Structural abnormalities of the X chromosome in non-Hodgkin's lymphoma.

It has recently been reported that additional X chromosomes occur in over 30% of B-cell non-Hodgkin's lymphomas (NHL), and that monosomy of the X chromosome occurs in 38% of female patients with T-cell leukaemia or lymphoma. These observations have suggested a possible role for the X chromosome in the evolution of NHL. We have now examined 280 cases of NHL, and have identified 19 examples of structurally altered X chromosomes in the malignant cells from 17 of these cases. These abnormalities were mainly characterized by either a translocation involving Xp22, or a translocation/deletion involving Xq28. The relevance of these observations is discussed with respect to other published reports, and together they suggest that lymphoma-associated oncogenes may exist on the X chromosome at bands p22 or q28.

Application of fluorescence in situ hybridisation to chromosome analysis of aged bone marrow smears.

AIMS: To evaluate the reliability of fluorescence in situ hybridisation (FISH) in the retrospective cytogenetic assessment of old bone marrow smears stored for periods of up to 20 years. METHODS: A series of bone marrow smears either Romanowsky stained, or frozen and unstained, and aged from one month to 20 years were hybridised with biotin labelled probes specific for the centromeric regions of human chromosomes X, 6, and 18. Sites of hybridisation were detected with fluoresceinated avidin. One hundred to 400 cells from each preparation were examined and the number of signals observed was recorded. RESULTS: All smears exhibited signals in most cells examined. In cytogenetically normal cases, an average 67.6% of cells (range 36%-90%) demonstrated the appropriate number of X centromere signals. In those samples known to contain extra chromosomes X, 6, or 18 the presence of cells with the abnormal copy number was clearly detected in each case. CONCLUSION: When applied in the way described, FISH can give consistent and accurate results with a variety of archival bone marrow smears, including aged prestained material. This will permit retrospective assessment of specific cytogenetic abnormalities in patients with leukaemia using their initial diagnostic slides even where these are several years old.

Cytogenetic analysis of a United Kingdom series of non-Hodgkins lymphomas.

We describe cytogenetic analyses of cells derived from 40 non-Hodgkins lymphoma (NHL) node biopsies, 23 of which were from patients who had not been treated before biopsy. We noted that the chromosomes most frequently gained were X (32%), 12 (27%), and 3 (24%). Monosomies were much less common; loss of chromosome 13 (13.5%) was most frequent. Structural abnormalities primarily involved chromosomes 14 (70%), 1 (40.5%), 18 (38%), 6 (35%), and 17 (22%). Low-and high-grade disease showed similar patterns of structural changes; however, a markedly greater number of chromosome gains were associated with low-grade disease. Biopsy samples from patients who had previously been treated showed an increased frequency of structural abnormalities, as well as a significantly larger number of chromosome gains. The importance of these observations, particularly with regard to possible oncogene involvement in lymphoma evolution, is discussed.

Analysis of 14q+ derivative chromosomes in non-Hodgkin's lymphomas by fluorescence in-situ hybridization.

The most common chromosomal abnormality observed in non-Hodgkin's lymphomas (NHL) involves the structural alteration of the q arm of chromosome 14. It is not always possible, however, to fully analyse such derivative chromosomes by Giemsa-banding. Therefore, we have applied the fluorescence in-situ hybridization (FISH) technique of chromosome painting to elucidate the origins of the der(14) chromosomes in 8 cases of NHL. In 2 NHL the der(14) appeared to be the product of the t(14;18)(q32;q21) translocation, but were not accompanied by the reciprocal der(18) chromosome. In 3 cases the breakpoint was at 14q32 but the translocated material appeared not to be from chromosome 18 and in 2 cases the breakpoint was centromeric to 14q32. One case with a t(14;18)(q32;q21) was also analysed as a control. Dual painting was carried out with paints for chromosome 14 and either chromosome 3, 8, 10, 11, 18 or 19. In the control and 2 other cases the translocated material was demonstrated to be from chromosome 18, in two cases it was from chromosome 3 and in 1 case there was an unusual insertion of chromosome 11 material. We were unable to identify the origins of the translocated material in 1 NHL and in the final case the apparent der(14) was demonstrated not to contain chromosome 14 material. These data demonstrated the utility of the FISH technique for analysing malignant cell karyotypes, and in particular indicated the potential of this approach for identifying cases containing putative NHL associated oncogenes that may have been translocated adjacent to the immunoglobulin locus at 14q32.

Activation of MYCN in a case of non-Hodgkin's lymphoma.

We have examined a series of non-Hodgkin's lymphomas (NHL) for evidence of expression of the MYC gene family. Northern blot analysis of RNA samples derived from 11 non-malignant reactive lymphoid tissues and 33 NHL was used to investigate expression of MYC, MYCL and MYCN. As expected MYC expression was detected in all samples. The levels of MYC expression were quantified by densitometry and appeared to be 3-8 fold higher in high grade NHL than in the low grade NHL or non-malignant lymphoid tissue. No expression of MYCL was detected in any sample. Expression of MYCN was however observed in one sample, which had been diagnosed as a T-cell high grade NHL. A detailed cytogenetic analysis of this sample proved difficult to obtain but, by using fluorescence in-situ hybridization (FISH), we were able to demonstrate that on one of the chromosomes 2 the MYCN gene was localised to a translocation breakpoint region. It therefore appears that in NHL it is possible for MYCN, like MYC in Burkitt lymphoma, to be activated as a result of a chromosome translocation event.

Cytogenetic analysis of non Hodgkin's lymphomas by ratio-painting and comparative genomic hybridization reveals unsuspected chromosomal abnormalities.

Cytogenetic analysis of cancer cells has proven to be a powerful tool in understanding malignant evolution and in providing clinically useful markers. In recent years the advent of new fluorescence in-situ hybridization (FISH) methods such as ratio-painting and comparative genomic hybridization (CGH) have enabled much more accurate karyotypes of malignant cells to be detected. In this study, we have examined the chromosomes present in malignant cells from a series of 6 low grade follicular centre and 2 high grade diffuse large cell non-Hodgkin's lymphomas (NHL) using conventional G-banding. In all cases chromosome abnormalities were observed, including the presence of marker chromosomes in six cases. The NHL cells were then subjected to the FISH method of ratio-painting. This provided a more accurate understanding of the origins of derivative chromosomes and identified the origins of all of the marker chromosomes. It also revealed hitherto unsuspected abnormalities. For example, in one case four abnormal chromosomes were demonstrated to contain material from chromosome 8, which had not been previously suspected from G-banding. Regions of amplification and deletion on the chromosomes were also investigated by CGH, which identified further unsuspected chromosomal abnormalities. For example, in case L124, trisomy of chromosome 7 was confirmed by CGH, but an unsuspected amplification of 3(p12) was also revealed. These approaches demonstrate the power of FISH technology in providing a more precise analysis of malignant cell chromosomes, and in doing so have produced comprehensive karyotypes of the NHL under study.

Gains on 9p are common genomic aberrations in idiopathic myelofibrosis: a comparative genomic hybridization study.

Ideopathic myelofibrosis (IMF) is a chronic myeloproliferative disorder resulting in bone marrow fibrosis as a consequence of growth factor release from clonal haematopoiesis. Conventional cytogenetic analysis identifies abnormalities in approximately a third of cases at diagnosis, although rarely uncovers unique, primary genetic events. We have used comparative genomic hybridization (CGH) to study 25 IMF cases and have compared the results with conventional cytogenetics. Metaphase cells were available for analysis in 13 cases, of which seven showed an abnormal karyotype. CGH chromosomal profiles showed imbalances in 21 of 25 cases. The most frequent aberrations were gains of 9p (12 cases), 2q (seven cases), 3p (seven cases), chromosome 4 (seven cases), 12q (seven cases), 13q (eight cases). The main losses were at 17q and occurred in six cases. The results for CGH and cytogenetics were matched for one case only. Investigation of IMF by CGH suggests that genomic aberrations are much more common than has been previously indicated by conventional cytogenetic analysis and occur in the majority of cases. Gains of 9p were the most frequent finding, occurring in 50% of patients and suggests that genes on 9p may play a crucial role in the pathogenesis of IMF.

Identification of a subclass of double minute chromosomes containing centromere-associated DNA.

In a study of abnormal chromosomes in non-Hodgkin's lymphoma (NHL) cells we have identified one case which contained extrachromosomal chromatin bodies that, on the basis of their morphology and negative C-banding, appeared to be double minute chromosomes (dmin). However, fluorescence in-situ hybridization (FISH) analysis using an X-specific centromeric alphoid repeat probe and a pan-centromere probe, clearly demonstrated the presence of centromere-associated DNA in these dmin. FISH analysis with the pan-centromere probe of the dmin in neuroblastoma and sarcoma cells failed to reveal the presence of centromere-associated DNA, but analysis of two cases of acute myeloid leukemia cells revealed centromere-associated DNA in 25% of their dmin. These data indicate the existence of dmin that contain centromere-associated DNA and suggest that such dmin might represent a new class of extrachromosomal chromatin bodies.

Chromosome analysis of non-Hodgkin's lymphomas by fluorescence in situ hybridization.

We have recently carried out a cytogenetic survey of a series of non-Hodgkin's lymphomas (NHLs) and identified chromosomal abnormalities in most of the samples studied. A third of these cases, however, had to be recorded as containing derivative or marker chromosomes, whose origins were either partially or completely unknown. In an attempt to further analyse such cases, we have adopted the fluorescence in situ hybridization (FISH) technique. The FISH technique has allowed us to map the myb gene to 6q23, and then to study its position on 6q- derivative chromosomes in the NHL cells. The related FISH technique of chromosome painting has enabled us to identify a marker chromosome in one of our cases as an abnormal X chromosome, and in other cases has allowed 14q+ derivative chromosomes to be further analysed. We have also applied the FISH technique to the analysis of interphase nuclei, and have been able to determine numerical chromosome changes in NHL interphase cells. The application of the FISH technique to the study of NHL cell chromosomes is likely to enable the identification of most chromosomal abnormalities present, and so may reveal the critical events leading to malignant transformation of lymphoid cells.

Semi-quantitative fluorescence in situ hybridization analysis indicates that the myc protein is consistently stabilized both before and after transformation of low-grade follicular center to high-grade diffuse large cell lymphoma.

We have investigated the expression of the MYC gene at both the mRNA and protein levels to determine how these parameters are related in lymphoma cells and in nonmalignant lymphoid cells. To do this we have adopted a multicolor fluorescence in situ hybridization methodology, which has allowed us to investigate the expression of different genes at the same time in the same cell. We have made use of the digital imaging capabilities of a charge-coupled device camera system to quantify the hybridization signals for the MYC gene and, by comparing these to the expression of a control gene (glyceraldehyde-3-phosphate dehydrogenase; GAPDH), have obtained relative quantitations of MYC mRNA and protein levels. In this study we have compared cells both within and outside the germinal centers in control tissues (reactive lymph nodes and tonsils) and in low-grade follicular center lymphomas, as well as cells in high-grade diffuse large cell lymphomas. The MYC/GAPDH mRNA hybridization signal ratios were calculated and found to be higher in cell populations containing a majority of malignant cells (p < 0.04). However, when the myc/GAPDH protein hybridization signal ratios were calculated, these were significantly higher in malignant cells from all lymphomas than the ratios observed in the nonmalignant cells (p < 0.0005). These observations indicate that the environment in a malignant cell may contribute to the stabilization of the myc protein, thus enabling it to function for a longer time period than in nonmalignant cells.

A simple method for PCR based analyses of immunohistochemically stained, microdissected, formalin fixed, paraffin wax embedded material.

Microdissection was performed on sections cut from formalin fixed, paraffin wax embedded archival material, which had been subjected to conventional immunohistochemistry. Crude DNA extracts, which were obtained from these microdissected samples by a simple microwave step, were then added directly to amplification reactions. Analyses using a range of polymerase chain reaction (PCR) based techniques, including microsatellite repeat polymorphism analysis at the NM23-H1 locus and sequencing of exons 5, 7, and 8 of the p53 gene, were performed successfully. Universal PCR amplification was also carried out on the microdissected material and probes suitable for use in comparative genomic hybridisation (CGH) were obtained in all cases. This technique will enable a range of effective genetic analyses to be carried out on specific subsets of cells that have been characterised previously by immunohistochemistry.

Copy number gain at 12q12-14 may be important in the transformation from follicular lymphoma to diffuse large B cell lymphoma.

The purpose of this study was to identify novel areas of genomic copy number change associated with transformation from follicular lymphoma (FL) to diffuse large B cell lymphoma (DLBL). DNA was extracted from tumour cells micro-dissected from paraffin- embedded tissue sections in 24 patients with FL and subsequent transformation to DLBL and 18 patients with de novo DLBL. Tumour DNA was compared to reference DNA using comparative genomic hybridization. Abnormalities common to all 3 groups were gains on chromosomes 4q, 5q, 7q, 11q and X and losses on 3p, 8p and 10q. Copy number changes seen in both transformed and de novo DLBL and not seen in FL were gains on 2p and losses on 1q, 15q and Xq. Gains on 2q, 6p, 7p and 17q and losses on 5p and 8q were specific to transformed DLBL cases. Gain on 12q12-14 was found in 52% of the transformed DLBL cases and was never seen in its follicular counterpart. Patterns of genomic copy number change associated with specific clinical events in NHL have been demonstrated and suggest that gains on 2q, 6p, 7p, 12q and 17q and losses on 5p and 8q may be important in the transformation from low to high-grade disease.

Investigation of the activation state of the X chromosome in non-Hodgkin's lymphomas.

Cytogenetic analysis of non-Hodgkin's lymphomas (NHLs) has previously revealed a high incidence of numerical abnormalities involving the X chromosome. We have now used a combination of fluorescence in situ hybridization (FISH) and Southern blot analysis of methylation to examine the activation state of additional X chromosomes in NHL. Although FISH analysis of X chromosome centromeres in interphase nuclei was complicated by a number of factors, such as cell-cycle position, there was evidence that more than one X chromosome was present in the active state in 4/9 NHL. Methylation studies were carried out using the M27 beta probe, which also suggested that more than one activated X chromosome was present in at least 2/7 NHL cases. The two approaches therefore provided evidence that in some cases of NHL, unlike sex-chromosome-syndrome individuals, additional X chromosomes may be present in the active state. These data support the suggestion that NHL-associated oncogenes might be located on the X chromosome.

Comparative genomic hybridization and pathological findings in atypical teratoid/rhabdoid tumour of the central nervous system.

The atypical teratoid/rhabdoid tumour (AT/RT) is an uncommon tumour of the central nervous system in children, characterized by the presence of a rhabdoid cell component associated with variable combinations of primitive neuroectodermal tumour, mesenchymal and epithelial differentiation. Immunohistochemistry reveals a complex pattern of antigen expression and cytogenetic studies have demonstrated losses from chromosome 22. We have performed comparative genomic hybridization (CGH) on paraffin-embedded material from three cases of AT/RT. Two cases showed losses from chromosome 22 associated with other chromosome imbalances including losses from 1p in both cases. The third case demonstrated a loss from 8p as the sole abnormality. While monosomy or deletion from chromosome 22 is a useful diagnostic marker for AT/RT, it is not present in all cases. The variation in cytogenetic patterns reported for this tumour type raises the possibility that different genetic pathways may underlie this tumour phenotype and warrants the further definition of the cytogenetic spectrum for this rare tumour.

Abnormalities of chromosomes 3 and 8 in posterior uveal melanoma correlate with prognosis.

Posterior uveal melanomas have nonrandom alterations affecting chromosomes 3, 6, and 8. Loss of chromosome 3 in uveal melanoma has been shown to act as a predictor of disease-free and overall survival. To confirm the significance of chromosome 3 loss and to extend the observations to include those of the associated alterations of chromosome 8, we have conducted a cytogenetic analysis on a series of 42 tumours from patients with primary uveal melanoma who were followed up for a median of 31 months (range = 8-96 months). Abnormalities of chromosomes 3 and 8 were the commonest changes and were confirmed in 10 tumours using fluorescence in situ hybridization. Monosomy of chromosome 3 was found in 21 (50%) of the tumours, and 23 (54%) tumours had additional copies of 8q. Alterations of chromosomes 3 and 8 were found occurring together in 19 (45%) of the tumours and were significantly associated with a ciliary body component (P < 0.0001). Prognostic indicators and changes of chromosomes 3 and 8 were analysed for correlation with patient survival. Of the chosen parameters, only ciliary body involvement (P = 0.003), monosomy of chromosome 3 (P = 0.0007), and additional copies of 8q (P = 0.003) correlated with reduced survival. Evaluation of the dosage effect of additional copies of chromosome arm 8q showed a significant association with reduced survival (P = 0.0001), which was also predictive of a decreased disease-free interval (P = 0.01). Thus, the cytogenetic analysis of uveal melanoma may provide a valuable predictor of prognosis.