RBE of X rays of different energies: a cytogenetic evaluation by FISH.

Mammography using 26-30 kVp X rays is routinely used in breast cancer screening. Discussion about the radiation-related risk associated with this methodology is ongoing. For radioprotection purposes, a quality factor of 1 has been assigned for all photon energies. However, the relative biological effectiveness (RBE) could increase as the photon energy decreases. Analyzing different biological parameters, for 30 kVp X rays, RBE values from 1 to 8 have been estimated. In the present study, a cytogenetic FISH evaluation of the RBE of 30, 80 and 120 kVp X rays has been done. Blood samples were irradiated with 10 doses from 0.05 to 3 Gy for each energy studied. The yields of translocations and dicentrics were determined by fluorescence in situ hybridization (FISH) using whole chromosome probes for chromosomes 1, 4 and 11 together with a pancentromeric probe. The alpha coefficients of the dose-effect curves for dicentrics, minimum number of breaks needed to produce exchange-type aberrations, and apparently simple translocations were used to estimate the RBE. Using the curves obtained for 120 kVp as a reference, the RBE values for dicentrics were 1.08+/-0.43 and 1.73+/-0.59 for 80 and 30 kVp X rays, respectively; for minimum number of breaks these values were 1.38+/-0.39 and 1.42+/-0.41, and for apparently simple translocations they were 1.26+/-0.40 and 1.51+/-0.47, respectively. Moreover, the induction of complex aberrations by these energies was compared. The percentage of complex aberrations relative to total aberrations showed a significant tendency to increase as X-ray energy decreased: 7.8+/-1.19, 9.8+/-1.6 and 14.1+/-1.9 for 120, 80 and 30 kVp, respectively (P<0.02).

Genetic abnormalities associated with the t(12;21) and their impact in the outcome of 56 patients with B-precursor acute lymphoblastic leukemia.

The ETV6/RUNX1 rearrangement is found in 20-30% of children with B-cell precursor acute lymphoblastic leukemia and is associated with a good outcome. To determine the cytogenetic and molecular abnormalities associated with the ETV6/RUNX1 rearrangement and the influence of this rearrangement in patients' evolution, we analyzed the molecular cytogenetic profiles of 56 children with this rearrangement and B-cell precursor acute lymphoblastic leukemia. Secondary changes detected with conventional cytogenetics and with fluorescence in situ hybridization were found in 71.4% of cases, the most frequent being the loss of the normal ETV6 allele, 12p aberrations, duplication of the fusion gene, and trisomy 21, as in replicating the results of previous studies. In this preliminary series, with a mean follow-up of 69.3 months, secondary abnormalities did not influence patients' outcome. It seems therefore that the prognostic value of the t(12;21) does not vary and that ETV6/RUNX1 rearrangement is an independent indicator of good prognosis.

Translocation yields in peripheral blood lymphocytes from control populations.

PURPOSE: To record the latest information on control levels of translocations in cultured human lymphocytes. MATERIALS AND METHODS: Control-level data from seven European laboratories that are using fluorescence in situ hybridization (FISH) techniques for retrospective biological dosimetry have been combined in a meta-analysis. After correction for the differing probe combinations used, tests of consistency are performed. The combined data have been used to test for individual variation, systematic variation with age, gender and smoking habits. RESULTS: There is a strong variation of translocation yield with age but no variation was detectable with gender or smoking habits. After correction for age, homogeneity tests showed that about 10% of individuals were outside the 95% confidence limits as opposed to 5% expected. From a total of 385, there is an excess of about 20 individuals most of whom have an unexpectedly high yield of translocations. CONCLUSIONS: For retrospective biological dosimetry purposes a generic age-dependent control level can be assumed. No other lifestyle factors such as smoking appear to have a significant effect on translocation yield.

Turner syndrome: evaluation of prenatal diagnosis in 19 European registries.

This study evaluated the prenatal diagnosis of Turner syndrome by ultrasound examination in an unselected population from all over Europe. Data from 19 congenital malformation registries from 11 European countries were analyzed. Turner syndrome was diagnosed in 125 cases (7.2%) in a total of 1,738 chromosome abnormalities. Sixty-seven percent of cases were detected prenatally by ultrasound examination due to the presence of congenital defects. The most frequent anomalies were cystic hygroma (59.5%) and hydrops fetalis (19%). The most frequent karyotype was 45,X (81.6%) followed by different types of mosaicism (16.8%). Significant differences in congenital defects (P = 0.0003) were observed between 45,X karyotypes and 45,X mosaicism cases. Prenatal counseling for 45,X mosaicism should take into account the expectation of a milder phenotype. In 78.6% of cases diagnosed by ultrasound examination due to congenital anomalies, the pregnancy was terminated. Prenatal detection of Turner syndrome by ultrasound examination was high in this unselected population.

ETV6/RUNX1 rearrangement in childhood B-precursor acute lymphoblastic leukemia with normal karyotypes or without cytogenetic results.

The ETV6/RUNX1 rearrangement (also known as TEL/AML1) was evaluated in 39 children with B-precursor acute lymphoblastic leukemia (ALL) who had a normal karyotype or lack of mitoses. Forty-one point six percent of patients with normal karyotypes and 66.6% of patients without mitoses presented with the ETV6/RUNX1 rearrangement. In addition to this rearrangement, eight patients showed loss of the normal ETV6 allele; of three patients without mitoses, two showed an extra signal of the RUNX1 gene and the third showed the fusion gene duplicated and loss of the normal ETV6 allele. One patient without the ETV6/RUNX1 rearrangement and without mitoses showed two extra signals of the RUNX1 gene.

Effect of DMSO on radiation-induced chromosome aberrations analysed by FISH.

The purpose of the present work was to determine if the described reduction in the frequency of radiation-induced chromosome aberrations by DMSO is homogeneous within different human chromosomes. Blood samples were irradiated with 4 Gy of X-rays in absence and presence of 0.5 M DMSO. FISH painting was carried out independently for human chromosomes 1, 2, 3, 4, 7, 11 and 12. The observed frequencies of apparently simple translocations and dicentrics for all these chromosomes, showed a homogeneous reduction when the irradiation was done in the presence of DMSO. Moreover, a better fit between the observed and expected frequencies was obtained when (DNA content)2/3 was used to calculate the expected frequencies, instead of just the DNA content. This result supports the idea that for exchange type aberrations, a better adjustment is obtained when the surface area of spherical chromosome territories is considered.

Prenatal detection of rare chromosomal autosomal abnormalities in Europe.

The aim of the present study was to evaluate the prenatal detection of rare chromosomal autosomal abnormalities by ultrasound (US) examination. Data were obtained from 19 congenital malformation registries from 11 European countries, between 01/07/96 and 31/12/98. A total of 664,340 births were covered and 7,758 cases with congenital malformations were recorded. Rare autosomal abnormalities were diagnosed in 114 cases (6.6%) from a total of 1,738 chromosome abnormalities. There were a wide variety of autosomal abnormalities: the most common were deletions (33 cases), duplications (32 cases), trisomies of chromosomes 8, 9, 10, 14, 15, and 16 (23 cases), and unbalanced rearrangements (19 cases). Out of these cases, 45.6% were detected prenatally by US examination due to the presence of congenital anomaly. As for the types of chromosomal anomaly, unbalanced rearrangements and deletions were the most frequently detected by US. A high percentage of cases with balanced rearrangements were associated with severe congenital anomalies. The most frequent congenital anomalies detected by US were cystic hygroma (20.6%), central nervous system defects (17.6%), cardiac defects (13.2%), and diaphragm defects (10.3%). This large series offers useful information about prenatal diagnosis by US of congenital defects associated with rare autosomal abnormalities and it provides a valuable knowledge about outcome. Fetal anomalies detected by US that were associated with rare autosomal abnormalities were significantly more frequent than those associated with common chromosomal abnormalities (45.6 vs. 34.7%). This study indicates the need to increase the detection of congenital anomalies by US.

AML1 amplification in a child with acute lymphoblastic leukemia.

We report a case of de novo acute lymphoblastic leukemia with tandem amplification of the AML1 gene located in a chromosome marker that originated from chromosome 21 and a long event-free survival.

DNA hypermethylation at the D17S5 locus is not a frequent event in human urothelial cancer.

OBJECTIVE: To analyse the DNA methylation status and the loss of heterozygosity (LOH) at the D17S5 locus (17p13.3) in urothelial cancer. MATERIALS AND METHODS: DNA methylation was assayed and LOH analysed by Southern blotting in a series of 33 transitional cell carcinomas of the bladder and renal pelvis. RESULTS: DNA hypermethylation and LOH at the D17S5 locus were detected in six (18%) and 17 (52%) of the tumours, respectively. The six cases with DNA hypermethylation were of the papillary type, and four also had LOH at this locus. CONCLUSION: In contrast to other epithelial tumours, DNA hypermethylation at the D17S5 locus is not a frequent event in human urothelial cancer.

Genetic evolution in the metastatic progression of human pancreatic cancer studied by CGH.

Metastases are thought to be derived from emerging clones within primary tumors. Although the concept of the clonal evolution of cancer is well defined, the genetic grounds and significance of this process in human cancer progression are still poorly understood. To gain insight into the genetic basis and clonal evolution underlying the metastatic progression of human pancreatic cancer in vivo, we analyzed by comparative genomic hybridization (CGH) chromosomal imbalances in seven metastases originated in nude mice and their three corresponding orthotopically xenografted human pancreatic tumors. All metastases were found to be closely related to the corresponding orthotopic implant, adding many additional changes to the already altered copy number profile of the pancreatic tumors. Recurrent metastasis-specific alterations included gains at 16cen-q22 and 17q21-qter. CGH results from paired specimens strongly suggest that the majority of additional genetic alterations present in metastases are likely to be present in subclones in the primary tumor.

Comparative genomic hybridization analysis of transitional cell carcinomas of the renal pelvis.

We used comparative genomic hybridization to analyze 10 primary tumor samples from patients with transitional cell carcinoma of the renal pelvis. The most frequent loss was located at 9q, that is, in 50% of the tumors. Gains of DNA sequences were most frequently observed in chromosome regions 1q21 approximately q23, 2p23 approximately p25, 8q21.1 approximately q22 and in the whole chromosome 20. High level amplifications at 1q21 approximately q25, 6p22 approximately p23, 8q21 approximately q22, 8q22 approximately q24.1, 11q13, and 12q14 approximately q21 were detected. Most of these regions have previously been reported to be involved in transitional cell carcinoma of the bladder, thus confirming the importance of an increasing number of chromosome imbalances in the development and progression of this type of tumors.

Detection of chromosomal imbalances in papillary bladder tumors by comparative genomic hybridization.

OBJECTIVES: To identify those genetic alterations that are associated with bladder cancer invasion and progression. METHODS: A total of 30 specimens of transitional cell carcinoma of the bladder were analyzed by comparative genomic hybridization. The results were compared and summarized with previously reported studies. RESULTS: The most frequent chromosome changes detected in our series of tumors were losses in 9q, 9p, 8p, and 11p and gains in 8q, 1q, 20q, and 11q. Three regions of deletion on chromosome 9 were delineated, at 9p21-p22, 9q13-q22, and 9q31-q34. Gains in 1q and losses on 11p were significantly more frequent in pT1G2 tumors than in superficial (pTa) ones. In our study, the most striking differences were seen between pT1G3 and pT1G2 tumors. Gains on 10p and 6p and losses at 5q, 6q, and 18q were significantly more frequent in the former. CONCLUSIONS: A summary of our results and those available from published reports suggest that several groups of chromosomal imbalances may be associated with specific steps along bladder cancer progression. These genetic changes assume two different patterns: those that are shared, but are more intensive in one stage than in the other, and those such as a gain on 3p that are unique to invasive tumors.

Cytogenetic analyses by fluorescence in situ hybridization (FISH) in hospital workers occupationally exposed to low levels of ionizing radiation.

An occupationally exposed population has been studied to evaluate the suitability of FISH painting techniques to detect chronic exposures to very low doses of ionizing radiation by the analysis of translocations. Whole-chromosome painting probes for chromosomes 1, 4 and 11 in combination with a pancentromeric probe have been employed. For comparison, a matched control population has also been studied. The mean genomic frequencies per 100 cells of total translocations in the control and exposed populations were 0.90 +/- 0.12 and 1.04 +/- 0.11, respectively. In the occupationally exposed population, no correlation between the frequencies of translocations and the doses received was found. When the two populations were compared, no significant differences were observed for the frequencies of the different chromosomal abnormalities examined. The absence of differences between control and exposed populations could be attributed to the very low-dose exposures recorded in the occupationally exposed population and to the wide range of individual frequencies of translocations observed.

Cytogenetic sensitivity of three Fanconi anemia heterozygotes to bleomycin and ionizing radiation.

It is well known that Fanconi anemia (FA) patients show a hypersensitivity to the effect of cross-linking agents such as mitomycin C and diepoxybutane, while the sensitivity of these patients to ionizing radiation is controversial. Fanconi anemia heterozygotes do not show a hypersensitivity to the above-mentioned agents. However, bleomycin it is used to identify mutagen sensitive individuals, especially among head and neck cancer patients. We present here a preliminary study in which the mean frequencies of bleomycin-induced chromatid breaks (ctb) from three FA heterozygotes (X = 0.90, range 0.80-1.01) and 11 controls (X = 0.40, range 0.21-0.66) differ significantly (P<.001), indicating a high sensitivity to bleomycin of G(2) lymphocytes from these three FA heterozygotes. An increased sensitivity was not observed after exposure of G0 lymphocytes to 2 Gy of ionizing radiation.

Follow-up by cytogenetic and fluorescence in situ hybridization analysis of allogeneic bone marrow transplantation in two children with Fanconi's anaemia in transformation.

Results of bone marrow transplantation (BMT) in patients with Fanconi's anaemia (FA) in transformation are very poor and only a few cases with favourable outcome have been reported. We present the follow-up of two FA-myelodysplastic syndrome (MDS) patients with monosomy 7 and complex karyotype implicating chromosome 1. Both relapsed with acute myeloid leukaemia (AML) following an allogeneic BMT from an HLA-identical brother. The patients showed clonal cytogenetic evolution coinciding with the leukaemic transformation. In one patient, fluorescence in situ hybridization using X and Y chromosome probes detected an increase of host cells before clinical relapse. Both patients received a successful second allogeneic BMT from the same donor using a more intensive treatment regimen and remain in clinical and cytogenetic remission more than 3 years later.

DNA copy number changes and evaluation of MYC, IGF1R, and FES amplification in xenografts of pancreatic adenocarcinoma.

We analyzed eight samples of xenografted human pancreatic tumors and two metastases developed in mice by comparative genomic hybridization (CGH). The most recurrent changes were: gains on chromosomes 8 (8q24-qter; 7/8 cases), 15 (15q25-q26; 6/8 cases), 16 (16p in 6/8 cases; 16q in 5/8 cases), 20 (20q; 6/8 cases), and 19 (19q; 5/8 cases); and losses on chromosomes 18 (18q21; 6/8 cases), 6 (6q16-q21 and 6q24-qter; 5/8 cases each), and 9 (9p23-pter; 5/8 cases). The two metastases maintained the aberrations of the original pancreatic tumor plus gain of 11q12-q13 and 22q. Loss of heterozygosity analysis was carried out for 10p14-pter, a region that was lost in 3/8 samples. All of them presented allelic imbalance for all the informative loci. Fluorescence in situ hybridization and Southern analysis were performed to test some candidate oncogenes in 8q24 (MYC) and 15q25-qter (IGF1R and FES). Two of seven tumors showed high-level amplification of MYC relative to the centromere (> 3-fold), another two tumors had low-level amplification (1.5- to 3.0-fold), and one displayed 5.5 MYC signals/cell. In relation to the FES gene, low-level amplification was found in three tumors. Southern analysis showed five cases with a low-level amplification of IGF1R. Our data suggest that either few extra gene copies may be enough for cancer progression or other genes located in these regions are responsible for the amplifications found by CGH.

Study of allelic losses on 3p, 6q, and 17p in human urothelial cancer.

Forty-eight transitional cell carcinomas of the bladder and three transitional cell carcinomas of the renal pelvis were examined for loss of heterozygosity (LOH) on chromosomes 3p, 6q, and 17p. The most frequent allelic loss was seen on 17p (18/36, 50%) followed by 6q (6/22, 27%), and 3p (5/22, 23%). In cases with LOH at more than one locus, the same DNA sample often varied in degree of signal reduction for missing alleles. This observation indicates that LOH studies can serve to detect intratumor heterogeneity. No correlation was found between allelic losses at these chromosome arms and tumor grade and stage. Allelic losses on 6q were associated with tumors having a solid growth pattern; in this kind of tumors, allelic losses on 3p were associated with invasion.

Follow-up of chimerism in children with hematological diseases after allogeneic hematopoietic progenitor cell transplants.

Using in situ hybridization with an X and Y chromosome probe mixture, 106 bone marrow samples from 38 patients with malignant and non-malignant hematological diseases who received sex-mismatched allogeneic hematopoietic progenitor cell transplants (PCT) in a single institution within short-term intervals (1, 3, 6, 12, 24 and >24 months) have been sequentially studied. The patients received either HLA-identical (n = 31) or non-identical (n = 7) PCT. Twenty-six children showed donor chimerism, 10 children showed mixed chimerism (MC) and two children presented autologous reconstitution. Chimerism status with different parameters has been related (age, sex, donor, disease status before PCT, conditioning regimen, GVHD prophylaxis, relapse, GVHD and survival). Our results indicate that female patients (P = 0.011) and a less intensive conditioning regimen (P = 0.039) are significantly associated with the MC status. Mixed chimerism is not, per se, significantly associated with leukemia relapse but an increase of the MC is indicative of clinical relapse.