Chromosome band 9p21 is frequently altered in malignant peripheral nerve sheath tumors: studies of CDKN2A and other genes of the pRB pathway.

Malignant peripheral nerve sheath tumors (MPNSTs) are frequently associated with the disease neurofibromatosis type 1. Only few recurrent cytogenetic changes have been reported, including rearrangements of the short arm of chromosome 9. By fluorescence in situ hybridization with a centromere 9 probe, and by allelic imbalance studies with seven 9p21-23 markers in nine familial and three sporadic MPNSTs, we found interstitial deletions that supported CDKN2A as a possible target gene. Nine MPNSTs showed aberrations of CDKN2A by Southern blot analyses, and in four of these, expression of CDKN2A could not be detected by Northern blot analysis. No mutations of CDKN2A were identified by sequencing of the coding region, and gene inactivation by promoter methylation was not found. In the 9p allelic imbalance studies, a novel allele was detected at one locus in one tumor. Analyses of additional markers (n = 8) excluded mismatch repair deficiency as an important mechanism in the genesis of these tumors. The tumors were analyzed further for alterations in other candidate cell cycle-associated genes. In total, 11/12 MPNSTs showed DNA changes in one or more of the genes CDKN2A, CDKN2B, RB1, CDK4, MDM2, and CCND2. The present study suggests that disruption of the pRB pathway is common in MPNST, and that dose reduction of CDKN2A is particularly frequent and contributes to MPNST development. Genes Chromosomes Cancer 26:151-160, 1999.

Cancer predictive value of cytogenetic markers used in occupational health surveillance programs: a report from an ongoing study by the European Study Group on Cytogenetic Biomarkers and Health.

The cytogenetic endpoints in peripheral blood lymphocytes: chromosomal aberrations (CA), sister chromatid exchange (SCE) and micronuclei (MN) are established biomarkers of exposure for mutagens or carcinogens in the work environment. However, it is not clear whether these biomarkers also may serve as biomarkers for genotoxic effects which will result in an enhanced cancer risk. In order to assess this problem, Nordic and Italian cohorts were established, and preliminary results from these two studies indicated a predictive value of CA frequency for cancer risk, whereas no such associations were observed for SCE or MN. A collaborative study between the Nordic and Italian research groups, will enable a more thorough evaluation of the cancer predictivity of the cytogenetic endpoints. We here report on the establishment of a joint data base comprising 5271 subjects, examined 1965-1988 for at least one cytogenetic biomarker. Totally, 3540 subjects had been examined for CA, 2702 for SCE and 1496 for MN. These cohorts have been followed-up with respect to subsequent cancer mortality or cancer incidence, and the expected values have been calculated from rates derived from the general populations in each country. Stratified cohort analyses will be performed with respect to the levels of the cytogenetic biomarkers. The importance of potential effect modifiers such as gender, age at test, and time since test, will be evaluated using Poisson regression models. The remaining two potential effect modifiers, occupational exposures and smoking, will be assessed in a case-referent study within the study base.

Chromosomal aberrations in lymphocytes predict human cancer: a report from the European Study Group on Cytogenetic Biomarkers and Health (ESCH).

Chromosomal aberrations (CAs), sister chromatid exchanges (SCEs), and micronuclei (MN) in peripheral blood lymphocytes have for decades been used as cytogenetic biomarkers to survey genotoxic risks in the work environment. The conceptual basis for this application has been the idea that increased cytogenetic damage reflects an enhanced cancer risk. Nordic and Italian cohorts have been established to evaluate this hypothesis, and analyses presented previously have shown a positive trend between CA frequency and increased cancer risk. We now report on a pooled analysis of updated data for 3541 subjects examined for CAs, 2703 for SCEs, and 1496 for MN. To standardize for interlaboratory variation, the results for the various cytogenetic end points were trichotomized on the basis of the absolute value distribution within each laboratory as "low" (1-33 percentile), "medium" (34-66 percentile), or "high" (67-100 percentile). In the Nordic cohort, there was an elevated standardized incidence ratio (SMR) for all cancer among subjects with high CA frequency [1.53; 95% confidence interval (CI), 1.13-2.05] but not for those with medium or low CA frequency. In the Italian cohort, a SMR in cancer of 2.01 (95% CI, 1.35-2.89) was obtained for those with a high CA frequency level, whereas the SMRs for those with medium or low did not noticeably differ from unity. Cox's proportional hazards models gave no evidence that the effect of CAs on total cancer incidence/mortality was modified by gender, age at test, or time since test. No association was seen between the SCEs or the MN frequencies and subsequent cancer incidence/mortality. The present study further supports our previous observation on the cancer predictivity of the CA biomarker, which seems to be independent of age at test, gender, and time since test. The risk patterns were similar within each national cohort. This result suggests that the frequency of CAs in peripheral blood lymphocytes is a relevant biomarker for cancer risk in humans, reflecting either early biological effects of genotoxic carcinogens or individual cancer susceptibility.

Allelic imbalance and cytogenetic deletion of 1p in colorectal adenomas: a target region identified between DIS199 and DIS234.

Both cytogenetic and molecular genetic analyses have shown that many colorectal adenomas carry an acquired deletion distally in the short arm of one chromosome 1, but the two methods have never been brought to bear on the same tumors. The major part of this study was the analysis of 53 previously short-term cultured and karyotyped colorectal adenomas for allelic imbalance at eight microsatellite loci in 1p. Allelic imbalances were detected in seven of the 12 adenomas that had cytogenetically visible abnormalities of chromosome 1, as well as in four adenomas that either had a normal karyotype (one case) or had clonal chromosome abnormalities that did not seem to involve chromosome 1 (three cases); i.e., 30% of the adenomas had abnormalities involving 1p by the combined approach. A minimal region of overlap seemed to map to between DIS199 and DIS234, suggesting that this is a relevant target region. This genomic area contains the human homologue of the tumor modifier gene Mom1 (1p35-36.1), which, in mice, modifies the number of intestinal tumors in multiple intestinal neoplasia (Min)-mutated animals. To evaluate whether the imbalances corresponded to interstitial deletions of 1p material, we performed fluorescence in situ hybridization with a pericentromeric probe (15 adenomas) and a telomeric probe (6 adenomas) on uncultured cells from the 16 adenomas with chromosome 1 abnormalities. Except for three adenomas that had already been shown by banding analysis to have a trisomic pattern, two centromere 1 signals were invariably found. In the cases hybridized with the 1p-telomeric probe, we found the same frequencies of telomeric and centromeric signals, in agreement with the interpretation that the deletions were interstitial. One of the 53 adenomas had genomic instability, seen as new alleles at five of eight microsatellite loci. A comparison of the genetic findings with clinicopathologic data indicated that adenomas in the rectum have 1p abnormalities more often than do adenomas of the sigmoid colon, and that adenomas with 1p changes are larger than adenomas without abnormalities of chromosome 1.

Cancer predictive value of cytogenetic markers used in occupational health surveillance programs.

It has not previously been clear whether cytogenetic biomarkers in healthy subjects will predict cancer. Earlier analyses of a Nordic and an Italian cohort indicated predictivity for chromosomal aberrations (CAS) but not for sister chromatid exchanges (SCES). A pooled analysis of the updated cohorts, forming a joint study base of 5271 subjects, will now be performed, allowing a more solid evaluation. The importance of potential effect modifiers, such as gender, age at testing, and time since testing, will be evaluated using Poisson regression models. Two other potential effect modifiers, occupational exposures and smoking, will be assessed in a case-referent study within the study base.

Chromosome analysis of peripheral lymphocytes from persons exposed to radioactive fallout in Norway from the Chernobyl accident.

Chromosome analysis of peripheral lymphocytes from two Norwegian populations (44 reindeer herding South samis from Røros and Snåsa, 12 sheep farmers from Valdres) exposed to fallout from the Chernobyl accident were made. The doses from caesium through the years 1987-1991 were calculated based on whole-body measurements 134Cs and 137Cs giving a total cumulative mean internal dose of 5.54 mSv for the total group of 56 persons. Chromosome aberrations were within the normal range when compared with historical controls with the exception of dicentrics (0.3% per cell, which is a 10-fold increase) and rings (0.07% per cell). A dose-dependent increase in dicentrics and rings based on caesium exposure was not observed.

Deletion of 1p loci and microsatellite instability in colorectal polyps.

Previous cytogenetic studies have indicated that a subset of large bowel adenomas have distal 1p deletions. We addressed this question by examining 70 sporadic polyps (63 adenomas, 5 hyperplastic polyps, and 2 polyps of undetermined histology) from 55 patients for alterations at eight loci on the short arm of chromosome 1 and found allelic imbalance (AI) or loss of one allele (LOH) in 14 (20%). The locus most frequently changed was MSI, which maps to 1p33-35. Fluorescence in situ hybridisation with centromeric and telomeric probes for chromosome 1, performed for 11 polyps, did not yield an abnormal number of signals, in accordance with the interpretation that the observed AI and LOH were the result of interstitial deletions in 1p. Whereas allelic imbalance at five other loci (mapping to 5q, 8p, 10p, 11p and 17q) was found less frequently, and then mainly in large (> 2 cm) tumours, the 1p alterations were equally distributed among small (< 1 cm) and large polyps. They were preferentially found in left-side tumours. Instability at microsatellite loci--the mutator phenotype--is demonstrated by shifts in the electrophoretic mobility of normal alleles. The mutator phenotype was first associated with hereditary nonpolyposis colorectal cancer but is also occasionally found in sporadic colorectal carcinomas; however, it is still uncertain when in the adenoma-carcinoma sequence in this type of genomic instability arises. We therefore looked for it at 12 dinucleotide repeat loci and found that seven tumours (six adenomas and one hyperplastic polyp) from seven patients had acquired new alleles not seen in the patients' corresponding normal DNA. Our results suggest that inactivation of a putative suppressor gene distally in chromosome arm 1p is an early event in colorectal tumourigenesis. They also show that microsatellite instability can be detected in large bowel polyps, indicating that this phenomenon, too, probably plays a pathogenic role for some colorectal tumours early in the adenoma-carcinoma sequence.

Alterations at chromosome 17 loci in peripheral nerve sheath tumors.

Little is known about the molecular genetic changes in malignant peripheral nerve sheath tumors (MPNST). Inactivation of the TP53 gene in 17p has been reported in a few tumors. The MPNST is one of the manifestations of neurofibromatosis 1 (NF1), suggesting that the NF1 gene in 17q might be important. We present a study of 15 neurofibromas and MPNST from nine individuals. Seven patients had NF1, and six of these developed MPNST. Genetic alterations at nine polymorphic loci on chromosome 17 were examined. Allelic imbalance was detected only in the malignant tumors from NF1 patients (4/6). Complete loss of heterozygosity of 17q loci was found in three of these tumors, all including loci within the NF1 gene. Two of the malignant tumors also showed deletions on 17p. No mutations were detected within exon 5-8 of the TP53 in any of the MPNST, and none of them were TP53 protein-positive using immunostaining with mono- and polyclonal antibodies against TP53. The numbers of chromosome 17 present in each tumor were evaluated by use of fluorescence in situ hybridization (FISH) on interphase nuclei with a centromere-specific probe. A deviation from the disomic status of chromosome 17 was observed in two of the MPNST from NF1 patients. These results support the hypothesis of inactivation of both NF1 gene alleles during development of MPNST in patients with NF1. In contrast to other reports, we did not find evidence for a homozygous mutated condition of the TP53 gene in the same tumors. Finally, FISH analysis was in accordance with the DNA analysis in the deduction of the numbers of chromosome 17 in these tumors.

Genetic alterations of chromosome 17 in human breast carcinoma studied by fluorescence in situ hybridization and molecular DNA techniques.

Fluorescence in situ hybridization (FISH) with a chromosome 17 centromere-specific probe was used together with Southern blot analyses and PCR amplification of a polymorphic segment to characterize 13 breast carcinomas. Cells with an abnormal number of chromosome 17 centromeres were found mixed with disomic cells in nine of the 13 tumors studied. Three of the four cases found to have a normal number of chromosome 17 centromeres had DNA alterations restricted to allelic imbalance of the two most distal markers on 17p; the fourth had no detectable alterations. In contrast, of the seven tumors found to have genetic alterations elsewhere on chromosome 17, all had more than one cell population when examined for numerical chromosome 17 alterations. The present data support the hypothesis that alterations at the distal short arm of chromosome 17 represent earlier events in the tumorigenic process than do the other chromosome 17 abnormalities observed. The results of molecular DNA studies interpreted as allelic imbalance of distal 17p markers in most cases probably reflect chromosomal rearrangements like loss of specific loci, rather than monosomies and large deletions. The report illustrates the usefulness of FISH when added to current molecular DNA techniques.

Cancer risk in humans predicted by increased levels of chromosomal aberrations in lymphocytes: Nordic study group on the health risk of chromosome damage.

Cytogenetic assays in peripheral blood lymphocytes (PBL) have been used extensively to survey the exposure of humans to genotoxic agents. The conceptual basis for this has been the hypothesis that the extent of genetic damage in PBL reflects critical events for carcinogenic processes in target tissues. Until now, no follow-up studies have been performed to assess the predictive value of these methods for subsequent cancer risk. In an ongoing Nordic cohort study of cancer incidence, 3182 subjects were examined between 1970 and 1988 for chromosomal aberrations (CA), sister chromatid exchange or micronuclei in PBL. In order to standardize for the interlaboratory variation, the results were trichotomized for each laboratory into three strata: low (1-33 percentile), medium (34-66 percentile), or high (67-100 percentile). In this second follow-up, a total of 85 cancers were diagnosed during the observation period (1970-1991). There was no significant trend in the standardized incidence ratio with the frequencies of sister chromatid exchange or micronuclei, but the data for these parameters are still too limited to allow firm conclusions. There was a statistically significant linear trend (P = 0.0009) in CA strata with regard to subsequent cancer risk. The point estimates of the standardized incidence ratio in the three CA strata were 0.9, 0.7, and 2.1, respectively. Thus, an increased level of chromosome breakage appears to be a relevant biomarker of future cancer risk.

Genotyping of the CYP2D6 gene in Norwegian lung cancer patients and controls.

Genotyping of five CYP2D6 alleles (wt, A,B,D,E) has been performed in 218 lung cancer patients and 289 healthy controls. PCR-methods were used to determine the CYP2D6A and CYP2D6B alleles. Both patients and controls were of Caucasian Norwegian origin. In the lung cancer group all major histological types were represented. The frequency of the CYP2D6B allele which is associated with poor metabolizers (PM), was 0.235 in the lung cancer population and 0.200 in the control population. The frequencies of the CYP2D6A allele, another mutant allele associated with the PM-phenotype, were 0.007 and 0.013 in the lung cancer and control population respectively. RFLP analysis using the restriction enzyme Xba I to determine the D and E alleles, was also performed on 178 of the patients and on 118 of the controls. The frequency of the deletion variant CYP2D6D (13 kb) was 0.025 in the lung cancer group and 0.012 in the control group. None of these frequencies in the lung cancer patients were statistically significantly different from the frequencies of the control population. When combining the PCR-typing results (CYP2D6A, CYP2D6B) 20 individuals were found to carry two PM-associated alleles in the lung cancer group (n = 204) compared with only six among the controls (n = 117). This corresponds to a PM frequency of 9.8% in the lung cancer population compared with 5.1% in the control population which is, however, not significantly different from each other. These results are not in concordance with previous results which suggest that the EM-phenotype is a susceptibility marker for lung cancer.

Chromosome 12 in human testicular cancer: dosage changes and their parental origin.

Cytogenetically, a marker chromosome interpreted as i(12p) is present in most testicular tumors of germ cell origin. In this study, 22 patients with testicular germ-cell tumors were investigated by Southern blot hybridization to characterize changes in chromosome 12. In comparison with normal DNA, tumor DNA of 18 patients showed increased dosages of 12p accompanied by a comparable or smaller increase or no change in the dosage of centromeric sequences of chromosome 12. A likely interpretation was that most testicular tumors had one or several isochromosomes for 12p that were formed by somatic division of the centromere and that the points of breakage and reunion in the centromeric region were different in different tumors. Allelic 12p fragments showing increased intensity were paternal in four and maternal in three of seven informative cases. Thus, there was no evidence of sex-limited parental imprinting. Furthermore, the observed patterns of allelic fragments suggested that the marker was an i(12p) formed by sister chromatids of one homolog number 12 rather than the result of interchange of genetic material between different homologues.

[Cytogenetic diagnosis in leukemia]. / Cytogenetisk diagnostikk ved leukemi.

Cytogenetic diagnosis of leukemia was taken into use at the Norwegian Radium Hospital in late 1988. The article presents results from the period 1989-90. The samples arrived the laboratory on the same day they were obtained from the patients, and the results indicate that the transport had no effect on outcome. The success rate (obtained by cytogenetic diagnosis) was 82%. Among the persons who were diagnosed successfully, 66% showed clonal abnormalities. Diagnostic and prognostic implications are discussed.

No evidence for constitutional chromosome instability in testicular cancer.

Chromosome aberrations in 20 lymphocytes of 20 patients with testicular germ cell tumors (TGCT) treated with surgery alone were compared with those of 20 cells from 20 healthy controls using standard G-banding technique. No increase in structural aberrations was found in the cancer group. An unexpected finding was that of more cells with losses of chromosomes being present in the control group. These losses predominantly affected small chromosomes in the control group, whereas the pattern of chromosome loss was different in the cancer group. The literature claiming increased chromosome instability in TGCT patients is reviewed. Point estimates and 95% confidence intervals to exclude such a hypothesis based on our results were calculated.

High resolution chromosome banding in search of germ line mutations applied on testicular cancer patients.

Patients with bilateral and/or familial testicular cancer are assumed to demonstrate germ line mutations if such mutations are instrumental in testicular cancer patients. Constitutional rearrangements have to our knowledge not been reported, while an increased level of constitutional chromosome instability has been claimed. Lymphocytes from 12 Norwegian patients with bilateral or familial testicular cancer have been studied by high resolution Giemsa banding. A possible germ line mutation would be expected to be seen in every cell. It is therefore sufficient to examine one pair of the individual chromosomes obtained, if necessary, from several cells. Based on our previous findings of loss of heterozygosity in the chromosome regions 3p and 11p in testicular tumors, these chromosome arms were paid special attention. However, no mutations were observed in these regions nor in any other chromosome. Applying a 95% confidence interval, we conclude that less than 23% of familial and/or bilateral testicular cancer patients are caused by germ line mutations large enough to be detected with high resolution banding of at least 450 G-bands per haploid genome. The presented analyses were used to standardize the resolution determination in our laboratory.

Chromosome 13 alterations in osteosarcoma cell lines derived from a patient with previous retinoblastoma.

Various sublines of cells established from an osteosarcoma that developed in a patient (O.H.) with previous bilateral retinoblastoma were examined for different restriction fragment-length polymorphisms of chromosome 13q, as well as for rearrangements of the retinoblastoma gene using a cDNA probe. The independently established sublines were used to help separate primary and secondary events taking place in tumorigenesis of the osteosarcoma of this patient. Information from the present DNA analysis, taken together with data from cytogenetic and enzymatic studies on chromosome 13 in the cell lines, revealed both common and distinct genetic changes on chromosome 13q. The common changes may indicate the nature of the first and second mutational events in the development of the osteosarcoma. The first, constitutional cancer predisposing mutation seemed to be a base mutation or a small deletion/insertion, and the second event involved a deletion of a larger part of the long arm of chromosome 13. The distinct genetic changes included other deletion and duplication events of chromosome 13q. The existence of multiple sublines with different genetic constitutions provides improved possibilities for gaining insight into the nature of the genetic lesions leading to tumor formation, as these may reflect the clonal variation present in the primary tumor. We also demonstrate the difficulty of inferring from single tumor cell isolates to properties of the primary tumor.

Karyotyping of a hematologic neoplasia developing shortly after treatment for cerebral extragonadal germ cell tumor.

Hematologic malignancies may be associated with mediastinal extragonadal germ cell tumors. It may be that the hematologic malignancy is a part of the natural history of the teratoma, one germ cell tumor line being able to differentiate into hematological cells, or the hematologic malignancy is related to the treatment, or the two malignancies develop independently. Cytogenetic analysis of bone marrow from a patient with a germ cell tumor in the brain and the almost simultaneous appearance of a hematologic neoplasia showed a rearranged karyotype in that all 15 analyzed cells had the same karyotype: 50,XY, +X, +del(1)(p21), +10, +11, -12, +der(12)t(12;?)(q?;?). Our findings were consistent with the interpretation that the hematologic malignancy was derived from the germ cell tumor.

Specific gain of chromosome 19 in preneoplastic mouse liver cells after diethylnitrosamine treatment.

Most attempts to elucidate primary cytogenetic events have been made on the basis of chromosome analysis of cells from the later stages of tumor development. Hence it is difficult to decide which, if any, of the observed chromosomal alterations are causative rather than consequential in neoplastic development. One approach to understand the role of chromosomal changes in neoplasia is to examine the chromosomal constitution of cells at early stages in the process of carcinogenesis. We have developed a method for direct preparation of metaphase plates of cells from preneoplastic liver nodules and we present herein the first report of nonrandom chromosome changes in solid tumor precursor cells. The results of the cytogenetic analysis demonstrated trisomies/polysomies for chromosome 11 and chromosome 19. A specific increase in the number of chromosome 19 was found in 43% of the 37 plates karyotyped and 25% of the plates demonstrated increase in the number of chromosome 11. The finding shows that gross chromosomal changes such as polysomies may play an important role in the early development of tumors. They further indicate the presence of a gene on chromosome 19 with an essential role either in the initiation or promotion of the neoplastic transformation of hepatocytes.