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A high level of microsatellite instability was associated with histological transformation of two cases of. FCL, but no mutations of the hMLH1 and hMSH2 genes ...
Leukemia (2000) 14, 2142–2148  2000 Macmillan Publishers Ltd All rights reserved 0887-6924/00 $15.00 www.nature.com/leu

Genetic instability is associated with histological transformation of follicle center lymphoma M Nagy1*, M Bala´zs2*, Z A´da´m2, Z Petko´2, B Ti´ma´r1, Z Szereday1, T La´szlo´1, RA Warnke3 and A Matolcsy1 1

Department of Pathology, Faculty of Medicine, Pe´cs University, Hungary; 2Department of Preventive Medicine, School of Public Health, University of Debrecen, Hungary; and 3Department of Pathology, Stanford University Medical Center, Stanford, CA, USA

Follicle center lymphoma (FCL) is an indolent B cell non-Hodgkin’s lymphoma (NHL) characterized genetically by the t(14;18) translocation. Histological transformation and clinical progression of FCLs are frequently associated with secondary genetic alterations at both nucleic acid and chromosomal levels. To determine the type and pattern of genomic instability occurring in histological transformation of FCLs and the role of DNA mismatch repair defects in this procedure, we have performed microsatellite analysis, comparative genomic hybridization (CGH) and mutational analysis of hMLH1 and hMSH2 genes on serial biopsy specimens from patients with FCL transformed to diffuse large cell lymphoma (DLCL). Paired biopsy samples of eight patients were analyzed for microsatellite instability and structural alterations for hMLH1 and hMSH2 genes, and tumor samples of five patients were subjected to CGH analysis. A high level of microsatellite instability was associated with histological transformation of two cases of FCL, but no mutations of the hMLH1 and hMSH2 genes were detected in any of the lymphoma samples. In the five cases subjected to CGH analysis, the histological transformation of FCLs was associated with genomic imbalances at 21 chromosomal regions. The genomic abnormalities found were rather heterogeneous and none of the genetic changes were overrepresented in the transformed DLCLs. These data suggest that histological transformation of FCLs to DLCL is frequently associated with genome wide instability at both nucleic acid and chromosomal levels, although mutations of the hMSH1 and hMLH2 genes are not involved in this process. Leukemia (2000) 14, 2142–2148. Keywords: follicular lymphoma; genetic instability; lymphoma transformation

Introduction Follicle center lymphoma (FCL) is a B cell non-Hodgkin’s lymphoma (NHL) with a relatively indolent clinical course.1,2 Histological transformation of FCL to aggressive diffuse large cell lymphoma (DLCL) occurs in about 25% to 30% of patients during the later stage of the disease.3,4 This transformation is usually associated with acceleration of the clinical course and shortened survival.5,6 Approximately 85–90% of FCLs carry the t(14;18)(q32;q21) chromosomal translocation that places the BCL-2 oncogene into juxtaposition with the immunoglobulin (Ig) heavy chain (H) gene.7 The t(14;18) translocation upregulates the expression of the BCL-2 gene-product that induces prolonged cell survival by blocking programmed cell death (apoptosis) (reviewed in Ref. 8). In the histological transformation of the FCL the neoplastic cells retain their t(14;18) translocation and frequently acquire additional genetic abnormalities, although these aberrations are heterogeneous and none of them seems Correspondence: A Matolcsy, Department of Pathology, Pe´cs University, Faculty of Medicine, H-7624 Pe´cs, Szigeti u´t 12, Hungary; Fax: (36–72) 216–732 *These authors contributed equally to this work. Received 12 July 2000; accepted 1 September 2000

to be a predominant genetic lesion responsible for the transformation. It has been demonstrated that secondary chromosomal defects,9–12 mutations of the c-MYC, p53, RAS, BCL-2 and BCL-6 genes,13–18 and allelic loss, mutation or hypermethylation of the p15INK4B and the p16INK4A genes19–21 may be associated with the transformed stage of FCL. The progressive accumulation of multiple genetic lesions in transformed stage of the FCLs strongly suggests that an acquired genome wide instability or mutator phenotype of the FCL cells predisposes and promotes the histological transformation. Several lines of evidence suggest that different types of genomic instability appear to be involved in tumor development and progression. In subsets of tumors, genetic instability is manifested at the level of nucleotide sequence as errors in DNA replication, usually detected as alterations in the length of short repetitive sequences (microsatellites). Microsatellite instability (MSI) is frequently associated with the somatic mutations of the DNA mismatch repair genes (reviewed in Refs 22 and 23). The other pathway of genomic instability is manifested at chromatin maintenance and segregation. In this pathway, genomic instability is evident by the presence of multiple chromosomal alterations including loss or gain of whole chromosomes, translocations, deletions and amplifications (reviewed in Ref. 24). To analyze whether histological transformation of FCL is associated with genomic instability at the nucleotide or chromosome level, we have performed longitudinal microsatellite analysis and comparative genomic hybridization (CGH) in paired samples of FCL and clonally related transformed DLCL. We have also analyzed the nucleic acid sequences of the hMLH1 and hMSH2 genes to reveal whether DNA mismatch repair defects are associated with the histological transformation of FCL. Our results indicate that both microsatellite instability and chromosomal imbalance are frequently associated with histological transformation of FCL, but these alterations are not related to the mutation of the hMLH1 or hMSH2 genes. Materials and methods

Pathological samples and DNA extraction Sequential lymph node biopsy samples of eight patients with FCL observed at Stanford University Medical Center and Faculty of Medicine, Pe´cs University were selected for this study based on the availability of frozen tissue for the molecular analyses. Diagnoses were based on histopathologic, immunophenotypic and immunogenotypic analyses according to the Revised European–American Lymphoma Classification.2 The histology of the first lymph node biopsy in four patients (cases 1–4) was FCL, provisional cytologic grade I, in three patients (cases 5–7) was FCL, provisional cytological grade II, and in one patient (case 8) was FCL, provisional cytological grade

Genetic instability in lymphoma transformation M Nagy et al

III. In all the patients, the histology of the second biopsy was classified as DLCL (Table 1). All samples included in this study displayed monoclonal IgH gene rearrangement and t(14;18) translocation. In each case, the subsequent biopsy samples showed identical Ig heavy chain gene rearrangements and identical breakpoint sequences of the t(14;18) translocation indicating the common clonal origin of the tumor samples of the first and the second biopsies. Lymph node biopsy samples of five patients with FCL, provisional cytologic grade I and normal peripheral blood samples of the same patients were also evaluated. Genomic DNAs were extracted from cryopreserved tissue samples, using the salting out technique.25

Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of the hMLH1 and hMSH2 genes

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PCR-SSCP analysis of the hMLH1 gene was performed on five exons (exons 9, 11, 14, 15 and 16). The selection of these exons for the mutational analysis of hMLH1 gene was based on the evidence that mutations of these sequences had been described in previous reports.26,27 The PCR-SSCP analysis of the hMSH2 gene was performed on nucleotide positions 2020–2225 of the cDNA and the flanking regions of the genomic DNA containing the intron/exon junctions.28 The PCRSSCP analysis of the hMLH1 and hMSH2 genes was performed as described previously.26,28

Analysis of microsatellite instability Comparative genomic hybridization Eight microsatellite repeat markers mapping to different chromosomes were studied, including five dinucleotide repeats (DCC, D6S262, D3S1262, D3S1261, MYC), one trinucleotide repeat (AR), and two tetranucleotide repeats (ACTBP2, FGA). Each microsatellite repeat was amplified by polymerase chain reaction (PCR). Primers were synthesized based on sequences derived from the Genome Data Bank (GDB, Baltimore, MD, USA) by Integrated DNA Technologies (Coralville, IA, USA). PCR reactions were performed in a final volume of 10 ␮l containing 100 ng of DNA template, 1–2 mmol/l MgCl2, 10 pmol of each primer, 2.5 ␮mol/l dNTPs, 1 ␮Ci of ␣-32P dCTP, 0.5 U Taq polymerase. Thirty-five cycles were performed consisting of denaturation (30 s at 94°C), annealing (annealing temperatures were optimized for each pair of primers), and extension (30 s at 72°C) using a Perkin-Elmer 2400 GeneAmp PCR system (Norwalk, CT, USA). Annealing temperatures were 55°C for D3S1261, DCC, MYC; 58°C for ACTBP2, FGA, D6S262, D3S1262; 62°C for AR. PCR products were separated by electrophoresis in 6% polyacrylamide-TBE denaturing gels for 2–4 h at 70 W. Gels were fixed in 10% acetic acid, air dried, and exposed to X-ray films at −70°C.

Table 1 Summary of clinical data of eight patients with FCL transformed to DLBL

Case No. 1 2 3 4 5 6 7 8

Sample

Date of biopsy

Histology

A B A B A B A B A B A B A B A B

1985 1988 1987 1988 1984 1986 1986 1987 1989 1990 1991 1992 1986 1991 1989 1990

FCL-I DLCL FCL-I DLCL FCL-I DLCL FCL-I DLCL FCL-II DLCL FCL-II DLCL FCL-II DLCL FCL-III DLCL

DLCL, diffuse large C cell lymphoma; FCL-I, follicle center lymphoma, follicular, cytological grade I; FCL-II, follicle center lymphoma, follicular, cytological grade II; FCL-III, follicle center lymphoma, follicular, cytological grade III.

Comparative genomic hybridization was performed as described by Kallioniemi et al29 with modification. Briefly, tumor DNA was directly labeled with SpectrumGreen-12dUTP (Vysis, Downers Grove, IL, USA) and normal DNA was labeled with SpectrumRed-5-dUTP (Vysis) by nick translation according to the protocol of the supplier. The experimental conditions were adjusted to allow DNA fragments of 600– 2000 bp to be obtained. The hybridization mixture, consisting of 200 ng each of labeled DNA and 10 ␮g of unlabeled human Cot-1 DNA (Gibco BRL, Life Technologies, Gaithersburg, MD, USA), was precipitated and dissolved in 10 ␮l of hybridization mixture. Probe mixture was denatured at 73°C and reannealed for 30–60 min at 37°C before being applied on to normal metaphase spreads (Vysis). Slides were denatured at 73°C, dehydrated through an ethanol series and air dried. Hybridization was carried out at 37°C in a moist chamber for 72 h. After hybridization, slides were washed three times in hybridization wash buffer at 45°C, once in 2 × SSC for 10 min at room temperature and air dried. Nuclei were stained with 0.15 ␮g/ml 4,6-diamino-2-phenylindole (DAPI; Vysis) in anti-fade solution. One negative (differentially labeled, normal DNA vs normal DNA) and a positive control (SpectrumGreen-12-dUTP labeled, cytogenetically wellcharacterized breast cancer cell line MPE-600) were included to monitor hybridization quality.

Digital image analysis A multicolor quantitative image processing system (ISIS; MetaSystem, Altlussheim, Germany) connected to a Zeiss fluorescence microscope was used for the acquisition and evaluation of the hybridized metaphases. Gray level images were acquired using a high sensitivity monochrome charge-coupled device (Compulog, IMAC-CCD-230, 8-bit resolution; Metasystem). After automatic interchromosomal background subtraction, chromosome segmentation was performed by thresholding the DAPI image. The fluorescence intensity profiles of green and red fluorescence were calculated by integrating fluorescence values across the chromosome width along the medial axis. The green to red ratios of each chromosome were plotted as a function of distance from the p telomeric region to the q telomeric region. Data from six to 10 chromosomes taken from three or more metaphases were used. The individual ratio profiles were combined to obtain the averaged ratio profiles that are displayed next to the ideograms together with significance intervals of 0.85 and 1.15. A gain of DNA Leukemia

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Table 2

Case No. 1 2 3 4 5 6 7 8

Microsatellite alterations associated with histological transformation FCL to DLCL

ACTBP2

AR

D3S1261

D3S1262

D6S262

DCC

FGA

MYC

− LOH MSI LOH − LOH MSI LOH

− LOH − − − − MSI −

− − − − − − − −



− MSI MSI − − LOH LOH −

− MSI − − − − MSI −

− − − − − MSI − MSI

− LOH − − − − LOH −

− − − − − −

LOH, loss of heterozygosity; MSI, microsatellite instability; −, no microsatellite alteration.

sequences was assumed at chromosomal regions where the hybridization resulted in a green (tumor) to red (normal) ratio of ⬎1.15. Over-representation defined by a sharp peak in a sub-region of a chromosome was considered as amplification (mean green/red ratio exceeding 1.5). A loss of DNA sequences was assumed at chromosomal regions where the hybridizations resulted in a green/red ratio of ⬍0.85. Ratio changes at the heterochromatic regions or the p-arms of acrocentric chromosomes were excluded from the analysis. Only images showing uniform high-intensity fluorescence were analyzed.

FCL and transformed DLCL samples. However, it could not be established whether microsatellite alteration had already occurred at the FCL stage of the disease or had developed only during tumor progression, since non-neoplastic control DNAs were not available in these eight cases. To analyze whether microsatellite alteration occurs in the FCL cells or develops exclusively in the transformed DLCL cells, in five patients eight microsatellite loci were compared to FCL and corresponding normal peripheral blood cells. In all the five patients, identical banding patterns of FCL and normal peripheral blood DNAs were detected (results are not shown).

Results

PCR-SSCP analysis of the hMLH1 and hMSH2 genes

Microsatellite analysis

PCR-SSCP analysis of hMLH1 and hMLH2 genes was performed in eight cases. In each case, the samples of the first and second biopsies were evaluated simultaneously. The PCRSSCP analysis revealed normally migrating bands exclusively, suggesting that nucleotide alterations of the hMLH1 and hMLH2 genes had not occurred in FCL and transformed DLCL cells (results are not shown).

In each case, FCL and corresponding transformed DLCL samples were evaluated in parallel using eight microsatellite markers. A locus was considered positive when the electrophoretic migration pattern differed in DNA from FCL compared with DNA from transformed DLCL samples. MSI was evident when DNA of DLCL displayed one or more new bands compared to their FCL counterparts. Loss of heterozygosity (LOH) was considered positive if bands of FCL samples were lost in DLCL samples. The results of the microsatellite analysis are summarized in Table 2 and representative cases are illustrated in Figure 1. The eight microsatellite loci of the eight patients displayed a total of 18 differences between FCL and transformed DLCL samples. Both MSI and LOH were detected in nine microsatellite loci. In one case (case 4) a single, in two cases (cases 3 and 8) two, in one case three (case 6) and in two cases (cases 2 and 7) five microsatellite loci showed alterations. In two cases (cases 1 and 5), the patterns of the microsatellites were identical in

Figure 1 case 7. Leukemia

CGH analysis The CGH analysis has been performed on FCL and transformed DLCL samples in five cases (cases 1–5). The chromosomal gains and losses are summarized in Table 3 and representative example of CGH profiles are shown in Figure 2. Chromosomal imbalance was found in one case of FCL but all the transformed DLCL samples displayed one or more chromosomal alterations. In the FCL sample two loci showed DNA amplification (1q13-qter and 17p12-qter), which was also present in the transformed DLCL sample of the patient.

Representative results of microsatellite analysis of paired serial biopsy specimens of FCL (A) and transformed DLCL (B) samples in

Genetic instability in lymphoma transformation M Nagy et al

Table 3 DNA gains and losses associated with FCL and transformed DLCL

Case No. 1 2 3 4 5

Sample

DNA gains

DNA losses

A B A B

− − − 2q14-q31, 6q, 13q22

A B A B

− 18 − 6p, 8, 13q31-qter, 18, 22q 1q31-qter, 17p12-qter 1q31-qter, 17p12-qter − 11q13, 11q23-qter

A B

− 2p14-pter, 13q31-qter

− 6q16-qter, 22q12-qter − 1p21-p12, 3q21-qter, 11q14-q22 − 9p21-pter, 13q13-q21, 14q

−, no DNA gain or loss.

In the transformed DLCL samples, the chromosomal gains and losses were highly heterogeneous. Overrepresenation of DNA sequences was detected at eight different sites (2p14-pter, 6p, 8, 11q13, 11q23-qter, 13q31-qter, 18 and 22q) and underrepresentation of DNA sequences was detected at 10 different loci (1p21-p12, 2q14-q31, 3q21-qter, 6q16-qter, 6q, 9p21-pter, 11q14-q22, 13q13-q21, 14q and 22q12-qter). Gain of chromosome 18 in two cases (cases 1 and 2) and gain of 13q31-qter in two cases (cases 2 and 5) were detected as recurrent genetic alterations.

Comparison of the results of microsatellite and CGH analyses The numbers of microsatellite and CGH alterations associated with the histological transformation of FCL are summarized in

Table 4. In five of the eight FCLs (cases 1–5) the results of both microsatellite and CGH analyses were available for comparison. In two cases (cases 1 and 3) the number of microsatellite and CGH alterations was relatively low or absent, in two cases (cases 4 and 5) the number of microsatellite alterations was low or absent but the number of CGH alterations was relatively high, and in one case (case 2) the number of both microsatellite and CGH alterations were high.

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Discussion The characterization of molecular mechanisms associated with histological transformation and clinical progression of FCL are critical issues in understanding the pathogenesis as well as for the diagnosis and management of these lymphomas. Our results demonstrate that histological transformation of FCL to DLCL is frequently associated with alterations of microsatellites and gains and/or losses of DNA copy number in chromosomes. These results suggest that genetic instability at both nucleic acid and chromosomal levels may be an important pathogenic factor in the histological transformation and clinical progression of FCLs. Although MSI has been found as a rare phenomenon in Table 4 Comparison of results of microsatellite and CGH analyses associated with histological transformation of eight cases of FCL

Case No. 1 2 3 4 5 6 7 8

Microsatellite alterations

CGH gains and loss

0 5 2 1 0 3 5 2

1 8 2 5 5 ND ND ND

ND, not done.

Figure 2 Representative CGH profiles of paired serial biopsy specimens of FCL (A) and transformed DLCL (B) samples in case 2. Numbers under each profile indicate the chromosomes and the number of chromosomes analyzed in order to draw averaged profiles. Lines on the left of the chromosomal ideograms indicate loss of chromosomal material and lines on the right indicate gain of chromosomal material. Leukemia

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Leukemia

non-Hodgkin’s lymphomas,30–33 several lines of evidence suggest that microsatellite instability may develop during the clonal evolution and clinical progression of different hemopoietic neoplasms. It has been reported that microsatellite alteration may occur in blastic crisis of chronic myeloid leukemia,34 Richter’s transformation of chronic lymphocytic leukemia,31 high-grade transformation of low-grade mucosaassociated lymphoid tissue (MALT) lymphoma,35 and in the progression of acute myeloid leukemia.36 In the present study we have provided evidence that histological transformation of FCL is also associated with microsatellite instability. In five of the eight FCLs that transformed to DLCL, the second biopsy showed altered microsatellites at two or more loci, but no microsatellite alteration was detected in those five cases where FCL showed no histological alteration. Although nonneoplastic tissue samples of the transformed FCL cases were not available in this retrospective study to analyze whether microsatellite instability had already occurred before the transformation of the FCLs, our results suggest that microsatellite instability is a late event in the histological transformation of FCL. Two of the eight FCLs demonstrated a high level of MSI in the histological transformation, since in these cases five of the eight microsatellite markers displayed alterations.37 It has been demonstrated that cases that meet the criteria of the definition of high-level MSI (more than 40% of markers are positive) most frequently developed through a pathway of DNA mismatch repair gene (hMLH1 or hMSH2) defects, whereas low-level MSI (less than 40% of markers are positive) correlated with normal mismatch repair gene expression.37 Here, we have studied the most frequently mutated exons of the hMLH1 and hMSH2 mismatch repair genes for mutations but no structural alterations were found. In agreement with our observation, other studies also reported that mutations of the mismatch repair genes have not been identified in all instances of micosatellite instability, suggesting that mutations of other mismatch repair genes (hMSH3, hMSH6 or hPMS2) or epigenetic pathways may generate microsatellite instability.38 Most recently, correlation between the methylation status of mismatch repair genes and microsatellite instability has been described, indicating that events that influence the gene expression may also cause mutator phenotype of neoplastic cells.39,40 The follow-up CGH analysis of the five patients with FCLs identified two sites of chromosomal gains in FCLs but 21 additional sites displayed DNA sequence copy number abnormalities in the transformed DLCL samples. These abnormalities included increased copy numbers at 10 and reduced copy number at 11 chromosomal regions. Most of the individual CGH alterations detected in the transformation of FCLs have already been observed in other types of NHL (reviewed in Refs 41, 42). The global profile of these alterations were heterogeneous suggesting that it is genome-wide instability and imbalance among several genes that are responsible for the transformation of FCLs rather than gain or loss of a single specific gene locus. Previous cytogenetic studies also revealed that histological transformation of FCL is frequently associated with complex karyotypic changes, although some of them are found to be recurrent with varying frequency. A loss of chromosome 6 or 6q and a gain of chromosome 7 have been found most frequently in FCLs with high-grade histology.9,12,42–44 In our patients, no alterations of chromosome 7 were observed but deletion of 6q or part of 6q and gain of chromosome 18 and 13q31-qter were detected in more than one transformed FCL. Interestingly, these alterations have also

been detected in other types of ‘de novo’ NHLs including blastoid variants of mantle cell lymphoma, Burkitt’s lymphoma, diffuse large cell lymphoma and in high-grade gastrointestinal lymphoma,41,45–47 suggesting that certain primary and secondary genetic defects can be similar in different types of NHL. The total number of DNA sequence copy number imbalance can be considered as a measure of the degree of genetic instability. Generally, unstable tumors accumulate more abnormalities as they proliferate than stable tumors. The number of abnormalities accumulated during histological transformation of FCLs is remarkable in this study, ranging from none to two for FCLs and from one to eight for transformed DLCLs. The accumulation of genetic aberration in the transformed DLCLs suggests that genetic instability is a rather late event in histological transformation of FCL, although we cannot rule out the possibility that a minor cell population with an altered copy number did exist in FCL samples but their proportion was too small to affect the overall copy number balance observed. In five cases of FCL we have compared the number of the microsatellite and CGH alterations associated with histological transformation. In one case, high numbers of microsatellite alteration associated with high numbers of CGH alterations, but in another two cases high numbers of CGH alterations were detected with low numbers of microsatellite alteration. However, the number of cases analyzed in this study is relatively low; these results are in agreement with other studies which suggest that chromosome instability and DNA sequence instability may occur independently in tumor progression (reviewed in Refs 24 and 48). In conclusion, this study provides evidence that genomic instability is developed at both nucleic acid and chromosomal levels in the clonal evolution of FCLs. However, the genetic and/or epigenetic events that may initiate genome wide instability and acquisition of multiple genetic abnormalities of the tumor clone in histological transformation of FCLs remain to be determined.

Acknowledgements This work was supported by grants from the Hungarian National Science Foundation OTKA T032572 (to AM), T022429 and T032587 (to MB), from the Hungarian Ministry of Health ETT 332/2000 (to AM) and from the National Institutes of Health CA34233 (to RAW).

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