Wilms tumors examined, 25 were informative for an ApaI polymorphism in the IGF2 gene, allowing analysis of allele-specific gene expression, and could be classified by pathologic subtype. Gene expression was measured quantitatively by real-time polymerase chain reaction, and pathologic analysis was blinded for genetic status. All statistical tests were two-sided. RESULTS: We observed LOI of IGF2 in nine (90%) of 10 Wilms tumors classified as having a pathologic subtype associated with a later stage of renal development and in only one (6.7%) of 15 Wilms tumors with a pathologic subtype associated with an earlier stage of renal development (P< .001). LOI was associated with a 2.2-fold increase (95% confidence interval [CI] = 1.6-fold to 3.1-fold) in IGF2 expression (P< .001). Children whose Wilms tumors displayed LOI of IGF2 were statistically significantly older at diagnosis (median = 65 months; interquartile range [IQR] = 47-83 months) than children whose tumors displayed normal imprinting (median = 24 months; IQR = 13-35 months; P< .001). CONCLUSIONS: These data demonstrate a clear relationship between LOI and altered expression of IGF2 in Wilms tumors and provide a molecular basis for understanding the divergent pathogenesis of this cancer. Analysis of LOI could provide a valuable molecular tool for the classification of Wilms tumor.

Multiple regions of allelic loss from chromosome arm 6q in malignant mesothelioma.

Our previous cytogenetic studies of malignant mesotheliomas (MMs) revealed losses from 6q15-21 in approximately 40% of cases, suggestive of recurrent loss of function of a putative tumor suppressor gene(s) located in this chromosome region. To more precisely define the critical region of molecular genetic loss within 6q, we have constructed a high-resolution deletion map of this chromosome arm in 46 MMs. We analyzed 32 microsatellite markers to detect loss of heterozygosity in tumor DNAs. Allelic losses from 6q were observed in a high percentage (61%) of cases. Partial deletions of 6q were identified in 11 cases, and these were used to define four nonoverlapping regions of chromosomal loss: a region involving 6q14-21 (approximately 9 cM; 7 of 11 cases with partial deletions), a region within 6q16.3-21 (approximately 8 cM; 9 cases), a region within 6q21-23.2 (approximately 10 cM; 8 cases), and a distal region located at 6q25 (approximately 13 cM; 9 cases). Most cases exhibited losses from more than one of these regions. We conclude from these data that genomic losses involving 6q in MM are more frequent than previously recognized cytogenetically and that the deletions fall into four discrete locations, suggesting the existence of multiple tumor suppressor loci in 6q that may contribute to the pathogenesis of this malignancy.

Isolation of MOAT-B, a widely expressed multidrug resistance-associated protein/canalicular multispecific organic anion transporter-related transporter.

Multidrug resistance-associated protein (MRP) and canalicular multispecific organic anion transporter (cMOAT) are closely related mammalian ATP-binding cassette transporters that export organic anions from cells. Transfection studies have established that MRP confers resistance to natural product cytotoxic agents, and recent evidence suggests the possibility that cMOAT may contribute to cytotoxic drug resistance as well. Based upon the potential importance of these transporters in clinical drug resistance and their important physiological roles in the export of the amphiphilic products of phase I and phase II metabolism, we sought to identify other MRP-related transporters. Using a degenerate PCR approach, we isolated a cDNA that encodes a novel ATP-binding cassette transporter, which we designated MOAT-B. The MOAT-B gene was mapped using fluorescence in situ hybridization to chromosome band 13q32. Comparison of the MOAT-B predicted protein with other transporters revealed that it is most closely related to MRP, cMOAT, and the yeast organic anion transporter YCF1. Although MOAT-B is closely related to these transporters, it is distinguished by the absence of a approximately 200 amino acid NH2-terminal hydrophobic extension that is present in MRP and cMOAT and which is predicted to encode several transmembrane spanning segments. In addition, the MOAT-B tissue distribution is distinct from MRP and cMOAT. In contrast to MRP, which is widely expressed in tissues, including liver, and cMOAT, the expression of which is largely restricted to liver, the MOAT-B transcript is widely expressed, with particularly high levels in prostate, but is barely detectable in liver. These data indicate that MOAT-B is a ubiquitously expressed transporter that is closely related to MRP and cMOAT and raise the possibility that it may be an organic anion pump relevant to cellular detoxification.

Common nonsense mutations in RAD52.

RAD51, RAD52, and RAD54 encode proteins that are critical to the repair of double-strand DNA breaks by homologous recombination. The physical interactions among the products of RAD51, BRCA1, and BRCA2 have suggested that the BRCA1 and BRCA2 breast cancer susceptibility genes may function, at least in part, in this DNA damage repair pathway. Given the observation that different genes within a common functional pathway may be targeted by mutations in human cancers, we analyzed RAD51, RAD52, and RAD54 for the presence of germ-line mutations in 100 cases with early-onset breast cancer and for somatic mutations in 15 human breast cancer cell lines. Two premature stop codons, Ser346ter and Tyr415ter, were identified in germ-line RAD52 alleles from 5% of early-onset breast cancer cases. Together, these two heterozygous mutations were also found in 8% of a healthy control population, indicating that they do not confer an increased risk for breast cancer. A rare germ-line missense mutation was identified in RAD54, whereas no sequence variants were found in RAD51. None of the three RAD genes demonstrated somatic mutations in breast cancer cell lines. We conclude that, despite their potential functional association with the BRCA gene products, RAD51, RAD52, and RAD54 are not themselves targeted by mutations in human breast cancer. The presence of common nonsense mutations in RAD52 within the population may have significance for other conditions associated with potential alterations in DNA damage repair pathways.

Comparative genomic hybridization and loss of heterozygosity analyses identify a common region of deletion at 15q11.1-15 in human malignant mesothelioma.

Comparative genomic hybridization analysis was performed to identify chromosomal imbalances in 24 human malignant mesothelioma (MM) cell lines derived from untreated primary tumors. Chromosomal losses accounted for the majority of genomic imbalances. The most frequent underrepresented segments were 22q (58%) and 15q1.1-21 (54%); other recurrent sites of chromosomal loss included 1p12-22 (42%), 13q12-14 (42%), 14q24-qter (42%), 6q25-qter (38%), and 9p21 (38%). The most commonly overrepresented segment was 5p (54%). DNA sequence amplification at 3p12-13 was observed in two cases. Whereas some of the regions of copy number decreases (i.e., segments in 1p, 6q, 9p, and 22q) have previously been shown to be common sites of karyotypic and allelic loss in MM, our comparative genomic hybridization analyses identified a new recurrent site of chromosomal loss within 15q in this malignancy. To more precisely map the region of 15q deletion, loss of heterozygosity analyses were performed with a panel of polymorphic microsatellite markers distributed along 15q, which defined a minimal region of chromosomal loss at 15q11.1-15. The identification of frequent losses of a discrete segment in 15q suggests that this region harbors a putative tumor suppressor gene whose loss/inactivation may contribute to the pathogenesis of many MMs.

Amplification of the ATP-binding cassette 2 transporter gene is functionally linked with enhanced efflux of estramustine in ovarian carcinoma cells.

An estramustine-resistant human ovarian carcinoma cell line, SKEM, was generated to explore resistance mechanisms associated with this agent. Cytogenetic analysis revealed that SKEM cells have a homogeneously staining region (hsr) at chromosome 9q34. Microdissection of the hsr, followed by fluorescence in situ hybridization to SKEM and normal metaphase spreads, confirmed that the amplified region was derived from sequences from 9q34. In situ hybridization with a probe specific for ABC2, a gene located at 9q34 that encodes an ATP-binding cassette 2 (ABC2) transporter, indicated that this gene is amplified approximately 6-fold in the estramustine-resistant cells. Southern analysis confirmed that ABC2 was amplified in SKEM, and Northern analysis indicated that the ABC2 transcript was overexpressed approximately 5-fold. The ABC1 gene located at 9q22-31 was not amplified in the resistant cells, and mRNA levels of several other ABC transporter genes were unaltered. Consistent with the concept that increased ABC2 expression contributes to the resistant phenotype, we observed that the rate of efflux of dansylated estramustine was increased in SKEM compared with control cells. In addition, antisense treatment directed toward ABC2 mRNA sensitized the resistant cells to estramustine. Together, these results suggest that amplification and overexpression of ABC2 contributes to estramustine resistance and provides the first indication of a potential cellular function for this product.

Phenol sulfotransferases: hormonal regulation, polymorphism, and age of onset of breast cancer.

In recent years, significant effort has been made to identify genes that influence breast cancer risk. Because the high-penetrance breast cancer susceptibility genes BRCA1 and 2 play a role only in a small fraction of breast cancer cases, understanding the genetic risk of the majority of breast cancers will require the identification and analysis of several lower penetrance genes. The estrogen-signaling pathway plays a crucial role in the pathophysiology of breast cancer; therefore, polymorphism in genes involved in this pathway is likely to influence breast cancer risk. Our detailed analysis of gene expression profiles of estrogen- and 4-OH-tamoxifen-treated ZR75-1 breast cancer cells identified members of the sulfotransferase 1A (SULT1A) phenol sulfotransferase family as downstream targets of tamoxifen. On the basis of the induction of SULT1A by 4-OH-tamoxifen and the known inherited variability in SULT1A enzymatic activity, we hypothesized that polymorphism in sulfotransferase genes might influence the risk of breast cancer. Using an RFLP that distinguishes an arginine to histidine change in exon 7 of the SULT1A1 gene, we characterized SULT1A1 genotypes in relation to breast cancer risk. An analysis of 444 breast cancer patients and 227 controls revealed no effect of SULT1A1 genotype on the risk of breast cancer (P = 0.69); however, it did appear to influence the age of onset among early-onset affected patients (P = 0.04). Moreover, individuals with the higher activity SULT1A1*1 allele were more likely to have other tumors in addition to breast cancer (P = 0.004; odds ratio, 3.02; 95% confidence interval, 1.32, 8.09). The large number of environmental mutagens and carcinogens activated by sulfotransferases and the high frequency of the SULT1A1*1 allele in human populations warrants additional studies to address the role of SULT genes in human cancer.

p16 alterations and deletion mapping of 9p21-p22 in malignant mesothelioma.

To determine whether p16 is altered in human malignant mesothelioma (MM), molecular analysis of multiple 9p loci was performed on 40 cell lines and 23 primary tumors from 42 MM patients. We identified homozygous deletions of p16 in 34 (85%) cell lines and a point mutation in 1 line. Down-regulation of p16 was observed in 4 of the remaining cell lines, 1 of which displayed a DNA rearrangement of p16. Homozygous deletions of p16 were identified in 5 of 23 (22%) primary tumors; no mutations or rearrangements were found in these specimens. Four cell lines displayed a single homozygous deletion proximal to or distal to p16; 4 others had 2 nonoverlapping deletions, one involving p16 and the other involving a region proximal to this locus. These data indicate that alterations of p16 are a common occurrence in MM cell lines and, to a lesser extent, in primary tumors. Furthermore, deletions of 9p21-p22 outside of the p16 locus may reflect the involvement of other putative tumor suppressor genes that could also contribute to the pathogenesis of some MMs.

Destabilization of CHK2 by a missense mutation associated with Li-Fraumeni Syndrome.

Li Fraumeni Syndrome (LFS) is a multicancer phenotype, most commonly associated with germ-line mutations in TP53. In a kindred with LFS without an inherited TP53 mutation, we have previously reported a truncating mutation (1100delC) in CHK2, encoding a kinase that phosphorylates p53 on Ser(20). Here, we describe a CHK2 missense mutation (R145W) in another LFS family. This mutation destabilizes the encoded protein, reducing its half-life from >120 min to 30 min. This effect is abrogated by treatment of cells with a proteosome inhibitor, suggesting that CHK2(R145W) is targeted through this degradation pathway. Both 1100delC and R145W germ-line mutations in CHK2 are associated with loss of the wild-type allele in the corresponding tumor specimens, and neither tumor harbors a somatic TP53 mutation. Our observations support the functional significance of CHK2 mutations in rare cases of LFS and suggest that such mutations may substitute for inactivation of TP53.

The retinoblastoma-related gene, RB2, maps to human chromosome 16q12 and rat chromosome 19.

A retinoblastoma-related human gene, referred to as RB2, has been cloned based on sequence homology of the E1A-binding domain of the retinoblastoma gene. Structural homology with the retinoblastoma gene suggests a possible function of RB2 as a tumor suppressor gene. In this study, we have mapped this gene to human chromosome 16q12.2 and rat chromosome 19, using fluorescence in situ hybridization and somatic hybrid cell analysis, respectively. Based on known syntenic relationships among human, rat and mouse, the data suggest that the mouse homolog resides on chromosome 8. Deletions of chromosome 16q have been found in several human neoplasias (including breast, ovarian, hepatic, and prostatic cancers) which is in support of an involvement of RB2 in human cancer as a tumor suppressor gene.

Early loss of heterozygosity on 17q in ovarian cancer. The Abe Ovarian Cancer Genetics Group.

We have studied 146 ovarian tumours (94 carcinomas, 22 tumours of low malignant potential and 30 benign tumours) for evidence of allele loss on chromosome 17p and 17q sufficient to imply the proximity of a tumour-suppressor gene. We have examined two polymorphic loci (YNZ22.2 and BHP53) on 17p13 and one on chromosome 17q (17q23-qter). Loss of heterozygosity (LOH) was detected in 34/63 (54%) informative malignant tumours at YNZ22.2 and 22/47 (47%) at BHP53; on 17q, 45/64 (70%) had LOH. Allele loss was detected in a small number of benign and borderline tumours. There was a statistically significant difference between the patterns of allele loss in serous and endometrioid groups of tumours, and allele loss occurred with significantly greater frequency on 17q than on 17p. Comparison of all malignant tumours presenting with either localized (FIGO stage I/II) or widespread (FIGO stage III/IV) disease showed that, particularly on 17q, allele loss increases in the more advanced stages. The p53 tumour-suppressor gene is implicated in ovarian carcinogenesis, and our findings suggest that an important tumour-suppressor gene may be located in the region 17q23-qter. Loss of function in this gene may be responsible for the frequently observed rapid progression of serous-type adenocarcinomas to an advanced stage.

Heterozygous germline ATM mutations do not contribute to radiation-associated malignancies after Hodgkin's disease.

PURPOSE: The successful treatment of Hodgkin's disease has been associated with an increased incidence of secondary malignancies. To investigate whether genetic factors contribute to the development of secondary tumors, we collected family cancer histories and performed mutational analysis of the ataxia-telangiectasia (AT) gene, ATM, in a cohort of Hodgkin's disease survivors with secondary malignancies. ATM was chosen for evaluation because of the increased radiosensitivity of cells derived from AT patients and obligate heterozygotes and the epidemiologic observation that AT carriers are at increased risk for radiation-induced breast cancer. PATIENTS AND METHODS: Fifty-two patients who developed one or more neoplasms after treatment for Hodgkin's disease participated in this study. Personal and family histories of cancer were obtained through patient interviews and review of medical records. ATM mutational analysis was performed using a yeast-based protein truncation assay. RESULTS: Seventy-six secondary neoplasms were observed in this cohort of 52 Hodgkin's disease survivors, with 18 patients (35%) developing more than one secondary neoplasm. Positive family histories of cancer were present in 11 (21%) of 52 patients, compared with three (4%) of 68 Hodgkin's disease patients in a comparison cohort who did not develop secondary neoplasms (P =.008; Fisher's exact test). No germline ATM mutations were identified, resulting in an estimated AT carrier frequency in this population of 0% (90% confidence interval, 0% to 4%). CONCLUSION: Analysis of the number of tumors per individual and the family history of cancer in our cohort suggests that genetic factors may contribute to development of secondary neoplasms in a subset of Hodgkin's disease survivors. Mutational analysis, however, does not support a significant role for heterozygous truncating ATM mutations. Future studies evaluating other genes involved in DNA damage response pathways are warranted.

Human annexin 31 genetic mapping and origin.

The cDNA encoding novel human annexin 31 was utilized for chromosomal mapping, structural comparison, and phylogenetic analysis to clarify its genetic relationship to other annexins. The ANX31 gene locus was mapped by fluorescence in situ hybridization to human chromosome 1q21, remote from ten other paralogous human annexins on different chromosomes but near the epidermal differentiation gene complex, the S100A gene cluster and a breast-cancer translocation region. Protein homology testing and characterization of incompletely processed expressed sequence tags identified annexin 2 as the closest extant homologue. Maximum likelihood analysis confirmed its most recent common ancestor with vertebrate annexin 2 and validated its classification, in order of discovery, as annexin 31. This subfamily was formed approx. 500-600millionyears ago, subsequent to the gene duplication that produced annexin 1. It has diverged relatively rapidly and extensively, and specifically in the well-conserved, functionally critical type II calcium-binding sites.

Advances in the analysis of chromosome alterations in human lung carcinomas.

A review of chromosomal analyses of human lung carcinomas is presented. Karyotypic studies have revealed multiple cytogenetic changes in most small cell lung carcinomas (SCLCs) and non-small cell lung carcinomas (NSCLCs). In SCLCs, losses from 3p, 5q, 13q, and 17p predominate; double minutes associated with amplification of members of the MYC oncogene family may be common late in disease. In NSCLCs, deletions of 3p, 9p, and 17p, +7, i(5)(p10), and i(8)(q10) often are reported. The recurrent deletions encompass sites of tumor suppressor genes commonly inactivated in lung carcinomas, such as CDKN2 (9p21), RB1 (13q14), and TP53 (17p13). Despite technical advances in cell culture, the rate of successful karyotypic analysis of lung carcinomas has remained low. Alternative molecular cytogenetic methods to assess chromosome changes in lung cancer, particularly comparative genomic hybridization (CGH) analysis, are discussed. Initial CGH studies confirm the existence of many of the karyotypic imbalances identified earlier in lung cancer and have revealed several recurrent abnormalities, such as 10q- in SCLC, that had not been recognized previously. The further application of such molecular cytogenetic approaches should enable investigators to define more precisely the spectrum and clinical implications of chromosome alterations in lung cancer.

Laryngotracheoesophageal cleft: the anterior approach.

Laryngotracheoesophageal cleft was reported by Richter in 1792 after he examined an infant at autopsy and found a common lumen of esophagus and laryngotrachea. The next case was not noted until 1949 by Finlay. A recent review shows 40 cases in the world literature to date. Successful surgical approaches to some of these problems have basically employed a lateral pharyngotomy technique. In the case presented herein, the exact pathology was obscured by a severe tetralogy of Fallot and recurrent pneumonitis from a suspected high H-type tracheoesophageal fistula. The length of this cleft prompted an anterior approach with laryngo-fissure, cricoidotomy, and division of four tracheal rings for a complete and successful repair. Thorough endoscopy of all infants suspected of any laryngeal anomaly would yield an earlier diagnosis and opportunity for reconstruction of the cleft.

Immunisation coverage in Christchurch in a birth cohort. Christchurch Immunisation Coordination Committee.

AIM: To ascertain the immunisation status of a birth cohort of infants at 8 months of age. METHODS: Data on all children born in Christchurch in June, July and August 1995 were matched with immunisation benefit claim information for the 6-week, 3-month and 5-month immunisation events. Those not listed as receiving all three immunisations by the age of 8 months were traced to ascertain their immunisation status. RESULTS: Immunisation status was established for 933 (93.1%) of the cohort of 1002 infants. Of these, 95% had received all three immunisations that were scheduled up to 5 months of age. This represents a coverage of at least 88.2% of the entire cohort. It was probable that many of those not traced would also have been fully immunised. Using a conservative estimate of coverage among this group, we calculated that 93% of the entire cohort had been fully immunised by the age of 8 months. CONCLUSION: There was a high immunisation coverage rate in Christchurch. This is in contrast to a commonly held view that children in New Zealand have a poor immunisation record. There was no evidence of a significant anti-immunisation stance among Christchurch parents. Immunisation data relevant to specific, defined populations can identify individual children and groups of children who may require assistance beyond general practitioner recall to achieve appropriate immunisation. The tracking process also allowed for immunisation reminders to be personally delivered and included some facilitation for achieving further immunisation. We suggest that a pilot programme, using a centralised register based on birth data, feeding into existing immunisation delivery and recall systems, and using tracking methods similar to those used in our study, should be the next step in addressing immunisation coverage surveillance and immunisation coordination in Christchurch.

MMR immunisation coverage in Christchurch. Christchurch Immunisation Coordination Committee.

AIM: To measure measles-mumps-rubella (MMR) immunisation status of a birth cohort at 18 months of age. METHOD: All children born in Christchurch in June, July and August 1995 who were alive at 18 months of age (n = 999), were matched with MMR immunisation benefit claims. Those not listed were traced. RESULT: The final immunisation coverage rate was estimated at 85%. CONCLUSION: An 85% coverage rate at 18 months fell well short of the Immunisation 2000 target of 95% coverage by two years of age.