RNA expression of the WT1 gene in Wilms' tumors in relation to histology.

BACKGROUND: On the basis of accumulating data, the recently isolated WT1 gene is a Wilms' tumor gene and a putative tumor suppressor gene. These findings include expression in developing fetal kidney, intragenic deletions in tumors, and germline mutations in predisposed individuals. Wilms' tumors, which exhibit a broad range of differentiation, are composed of three cell types: blastema, epithelium, and stroma. PURPOSE: The purpose of this study was to investigate the relationship between WT1 gene expression and histologic composition in Wilms' tumors in an effort to elucidate how the WT1 gene functions in proliferation of these histologic components. METHODS: We used Northern blot hybridization to study WT1 gene expression by messenger RNA (mRNA) accumulation in 20 tumors of varying histology and in adjacent uninvolved kidney tissue. In two patients, tumors were also compared before and after therapy. RESULTS: Tumors that were predominantly blastemal expressed high amounts of WT1 mRNA, whereas predominantly stromal tumors expressed either low or undetectable amounts. Blastemal tumors that were predominantly poorly differentiated expressed WT1 mRNA at higher levels than those that were more well differentiated. Although we expected that a putative tumor suppressor gene like WT1 would generally be expressed at lower levels in tumor than in normal kidney, this was true only in predominantly stromal cells. One of the two patients studied before and after therapy had a dramatic response to therapy accompanied by a decline in WT1 gene expression and disappearance of blastemal and epithelial elements. CONCLUSIONS: A correlation was observed between WT1 gene expression and histology of the tumors. Level of expression was inversely related to the degree of differentiation in blastemal tumors and in the patient with a dramatic response to therapy. These results, in conjunction with the observation that WT1 mRNA is abundant in normal fetal kidney, suggest that WT1 gene expression is related to kidney development, especially in differentiation of blastemal components. IMPLICATIONS: Further studies to search for alterations of the WT1 gene in tumors and to identify regulatory factors in gene expression will increase understanding of the role of this gene in normal development and tumorigenesis.

BRCA2 is required for ionizing radiation-induced assembly of Rad51 complex in vivo.

Mutations in BRCA1 and BRCA2 account for the majority of familial breast cancers. Cells with mutated BRCA1 or BRCA2 are hypersensitive to ionizing radiation (IR) and exhibit defective DNA repair. Both BRCA1 and BRCA2 have been reported to bind Rad51, a protein essential for homologous recombination and the recombinational repair of DNA double-strand breaks. In normal cells, a redistribution of Rad51 protein, manifested as formation of Rad51 nuclear foci, is seen upon IR treatment. Here we demonstrate that IR-induced Rad51 foci formation is aberrant in BRCA2- but not BRCA1-deficient tumor cells. In Capan-1 cells, which do not express functional BRCA2, there was little Rad51 foci formation in response to a wide range of radiation dosages. Moreover, forced expression of a fusion protein containing green fluorescent protein and the first Rad51-binding BRC repeat of BRCA2 in cells with wild-type BRCA2 rendered them hypersensitive to IR and cisplatin and compromised IR-induced Rad51 foci formation. In HCC1937 cells, which harbor mutation of BRCA1, IR-induced Rad51 foci were readily detected. This study suggests a requirement of BRCA2 protein for the IR-induced assembly of Rad51 complex in vivo.

Gamma-radiation-induced G2 delay, apoptosis, and p53 response as potential susceptibility markers for lung cancer.

Gamma-radiation results in cell cycle arrest and apoptosis in a wide variety of cells. Cell cycle arrest provides time for the cell to repair damaged DNA before entering the next phase of the cycle. If the damage is severe and cannot be repaired, the cells undergo apoptosis. However, if the damaged cells continue to grow without repair or apoptosis, then carcinogenic transformation may occur. We hypothesized that individuals with inherited disruption in cell cycle control and/or apoptosis and/or DNA repair may be susceptible to lung cancer development. The cells from susceptible individuals would have a shorter G2 period and less apoptosis compared with cells from normal individuals upon exposure to gamma-radiation. To test this hypothesis, the following methods were used: (a) fluorescence-activated cell sorting method was used to measure apoptosis and G2 cell cycle delay; (b) the ELISA method was used to measure p53 protein expression levels in these cell lines; and (c) gamma-radiation-induced chromatid breaks were counted as a marker for DNA damage or repair. Next, gamma-radiation-induced G2 delay and apoptosis were tested in three lymphoblastoid cell lines to determine the dose response effect and optimal time points of gamma-radiation. Finally, these assays were tested in lymphoblastoid cell lines from 30 lung cancer patients and 22 healthy controls. We found a dose-response relationship for gamma-radiation-induced G2 delay and apoptosis. The optimal time points to detect differential G2 delay and apoptotic index were 10 h and 48 h after gamma-radiation, respectively. The mean G2 delay was 22.5% +/- 10.5% for the control cell lines and 14.71% +/- 8.8% for case cell lines (P < 0.01). The mean apoptotic index was 20.4% +/- 11.7% for the controls and 14.3% +/- 7.8% for the cases (P < 0.05). The controls had a significantly higher p53 response ratio and fewer chromatid breaks than the cases. We also found that a p53 increasing ratio was strongly related to cell cycle G2 delay (gamma = 0.413; P = 0.002) and chromatid breaks (gamma =0.384; P = 0.028). Therefore, we concluded that gamma-radiation-induced G2 delay, apoptosis, p53 increasing ratio, and chromatid breaks might potentially be used as susceptibility markers for lung cancer risk. A large epidemiology study is in progress to confirm these findings.

Benzo[a]pyrene diol epoxide-induced 3p21.3 aberrations and genetic predisposition to lung cancer.

3p deletion, a common chromosome defect in lung cancer, occurs more frequently in the lung tumor tissues of smoking patients than it does in those of nonsmoking patients. This pilot study evaluated whether 3p aberrations induced by benzo[a]pyrene diol epoxide (BPDE), the metabolic product of benzo[a]pyrene, a constituent of tobacco smoke, were more common in the peripheral blood lymphocytes of 40 lung cancer patients than they were in those of 54 matched controls. Our hypothesis was that 3p sensitivity to BPDE reflects the susceptibility of a specific locus to damage from carcinogens in tobacco smoke. BPDE-induced chromosome 3p21.3 aberrations were significantly more frequent in cases (34.1 per 1000) than they were in controls (22.1 per 1000; P < 0.0001). However, no such difference was observed for 6q27, a control locus. Using the median value in the controls (20 per 1000) as a cutoff point to classify BPDE-induced sensitivity at 3p21.3 and after adjustment by age, sex, ethnicity, and smoking status, 3p BPDE sensitivity was associated with an elevated risk of 14.1 (95% confidence interval: 3.5, 56.2) for lung cancer. There was also a dose-response relationship between the degree of BPDE sensitivity at 3p21.3 and increased risk for lung cancer. Therefore, 3p may be a molecular target for BPDE damage in lung cancer cases.

Analysis of the FHIT gene and FRA3B region in sporadic breast cancer, preneoplastic lesions, and familial breast cancer probands.

The FHIT gene, which spans the FRA3B fragile site at chromosome 3p14.2, is a candidate tumor suppressor gene in breast and other cancers. We investigated FHIT and FRA3B for loss of heterozygosity (LOH); homozygous deletions; abnormal transcripts; and acquired/germ-line point mutations in breast cancer cell lines (n = 32), breast epithelial and stromal cell cultures (n = 18), microdissected invasive (n = 16) and ductal in situ carcinomas (n = 6), and their accompanying normal and abnormal epithelial foci (n = 14). LOH at 3p14.2, especially at FHIT intragenic marker D3S1300, was found in 6 of 16 microdissected invasive tumors and 3 of 6 ductal in situ carcinomas. In accompanying preneoplastic foci, LOH occurred in two of eight intraductal hyperplasias but not in histologically normal ductal epithelium (n = 6). Three of 32 (9%) breast cancer cell lines demonstrated homozygous deletions of FHIT exon 4 (two cases) and exon 5 (one case), which correlated with exon 4-deleted transcripts and loss of the cDNA transcript containing the coding exons 5-9, respectively. Normal mammary cultures and 31 of 32 tumor cell lines (97%) expressed wild-type coding transcripts as well as a minor exon 8-deleted message. Single-strand conformation polymorphism analysis of the coding exons in the 32 tumor and 18 normal breast cell lines and their sequencing revealed four silent polymorphisms and a germ-line histidine triad point mutation (651 G-->T) in a tumor arising in a 70-year-old woman. This mutation was also present in one of her two thus far unaffected daughters. Analysis of additional DNAs from 280 probands of high-risk breast cancer families for other FHIT exon 8 mutations detected an intronic point mutation 13 bases upstream of exon 8. Thus, we have demonstrated relatively early abnormalities of the FHIT/FRA3B region in breast cancer and discovered two rare FHIT germ-line mutations. The expression of a transcript containing the coding exons in nearly all cell lines, including those with germ-line mutations, suggests the possibility that another gene in the FRA3B region may be involved in the pathogenesis of breast cancer.

Characterization of a breast cancer cell line derived from a germ-line BRCA1 mutation carrier.

A tumor cell line, HCC1937, was established from a primary breast carcinoma from a 24-year-old patient with a germ-line BRCA1 mutation. A corresponding B-lymphoblastoid cell line was established from the patient's peripheral blood lymphocytes. BRCA1 analysis revealed that the tumor cell line is homozygous for the BRCA1 5382insC mutation, whereas the patient's lymphocyte DNA is heterozygous for the same mutation, as are at least two other family members' lymphocyte DNA. The tumor cell line is marked by multiple additional genetic changes including a high degree of aneuploidy, an acquired mutation of TP53 with wild-type allele loss, an acquired homozygous deletion of the PTEN gene, and loss of heterozygosity at multiple loci known to be involved in the pathogenesis of breast cancer. Comparison of the primary tumor with the cell line revealed the same BRCA1 mutation and an identical pattern of allele loss at multiple loci, indicating that the cell line had maintained many of the properties of the original tumor. This breast tumor-derived cell line may provide a useful model system for the study of familial breast cancer pathogenesis and for elucidating BRCA1 function and localization.

Analysis of possible WT1 RNA processing in primary Wilms tumors.

WT1 RNA processing abnormalities have been suggested to play a role in the development of Wilms tumor by reports of editing at codon 280 in the rat WT1 transcript (codon 281 in humans) and aberrant splicing of exon 2 in WT1 transcripts from Wilms tumor xenograft cell lines. Both events result in a functionally changed WT1 protein and are potential mechanisms of altering normal protein function in the absence of WT1 DNA mutations. To determine whether either of these RNA processing events occurs in primary Wilms tumors, we analysed WT1 mRNA from 15 primary tumors. There was no evidence of WT1 RNA editing at codon 281, and only one primary tumor displayed aberrant splicing of exon 2. Sequence and Southern analysis of DNA from this tumor did not reveal any alteration in or around exon 2. These results suggest that neither RNA editing at codon 281 nor aberrant exon 2 splicing is a frequent mechanism of WT1 alteration during tumorigenesis.

Searching for microsatellite mutations in coding regions in lung, breast, ovarian and colorectal cancers.

RepX represents a new informatics approach to probe the UniGene database for potentially polymorphic repeat sequences in the open reading frame (ORF) of genes, 56% of which were found to be actually polymorphic. We now have performed mutational analysis of 17 such sites in genes not found to be polymorphic (<0.03 frequency) in a large panel of human cancer genomic DNAs derived from 31 lung, 21 breast, seven ovarian, 21 (13 microsatellite instability (MSI)+ and eight MSI-) colorectal cancer cell lines. In the lung, breast and ovarian tumor DNAs we found no mutations (<0.03-0.04 rate of tumor associated open reading frame mutations) in these sequences. By contrast, 18 MSI+ colorectal cancers (13 cancer cell lines and five primary tumors) with mismatch repair defects exhibited six mutations in three of the 17 genes (SREBP-2, TAN-1, GR6) (P<0.000003 compared to all other cancers tested). We conclude that coding region microsatellite alterations are rare in lung, breast, ovarian carcinomas and MSI (-) colorectal cancers, but are relatively frequent in MSI (+) colorectal cancers with mismatch repair deficits.

Programs for adult survivors of childhood cancer.

PURPOSE: The potential for late effects of treatment necessitates long-term monitoring of adult survivors of childhood cancer. The purpose of this study was to determine how institutions follow up young adult survivors of pediatric malignancy. Specifically, we were interested in the types of health care providers who follow up these patients, how the follow-up is administered, and what barriers to follow-up have been encountered. METHODS: A 16-item questionnaire was mailed to the 219 members of the Children's Cancer Group (CCG) and the Pediatric Oncology Group (POG). The survey consisted of four categories of questions that asked for information regarding the existence of a program to follow up young adults, the setting of the program, routine activities of the program, and commonly encountered barriers to care. RESULTS: One hundred eighty-two members returned the survey (83% response rate). Fifty-three percent of the respondents have a long-term follow-up clinic at their institution. Whereas 44% have a mechanism for following up adult survivors, only 15% of the programs have established a formal database for adults. Nearly all the programs (93%) use a pediatric oncologist. Although an adult oncologist assists in 13% of the programs, primary care physicians are uncommonly (8%) involved. CONCLUSION: Few programs focus on the long-term health care needs of adult survivors of childhood cancer. The majority of existing programs are in pediatric institutions, without significant input from adult-oriented, generalist health care providers.

Childhood acute lymphoblastic leukemia with both t(1;19) and t(9;22).

The chromosomal rearrangements t(1;19)(q23;p13.3) and t(9;22) (q34;q11.2) are independent abnormalities commonly observed in the blast cells of children with acute lymphoblastic leukemia (ALL). We report three children whose leukemic cells contained both translocations at diagnosis. The patients, two males aged 3 and 8 years and a female aged 14 years, all presented with central nervous system involvement. One patient exhibited a pre-B leukemic phenotype (cytoplasmic immunoglobulin, cIg, positive), while two had an early pre-B phenotype (cIg negative). All three patients received radiotherapy and multiagent chemotherapy which included an epipodophyllotoxin in two patients. Two patients suffered relapses of ALL, in both cases with disappearance of t(1;19)-containing clones but persistence of t(9;22). The two patients who received an epipodophyllotoxin as part of their chemotherapeutic regimen both developed secondary myeloid leukemia with entirely new cytogenetic findings, including abnormalities of chromosome band 11q23. These patients are the first to be described with this unusual combination of cytogenetic abnormalities.

Renal cell carcinoma with translocation (X;1). Further evidence for a cytogenetically defined subtype.

A renal cell carcinoma from a 15-year-old male had a 49,Y,t(X;1)(p11.2;q21), +der(X)t(X;1) (p11.2;q21), +5, -16, +17, +18 karyotype. This is the third report of a translocation involving a breakpoint at Xp11.2 in a renal cell carcinoma in a child. A total of nine cases of renal cell carcinoma involving Xp11, including this case, have been reported. Of the eight cases for which there are genetics reports, all are male. Patients with renal cell carcinoma with abnormalities at Xp11 appear to be younger than renal cell carcinoma patients overall.

Cytogenetics of a renal cell carcinoma in a 17-month-old child. Evidence for Xp11.2 as a recurring breakpoint.

A renal cell carcinoma from a 17-month-old boy with a history of maternal hydrocarbon exposure was found to have a 46,Y,t(X;17)(p11.2;q25) karyotype. Although this translocation has not previously been reported, other translocations involving Xp11.2 have been described, suggesting that this may represent a non-random breakpoint involved in the pathogenesis of childhood renal cell carcinoma. Both chromosomes 3 in the tumor were normal by both karyotype and RFLP analysis.

Ultrastructural, immunocytochemical, and cytogenetic characterization of a large congenital fibrosarcoma.

Cytogenetic, immunocytochemical, and ultrastructural studies were performed on a large congenital fibrosarcoma. To our knowledge, this is the first report of a congenital fibrosarcoma characterized by all of these techniques. The findings of immunochemical and electron microscopic studies supported the suggestion that the tumor is of fibroblastic origin. The karyotype of the tumor (48,XY,+11,+20) is similar to that observed in previously reported cases and substantiates the hypothesis that congenital fibrosarcoma is characterized by nonrandom gain of chromosomes.

A gnathodynanometer as an objective means of pain assessment following wisdom tooth removal.

There appears to be no information concerning changes in bite strength following wisdom tooth removal. A gnathodynanometer was constructed, calibrated and then tested on 20 normal subjects. Sixty five patients scheduled for third molar surgery were assessed by this device immediately before operation and on the seventh post-operative day. Post-operatively, they were asked to complete a McGill pain questionnaire (MPQ) and indicate their present pain on a numerical rating scale (NRS). Bite strength was reduced in all cases. The side which was most painful tended to have the larger reduction. The reduction in bite strength was found to have a highly significant correlation with the numerical rating scale and a significant correlation with the sensory descriptors of the McGill pain questionnaire. It would appear, therefore, that the gnathodynanometer holds promise as a research tool in oral surgery.

Providing primary care for long-term survivors of childhood acute lymphoblastic leukemia.

Primary care physicians will be providing longitudinal health care for long-term survivors of childhood acute lymphoblastic leukemia (ALL) with increasing frequency. Late effects (sequelae) secondary to treatment with radiation or chemotherapeutic agents are frequent and may be serious. Depending on treatment exposures, this at-risk population may experience life-threatening late effects, such as cirrhosis secondary to hepatitis C or late-onset anthracycline-induced cardiomyopathy, or life-changing late effects, such as cognitive dysfunction. Many survivors of childhood ALL will develop problems such as obesity and osteopenia at a young age, which will significantly affect their risk for serious health outcomes as they grow older. The goal of our review is to assist primary care physicians in providing longitudinal health care for long-term survivors of childhood ALL. We also highlight areas needing further investigation, including the prevalence of different late effects, determination of risk factors associated with a late effect, a better understanding of the potential impact of late effects on the premature development of common adult health problems, and the value and timing of different tests for screening asymptomatic survivors.