Common fragile sites in chromosomes of bone marrow cells and peripheral blood lymphocytes from healthy persons and leukemia patients

Frequency and distribution of aphidicolin-induced fragile sites (c-fra) on chromosomes of both peripheral blood lymphocytes (PBL) and bone marrow (BM) from 15 leukemia patients were studied in comparison with 22 PBL and six BM samples from healthy volunteers. In normal controls, the most frequent c-fra was 3p14 in PBL, but it was 4q21-25 in BM. The second most frequent site was 16q23 in PBL, but it was 7q11.2 in BM. These differences in fragile sites between PBL and BM may be related to distinct functions of cells in different tissues. The total number of breaks in PBL and BM showed a significant difference among individuals, but the sites were generally common. The frequency of breaks in PBL from leukemia patients was higher than in controls when the leukemic cells had any karyotypic abnormalities. In leukemia without karyotypic abnormalities and acute myeloid leukemia (AML) with (15:17), the frequency of breaks fell within normal or slightly above normal ranges. Breaks at 3p14 (22.0% of total breaks), 16q23 (7.3%), 7q32 (4.3%), Xp22 (3.7%), and 6q26 (2.9%) were frequent in PBL from seven AML patients. Breaks at 4q21-25 (2.1%), 7q22 (2.2%), 7q32, and Xp22 were more frequently induced than in controls, and 1p32 (0.1%), 3p14, 6q26, and 16q23 were less often expressed than in controls. On the other hand, PBL from acute lymphoblastic leukemia patients showed a higher frequency of breaks only at 1p22 (3.4%) and the frequency of breaks at 3p14 (30.2%) decreased (p less than 0.05). The PBL from AML patients with t(8;21) (q22;q22) showed breaks at 8q22 and 8q24, and the frequencies were significantly higher than those of other types of leukemia or in controls (p less than 0.001). The results of this study suggest that fragility of chromosomes may be related to the chromosomal rearrangement in or predisposition to leukemia.     

Distamycin A-inducible fragile sites and cancer proneness

To determine the baseline frequency of autosomal rare fragile sites in cancer patients, we conducted a population cytogenetic study of 370 patients with leukemias, solid tumors, and other neoplastic disorders. Twenty carriers of rare fragile sites were detected in this patient group. The rare autosomal fragile sites were at fra(8)(q24), fra(11)(p15), fra(16)(p12.1), fra(16)(q22), and fra(17)(p12). All of these fragile sites were found to be distamycin A inducible. Compared with a population incidence in healthy subjects (44 of 845, 5.21%), the overall incidence of distamycin A-inducible fragile sites was not higher in the patient group (20 of 370, 5.41%). Analysis of these individual fragile sites and particular diseases, however, suggests that the distamycin A-inducible fragile sites may play a role in the etiology of leukemia, myeloproliferative disorders, and benign tumors.     

Fragile site expression in families with von Hippel-Lindau disease

Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder that predisposes to diverse tumors including renal cell carcinoma. Six affected and four unaffected subjects from five families were studied to determine the frequency of fragile site expression. Peripheral lymphocyte cultures from each subject were treated with low folate, 5-fluorodeoxyuridine (FUdR), and FUdR plus caffeine for fragile site induction. A site was considered to be fragile if it was expressed at least two times in half of the affected or unaffected subjects. Of the established sites, four were expressed in the unaffected group (3p14, 6p22, 8q22, and Xp22) and six were expressed in the affected group (3p14, 4q31, 5q31, 7q32, Xp22, and Xq22). Only 3p14 and Xp22 were expressed in both groups. There were four new sites: three (3q26, 6p21, 7p15) in the unaffected group and one (16q24) in the affected group. The 3p14 site was expressed twice as frequently in affected versus unaffected subjects. This finding is of interest because of reports of the involvement of 3p14 in hereditary renal cell carcinoma and in VHL.     

Constitutive fragile sites 1p31, 3p14, 6q26, and 16q23 and their use as controls for false-negative results with the fragile(X)

Fragile(X) estimations in fragile(X)-mental retardation hemizygotes or heterozygotes can become falsely negative in stored blood (lymphocytes). This was shown in blood stored (before culture) at 4°C, room temperature (25°C), 37°C, and 39°C for 1–4 days. After storage, blood was cultured in Ham's F10-5% FC serum with 0.1 μM FUdR and scored for fra(X) and the constitutive fragile sites at 3p14 and 6q26. It was found that the proportion of cells expressing the fragile(X) and the 3p14 site varied inversely with the temperature and time of storage. In addition, 50 patients and controls were scored for the three latter sites after routine 72–96-hr culture in F 10–0.05 or 0.1 μM FUdR. The 3p14 site was detected in every individual tested in a mean ± S.D. of 11.3 ± 3.2% of cells (0.1 μM FUdR). It was found that this site was FUdR dose dependent whereas the 6q26 site was not. The 3p14 (but not the 6q26) site is therefore suitable as a control site for the FUdR effect. It is proposed that repeat studies are necessary when less then 4% 3p14 sites are present in specimens from males referred for fra(X) estimation. Other constitutive fragile sites (eg, 1p31 and 16q23) can also be used.     

Atypical (7;19) translocation in acute myelomonocytic leukemia.

Chromosome studies were carried out after a 24-hour harvest of unstimulated bone marrow aspirate cell cultures from a 75-year-old male with a clinical diagnosis of acute myelomonocytic leukemia (FAB M4). Analysis of nine cells after trypsin-Giemsa banding (GTG) revealed two cell lines with a mosaic chromosome pattern, 46,XY/46,XY,t(7;19)(q22;p13.3). A review of the recent literature reveals one case of childhood ALL with a 46,XY/46,XY,t(7;19)(q11;q13) chromosome pattern [1] and a 46,XY,t(3q;11q),t(7q;19p),t(15;17)(q26;q22) in one patient with ANLL (FAB M3) [2]. The t(7;19)(q22;p13.3) seen in our case has not been reported as the sole specific clonal chromosome rearrangement in myeloid neoplasia. Interestingly, the plasminogen activator inhibitor type I, multi-drug resistance, and erythropoietin genes are located at band 7q22 and the insulin receptor gene is located at band 19p13.3. Both sites contain fragile site loci. The possible role of these fragile sites, genes, or other genes in the rearrangement can only be surmised.     

Fragile sites, Alzheimer's disease, and aging.

The fragile site expression under conditions of folate deprivation was compared in the chromosomes from 5 Alzheimer's disease (AD) female patients, 5 healthy elderly females and 5 healthy young females. Although different fragile sites were observed in the three groups, nevertheless, more similarities were found between the AD patients and elderly normal donors. The only fragile site common to all groups was 3p14. This site was the most frequent in the young donors group. In both AD and elderly control groups we observed a higher frequency of fragility in 6p21, but not in the young controls. Other interesting fragility points observed in these two groups were: 6q21 and 14q24 (in the AD patients) and 9q13, 14q24 and 17q21 (in the healthy aged). 6p21 and 17q21 have been proposed as 'new' fragile sites. We confirm the existence of these fragile sites and comment that in these bands the genes MTBT2 and MTBT1, which are microtubule (beta) associated protein tau-like and tau 1, respectively, are mapped. The tau protein is a component of paired helical filaments which accumulate in degenerating neurons in the brain of patients with AD and with less intensity of normal elderly individuals.     

Aphidicolin-sensitive specific common fragile sites: A biomarker of exposure to pesticides

In this work, we analyzed the aphidicolin-sensitive common fragile sites in seven females and four males occupationally exposed to pesticides and in ten controls. The same males had been monitored one year earlier in a previous study by the same authors. Results showed enhanced expression in exposed subjects at eight bands, namely, 6q25, 7p22, 7q22, 7q32, 13q14, 14q24, 16q22, and 16q23. Most of these bands were fragile sites andbreakpoints involved in chromosome rearrangements found in hematopoietic tumors. Moreover, six of these bands were already detected, with enhanced expression, in the first monitoring carried out on male subjects. These results indicated that fragile sites analysis is a reproducible cell response to human exposure to pesticides. Environ. Mol. Mutagen. 29:250-255, 1997 © 1997 Wiley-Liss, Inc.     

Intratumoural cytogenetic heterogeneity of sporadic colorectal carcinomas suggests several pathways to liver metastasis

Much has been learned about the chromosomal abnormalities of colorectal carcinomas but the cytogenetic relationship between the neoplastic clones present in primary versus metastatic tumour samples remains unclear. We analyse the frequency of abnormalities for 47 chromosome regions using the interphase fluorescence in situ hybridization technique in a group of 48 tumours, including 24 primary colorectal tumours and 24 paired liver metastases. All tumours showed complex karyotypes with numerical/structural abnormalities for seven or more different chromosomes/chromosome regions both in the primary tumours and in their paired metastases. Chromosome 8 was the most frequently altered (22/24 primary tumours), consistently showing del(8p22) and/or gains/amplification of 8q24, followed by abnormalities of the entire chromosome 7 (21/24 primary tumours) and of chromosomes 17p and 20q (20/24 primary tumours). Simultaneous staining for multiple chromosome probes revealed the presence of two or more tumour cell clones in 23/24 cases (46/48 tumour samples). Interestingly, the liver metastases typically contained tumour cell clones similar to those found in the primary tumours, suggesting the absence of selective selection of specific tumour clones. Despite this, additional chromosomal abnormalities were detected in 23/24 metastatic tumours, which preferentially consisted of del(17p13) and gains/amplification of 11q13 and 20q13; moreover, compared to primary tumours, metastases showed an increased number of abnormalities of chromosomes 1p, 7q, 8q, 13q, and 18q, and new chromosomal abnormalities involving chromosomes 6, 10q23, 14q32, 15q22, and 19q13. Owing to the high frequency of numerical abnormalities of the entire chromosome 7 and loss and/or gain/amplification of specific regions of chromosome 8, eg del(8p22) and/or gains/amplification of 8q24 in primary colorectal tumours with associated metastases, it is suggested that their assessment at diagnosis could be of great clinical utility for the identification of colorectal cancer patients at higher risk of developing liver metastases. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Variant Philadelphia translocations in CML: correlation with fragile sites

Of 175 CML patients studied, 14 variants were found, seven of which are presently described. The breakpoints involved in the translocation, other than 9q34 and 22q11, are 3p21, 5q13, 6p21, 7q22, 10q22, and 11p13. Fragile sites were investigated in some of these patients. In two cases a coincidence between fragile site location and breakpoint of the third chromosome involved in Philadelphia formation was found. This observation suggests that the fragile sites can lead to Ph variants in patients developing CML.     

Chromosomal aberrations associated with invasion in papillary superficial bladder cancer

Non-invasive and invasive papillary transitional cell carcinomas of stages pTa and pT1 represent the first steps of tumour progression in bladder cancer. In order to analyse different chromosomal alterations of pTa and pT1 superficial bladder cancer, 46 tumour specimens were examined by comparative genomic hybridization (CGH). Losses of chromosome 9 material (11/20) and gains of chromosome 17 material (6/20) were frequently found in pTa tumours. Stage pT1 tumours were characterized by gains of chromosome 1q (14/26; including amplification at 1q21–q24 in three cases) and chromosome 17 material (15/26), as well as by losses of 11p (15/26) and 11q (13/26). Other loci frequently showing losses in pT1 tumours were 2q (9/26), 4q (10/26), 5q (9/26), 8p (10/26), 9p (9/26), 9q (12/26), 10q (8/26), 17p (7/26), and 18q (8/26). Amplifications were detected at 8q21/22, 5q21, 7q36, 10p14, 10p12, 10q25, 12q12, and 12q14. The most striking differences between grade 2 pTa and pT1 tumours were gains of 1q (P<0·01) and losses at 2q (P<0·025), 10q (P<0·05), 11p (P<0·01), 11q (P<0·01), and 17p (P<0·05), as well as the total number of aberrations (pTa grade 2: 4·1; pT1 grade 2: 8·6 aberrations per tumour). These data show characteristic chromosomal aberrations associated with invasion in superficial bladder cancer. © 1998 John Wiley & Sons, Ltd.     

Fragile sites: overview, occurrence in acute nonlymphocytic leukemia and effects of caffeine on expression

Interest has recently grown in the possible role of chromosomal fragile sites as factors predisposing to chromosome rearrangements characteristic of specific human cancers. Data from two series of experiments relating to this hypothesis are presented. First, the effects of caffeine and theophylline on expression of the fragile X and common fragile sites was studied in lymphocytes from three subjects. Caffeine and theophylline did not enhance fragile X expression under the conditions employed but did greatly enhance expression of the common fragile sites. Second, three patients with acute nonlymphocytic leukemia-M4 and inv(16)(p13q22) in leukemic cells were tested for the presence of fra(16)(q22) in normal cells. The fragile site was not seen in any of the patients in this study.     

Distribution of autosomal fragile sites in specimens cultured for prenatal fragile X diagnosis

We reviewed the distribution of autosomal fragile sites (FS) and spontaneous chromosome breaks or gaps (CB) at chromosome locations other than those recognized as FS from 100 amniotic fluid samples (AF), 19 chorionic villus samples (CVS), and 5 percutaneous umbilical blood samples (PUBS) referred for fragile X [fra(X)] analysis. We present data on the degree of expression of autosomal fragility in AF, CVS, and PUBS samples, and the relationship between degree of expression and induction system. The most common observed FS were: 3p14, 9p32, and 6q26 in AF; 9q32, 3q27, and 8q22 in CVS; and 3p14, Xq22, and 16q23 in PUBS cases. Distribution of FS and CB, when compared by induction system, was not found to be identical. Our data also indicate that the presence of any particular FS cannot be used as an indicator for the effectiveness of the fra(X) induction system in prenatal samples.     

Chromosome abnormalities and rare fragile sites detected in azoospermia patients

Summary We have examined constitutional chromosome abnormalities and fragile sites in 40 patients with azoospermia. Chromosome abnormalities were found in four cases. Three cases showed a deletion of the long arm of the Y chromosome 46,X,del(Yq) and the other case had a ring of G group chromosome 46,XY,r(G). In a rare fragile sites test, four fragile site carriers were detected and three rare autosomal fragile sites were identified; fra(8)(q24.1), fra(11)(p15.1), and fra(17)(p12). The expression of these fragile sites were induced specifically by AT-specific DNA ligands, such as distamycin A and Hoechst 33258. In addition, one patient was found to be the case of double ascertainment of fragile sites, fra(8)(q24.1) and fra(17)(p12). The overall frequency of distamycin A-inducible fragile sites in azoospermia patients appeared to be higher than those reported for Japanese healthy subjects and cancer patients. However, no significant relation among fragile sites, clinical and histological findings has been detected so far.     

Variant Ph translocations in chronic myeloid leukemia

Variant translocations were found in eight of 142 consecutive patients with Ph-positive, chronic myeloid leukemia encountered in our laboratory during the last decade. Two patients had simple, two-way variant translocations: t(17;22)(p13;q11) and t(16;22)(q24;q11). Both of these patients had an additional translocation involving chromosomes #9: t(7;9)(q22;q34) and t(9;17)(q34;q21), respectively. Complex variant translocations were found in four cases: t(2;9;22)(p23q12;q34;q11), t(3;9;22)(p21;q34;q11), t(9;12;22)(q34;q13;q11q13), and t(13;17;22)(p11;p11q21;q11). In two cases, the only discernable cytogenetic aberration was del(22)(q11). A review of the chromosomal breakpoints involved in this series and in 185 cases of variant Ph translocations previously reported in the literature reveals that a disproportionately large number of breakpoints are located in light-staining regions of G-banded chromosomes. Furthermore, the breakpoints in simple variant translocations are more often located in terminal chromosomal regions, whereas, the breakpoints in complex translocations typically affect nonterminal bands. No obvious correlation was detected between variant Ph translocation breakpoints and either fragile sites, oncogene locations, or consistent chromosome breakpoints in other malignancies.     

Common fragile sites: their prevalence in subjects with constitutional and acquired chromosomal instability

Chromosomal fragile sites that are inducible by methotrexate and aphidicolin are frequent in the human population. To assess the frequency and distribution of these common fragile sites, we performed a cytogenetic survey on lymphocytes from subjects known to be particularly prone to breakage because of constitutional chromosomal instability, the possession of a rare fragile site, or Fanconi anemia. Furthermore, a group of cancer patients was included in this study in view of possible acquired chromosomal instability. Lymphocyte chromosomes from several healthy donors were analyzed under identical conditions. We found that methotrexate- and aphidicolin-induced fragile sites are widespread in the general population, showing a similar breakpoint distribution. Ten fragile sites (3p14, 16q23, 2q32, 6q25, 4p16, 4q31, 14q24, 1p31, 20p12, 7q21) were observed in at least 40% of the individuals among the different groups. Our data point out a significantly increased breakage induced by aphidicolin in lymphocytes from cancer patients and, to a lesser extent, from rare fragile sites carriers. These results suggest that common fragile sites are enhanced in some constitutional and acquired conditions.     

Genetic imbalances with impact on survival in colorectal cancer patients

AIMS: To investigate the prognostic significance of chromosomal alterations in colorectal cancer patients. Histopathological tumour classification is still considered to be the gold standard for the characterization of solid tumours. However, it is well known that such established parameters do not satisfactorily predict the clinical outcome in individual cases. Markers that reliably predict survival are needed. These markers should guide the clinical treatment of neoplastic disease. METHODS AND RESULTS: Chromosomal imbalances in 61 colorectal carcinoma specimens in 37 patients determined by comparative genomic hybridization were correlated with patient survival using custom-made computer software which enabled the assessment of individual chromosomal loci. Kaplan-Meier analysis revealed that over-representations of 2p14-15, 6q23-6q24, 15q22-15q23, 22q11.2 and deletions of 1p36.1-36.2, 4q31.3, 4q35, 8q12-q21, 8p11.2 and 9p22 were significantly associated with shorter disease-specific survival, whereas over-expression of 20q13.3 and deletion of 18q11.2 were significantly associated with longer disease-specific survival in this collection of colorectal cancers. Multivariate Cox proportional hazards regression models consistently identified gains of 2p14-15, 15q22-23, 22q11.2 and losses of 1p36.1-36.2 and 4q35 as independent markers of shorter patient survival carrying greater significance than the classical clinicopathological parameters of nodal status and tumour grade. CONCLUSIONS: These five markers allow a molecular categorization of patients into high and low clinical risk groups. Thus, the genomic data have refined the histopathological classification highlighting the necessity for a supplementary genetically based stratification of colorectal cancer.     

Identification of extensive genomic loss and gain by comparative genomic hybridisation in malignant astrocytoma in children and young adults

Although astrocytomas are the most common central nervous system tumours in all age groups, there is substantial evidence that tumours arising in young patients (< 25 years of age) do not have the same genetic abnormalities that are characteristic of tumours in older patients. Furthermore, novel, consistent changes have not been identified in astrocytomas in children and young adults. We analysed 13 malignant astrocytomas from young patients using comparative genomic hybridisation. Regions of genomic imbalance were identified in 10 cases. The most common recurrent copy number aberrations were loss of 16p (54% of cases), 17p (38%), 19p (38%), and 22 (38%) and gain on 2q (38%), 12q (38%), 13 (38%), 4q (31%), 5q (31%), and 8q (31%). Seven regions of high copy number amplification were observed at 8q21-22 (three cases), 7q22-23 (two cases), and 1p21-22, 2q22, 12q13-pter, 12q15-21, and 13q11-14 (one case each). This study provides evidence of new characteristic chromosomal imbalances from which potential candidate genes involved in the development of malignant astrocytoma in children and young adults may be identified.     

Investigation of genetic susceptibility to non-small cell lung cancer by fragile site expression

Fragile sites are non-staining gaps and breaks in specific points of chromosomes that are inducible by various culture conditions. Previous studies have shown that various clastogenic agents increase expression of fragile sites. In this study, the expression of common fragile sites induced by aphidicolin was evaluated on prometaphase chromosomes obtained from peripheral blood lymphocytes. Chromosomal aberrations and fragile site expression of 60 individuals, including 20 patients with non-small cell lung cancer (NSCLC), 20 of their clinically healthy family members, and 20 age-matched normal healthy controls without history of any cancer type were studied. Both the proportion of damaged cells (P < 0.001) and the mean number of gaps and breaks per cell (P < 0.001) were significantly higher in both the patients and relatives' groups when compared with the control group. However, they were insignificant when the patients were compared to their relatives (P > 0.05). We determined four aphidicolin type common fragile sites in our study. These sites in patients with NSCLC and relatives were the following: 1p21, 2q33, 3p14, and 16q23. In these fragile sites, 2q33, 3p14, and 16q23 sites were statistically significant when compared with control group (P < 0.001, P < 0.0005, and P < 0.05, respectively). Consequently, we believe that fragile site studies may be helpful to detection of cancer risk.     

Helicobacter pylori-related and -non-related gastric cancers do not differ with respect to chromosomal aberrations

Gastric carcinogenesis is strongly associated with Helicobacter pylori infection, but the underlying genetic mechanisms are largely unknown. The aim of this study was to correlate chromosomal aberrations in gastric cancer to H. pylori status and its different strains, as well as to histological type and other clinico-pathological variables. DNA from 46 gastric cancers (male/female 35/11, age 27-85 years) was extracted from formaldehyde-fixed, paraffin-embedded material and tested for chromosomal gains and losses by comparative genomic hybridization (CGH). Chromosomal aberrations with frequencies of 20% or higher were considered to be non-random changes associated with gastric cancer. The mean number of chromosomal events per tumour was 9.7 (range 0-27), with a mean of 3.2 gains (range 0-16) and 6.5 losses (range 0-15). Gains were most frequently found at chromosomes 8q and 13q (24% and 26%, respectively). Losses were predominantly found on chromosome arms 2q, 9p, 12q, 14q, 15q, 16p, 16q, 17p, 17q, 19p, 19q, and 22q (22%, 30%, 43%, 22%, 33%, 50%, 28%, 50%, 39%, 33%, 39%, and 37%, respectively). Common regions of overlap narrowed down to 2q11-14, 8q23, 9p21, 12q24, 13q21-22, 14q24 and 15q11-15. The mean number of gains was higher in tumours with metastases than in localized tumours (4.1 vs. 1.9, p=0.04). Tumours with a loss at 17p showed a higher number of losses than tumours without a 17p loss (9. 5 vs. 4.7 on average, p<0.001). Neither H. pylori status (+, n=25; -, n=21) nor H. pylori strain was correlated to the total number of events or to any specific chromosomal aberration, nor were there differences between intestinal (n=30) and diffuse (n=15) cancers or any other clinico-pathological variable tested. In conclusion, a complex of chromosomal aberrations is involved in gastric cancer, but their pattern does not depend on H. pylori status or strain, nor on the histological type of the tumour. The exact biological meaning of these aberrations in carcinogenesis needs further clarification.     

Comparative genomic hybridization reveals genetic progression of oral squamous cell carcinoma from dysplasia via two different tumourigenic pathways

To clarify the genetic pathway(s) involved in the development and progression of oral squamous cell carcinoma (OSCC), as well as the relationship between genetic aberrations and biological characteristics of OSCC tumours, comparative genomic hybridization was used to analyse genetic alterations in both primary OSCCs and adjacent dysplastic lesions of the same biopsy specimens from 35 patients. Gain of 8q22-23 was the most frequent alteration in both OSCC and mild dysplasia, and was considered the earliest event in the process of oral tumourigenesis. The average number of DNA sequence copy number aberrations (DSCNAs) increased with progression from mild dysplasia to invasive carcinoma (r = 0.737, n = 70, p < 0.001). OSCC samples were classified as having a large or small number of DSCNAs (OSCC-L, 21.4 +/- 4.7 DSCNAs or OSCC-S, 10.0 +/- 1.7 DSCNAs, respectively; p < 0.0001). Gains of 3q26-qter, 8q, 11q13, 14q, and 20q and losses of 4q, 5q12-22, 6q, 8p, 13q, and 18q22-qter were common to OSCC-L and OSCC-S. Gains of 5p15, 7p, 17q11-22, and 18p and losses of 3p14-21, 4p, and 9p were detected exclusively in OSCC-L. The average number of DSCNAs depended on whether the samples showed OSCC- L or dysplasia plus OSCC-L, or showed OSCC-S or dysplasia plus OSCC-S (p = 0.001). Gain of 5p15 and losses of 4p and 9p were detected even in dysplastic lesions adjacent to OSCC-L samples. Loss of 4p was associated with node metastasis by multivariate analysis (p = 0.013). OSCC-L tumours were more often T3-T4 stage tumours than T1-T2 stage tumours (p = 0.03). These findings suggest that two different types of OSCC, OSCC-L associated with high-stage cancer and OSCC-S associated with low-stage cancer, arise from different types of dysplasia via different genetic pathways.