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Keywords:

  • NK/T-cell lymphoma;
  • CGH;
  • LOH;
  • chromosome 6;
  • P53 mutation

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

BACKGROUND

Nasal-type NK/T-cell lymphoma is a rare type of non-Hodgkin's lymphoma. The genetic changes associated with pathogenesis have not been well defined. This study investigates the nonrandom genetic alteration of nasal-type NK/T-cell lymphoma. Methods: Nine cases were studied. Comparative genomic hybridization (CGH) was carried out using fresh tumor tissues of seven nasal-type NK/T-cell lymphomas. To complement the data by CGH, loss of heterozygosity (LOH) of chromosomes 6q, 1p, and 17p using polymorphic markers and p53 gene mutation by polymerase chain reaction–single-strand conformation polymorphism (PCR-SSCP) were analyzed. Results: The DNA copy number changes of seven nasal-type NK/T-cell lymphomas were gains on chromosomes 2q(5), 13q(4), 10q(3), 21q(2), 3q(2), 5q(2), and 17q(2), and losses involving chromosomes 1p(4), 17p(4), 12q(3), 13q(2), and 6q(1). One of six cases informative for at least two markers for chromosome 6q showed LOH at D6S300, D6S1639, D6S261, D6S407, and D6S292. Two cases showing loss of 1p and 17q by CGH revealed LOH at D1S214, D1S503, and D17S559. P53 mutation was detected in exon 8 in one of nine cases. Conclusion: Frequent DNA losses at 1p, 17p, and 12q and gains at 2q, 13q, and 10q suggested that these regions could be targets for further molecular genetic analysis to investigate tumor suppressor genes or oncogenes associated with tumorigenesis of NK/T-cell lymphoma. Infrequent alteration of 6q contrary to previous studies raises doubt about an implication of 6q loss in the pathogenesis of early-stage NK/T-cell lymphoma. Further studies on more defined cases are required to verify their association. Cytometry (Comm. Clin. Cytometry) 46:85–91, 2001. © 2001 Wiley-Liss, Inc.

Among lymphoid malignancies of NK-lineage, nasal NK/T-cell lymphoma is the most well known. It presents with a destructive lesion in the nasal cavity and nasopharynx and is highly associated with Epstein-Barr virus (EBV). Tumors with similar histologic and immunophenotypic features to those of nasal lymphoma occur less frequently in extranasal sites including skin, gastrointestinal tract, and soft tissue (1, 2). These nasal and extranasal NK/T-cell lymphomas were classified as extranodal NK/T-cell lymphoma, nasal type, in the World Health Organization (WHO) classification (3).

Although the clinicopathologic features of nasal-type NK/T-cell lymphoma have been well defined, little is known about the pathogenetic mechanism associated with lymphomagenesis. A few reports describe the common deletion of chromosome 6q by conventional cytogenetic study and p53 alteration detected by immunohistochemistry and molecular methods (4–8). Lymphomas arise from a clonal expansion of lymphoid cells that are transformed by the accumulation of genetic lesions affecting oncogenes and tumor suppressor genes. During the past decade, recurring cytogenetic abnormalities closely associated with morphologically and clinically distinct subsets of lymphoma led to the identification of the genes involved in lymphomagenesis.

In NK/T-cell lymphoma, the cytogenetic study has been hampered because of the rarity of this type of lymphoma and the difficulty in applying conventional banding technique on the small biopsy from the nasal cavity. Comparative genomic hybridization (CGH) provides an opportunity to scan the entire human genome and localize genetic alterations to a specific chromosomal region in a single experiment using a small amount of tumor tissue without cell culture (9).

In this study, we applied CGH to seven nasal-type NK/T-cell lymphomas to define the chromosomal regions that are putatively involved in lymphoma development. Additionally, analyses for loss of heterozygosity (LOH) of chromosomes 6q, 1p, and 17p and p53 gene mutation were carried out to confirm their association with nasal-type NK/T-cell lymphoma.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Case Selection

The pathologic diagnosis of nasal-type NK/T-cell lymphoma was based on the criteria described by Jaffe et al. (10). Tumors presenting with nasal or midfacial destructive lesions were diagnosed when they expressed cCD3 and CD56 on paraffin sections in the absence of B-cell markers. Tumors in extranodal sites, excluding the nasal cavity and nasopharynx, were included when they showed characteristic histologic features of nasal-type NK/T-cell lymphoma with expression of cCD3 and CD56, lack of clonal T-cell receptor gene rearrangement, and positivity for EBV by in situ hybridization (ISH). Nine nasal-type NK/T-cell lymphomas were included for study. All were primary tumors. Their clinical and genotypic findings are summarized in Table 1.

Table 1. Clinical Data of Nine Patients Included in the Study*
CaseAge/sexPrimary siteDiagnosisEBV ISHTCRγStageTxPrognosis
  • *

    ES, ethmoid sinus; DOD, died of disease; AOD, alive without disease; TCRγ, gene rearrangement; G, germline; R, rearranged; ChemoTx, chemotherapy; RT, radiotherapy.

140/MNoseNasal-type+GIIChemoTxDOD
233/MNoseNasal-type+GIChemoTxAOD
376/FCalfNasal-type+GIExcisionAOD
455/FCecumNasal-type+GIExcisionAOD
548/FNose/ESNasal-type+GIChemoTxRecur
660/MNose/ESNasal-type+GIChemoTxRecur
753/MNoseNasal-type+GIRTProgress
856/MNoseNasal-type+RIChemoTxDOD
949/FNoseNasal-type+GIChemoTxAOD

Immunohistochemistry

Immunophenotyping was performed using a panel of monoclonal antibodies including polyclonal CD3 (Dakopatts, Copenhagen, Denmark), CD20 (Dakopatts), and CD56 (Monosan, Uden, The Netherlands).

EBV ISH

EBV RNA was detected by ISH technique using fluorescein isothiocyanate (FITC)-conjugated EBV oligonucleotides (Dakopatts) complementary to the nuclear RNA portion of the EBER 1 and 2 genes.

Gene Rearrangement Study

For PCR amplification of the TCRγ locus, a seminested polymerase chain reaction (PCR) followed by heteroduplex analysis was performed as described (11, 12).

CGH

Probe DNA was isolated from tumor tissue and reference DNA was isolated from peripheral blood lymphocytes of healthy males and females using the genome DNA kit (BIO 101, USA.) as described by the manufacturer. Paraffin and frozen sections of the tumor sample were reviewed before CGH analysis. The proportion of tumor cells in each sample was at least 50%.

Metaphase slides for hybridization were purchased from Vysis. CGH was performed as described (9). Briefly, tumor DNA was directly labeled with SpectrumGreen dUTP and reference DNA was labeled with SpectrumRed dUTP of the CGH nick translation kit (Vysis). The DNase concentration in the labeling reaction was adjusted to reveal an average fragment size of 300–2,000 bp. Mixtures of 150 ng of labeled reference DNA, 300 ng of tumor DNA, and 20 μg of unlabeled human Cot-1 DNA were precipitated and resolved in 10 μl of hybridization buffer. The hybridization mixture was denatured at 75°C for 5 min and preannealed at 37°C for 1 h. Normal metaphase slides were denatured in formamide solution(70% formamide in 2 × SSC, pH 7.0) for 2 min and dehydrated in graded ethanol. The probe mixture was applied to the slides under a coverslip, sealed with glass coverslip sealant (Oncor), and hybridized for 3 days at 37°C. The slides were washed three times in 50% formamide/2 × SSC (pH 7.0), twice in 2 × SSC, and once in 0.1 × SSC at 45°C. After air drying, the slides were counterstained with DAPI in an antifade solution. The experiment included a negative control (peripheral blood DNA from a healthy donor) and a positive control (tumor DNA with known copy number changes).

Digital Image Analysis and Interpretation of CGH Results

The hybridizations were analyzed using an Olympus fluorescence microscope and an automated CGH software package (Applied Imaging, CA). Three-color images (red for reference DNA, green for tumor DNA, blue for counterstaining) were acquired from 8–10 metaphases per sample. Only metaphases of good quality with strong, uniform hybridization were used in the analysis. As tumor specimens and normal DNA samples were not sex matched, X and Y chromosomes were excluded. Also excluded were centromeric and satellite regions of the acrocentric chromosomes and chromosome 19, because of the abundance of highly repetitive DNA sequences and frequent occurrence of false positive CGH results. Chromosomal regions were interpreted as overrepresented when the corresponding ratio exceeded 1.25 (gains) or 1.5 (high-level amplifications) and as underrepresented (losses) when the ratio was less than 0.75. The results were confirmed using a 99% confidence interval.

LOH Assay

Microsatellite PCR to detect LOH of chromosome 6q was performed in the seven cases for which tumor DNA and reference DNA were available. Eleven polymorphic markers that covered the regions from 6q21 to 6q24 according to the Entrez data base (National Center for Biotechnology information, NIH, Bethesda, MD) were used. The markers were D6S300, D6S424, D6S468, D6S407, D6S278, D6S261, D6S287, D6S262, D6S292, D6S1639, and D6S1699 (Research Genetics, Huntsville, AL). The LOH at chromosomes 1p and 17p was analyzed in four cases. The following 11 polymorphic markers were used: D1S2795, D1S214, D1S503, D1S507, D1S482, D1S211 (all located on chromosomes 1p32.3-36.3), D1S2861 (located on chromosome 1p), and D17S513, D17S1176, D17S525, D17S559 (all located on chromosome 17p13; Research Genetics).

Reference DNA was obtained from the uninvolved peripheral blood (case 5) or from bone marrow biopsy for staging (cases 1–4,8,9). Microsatellite PCR was performed on the genomic DNA samples using the following conditions: 5 ng DNA, 10 pmol of each primer, 2 nmol of each of four dNTP, 0.5 U Taq DNA polymerase, 2 μCi [32 P] dCTP in 20 μl of the PCR buffer with 1.5 mM MgCl2 (Boehringer Mannheim, Indianapolis, IN). After denaturation at 95°C for 2 min, PCR was carried out for 35 cycles as follows: denaturation at 94°C for 45 s, annealing at 55°C for 60 min, and synthesis/extension at 72°C for 1 min. After amplification, 1.5 μl of PCR products was electrophoresed through a 6% denaturing polyacrylamide gel containing 8.3 M urea. Subsequently, the gels were dried and subjected to autoradiography using Kodak XAR film at -80°C.

All samples in which two distinct alleles of similar intensity were present in the normal DNA were considered to be informative. LOH was scored as positive when a clear reduction in signal intensity was detected in one of the alleles of the tumor DNA compared with the paired normal DNA.

Immunohistochemistry for p53 Protein

Formalin-fixed paraffin-embedded tissue was cut into 3-μm thick sections, dewaxed, rehydrated. The slides were treated in a microwave oven for 15 min in 10 mM citrate buffer for antigen retrieval. A monoclonal antibody (DO-7; Dakopatts) was applied at a dilution of 1:10. Reaction was detected by the avidin-biotin complex system and counterstained with Meyer's hematoxylin.

Analysis of p53 Mutation

All samples were analyzed for p53 mutations by using single-strand conformation polymorphism (SSCP)-PCR. The PCR primer pairs for the amplification of the p53 gene exon 5 through 8 were 5′-GTACTCCCCTGCCCTCAACA-3′ and 5′-CTCACCATCGCTATCTGAGCA-3′ for exon 5, 5′-TTGCTCTTAGGTCTGGCCCC-3′ and 5′-CAGACCTCAGGCGGCTCA- TA-3′ for exon 6, 5′-TAGGTTGGCTCTGACTGTACC-3′ and 5′-TGACCTGGAGTC- TTCCAGTGT-3′ for exon 7, and 5′-AGTGGTAATCTACTGGGACGG-3′ and 5′-ACCTCGCTTAGTGCTCCCTG-3′ for exon 8. PCR was performed as follows: After denaturation at 95°C for 5 min, 30 cycles of denaturation at 95°C for 45 s, annealing at 60°C for 45 s, and extension at 72°C for 1 min were carried out. The amplified products were electrophoresed on a 12% polyacrylamide gel containing 7 mol/L urea.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

CGH

Seven cases for which fresh tumor tissue was available were analyzed by CGH. There were seven nasal-type NK/T-cell lymphomas from the nasal cavity (five), the cecum (one), and calf muscle (one). In all seven nasal-type NK/T-cell lymphomas analyzed by CGH, chromosomal gains or losses were identified. An example of a hybridization experiment is shown in Figure 1. An average of seven chromosomal changes per tumor were found. Overrepresentations of chromosomal material were more frequent than underrepresentations (32 gains versus 17 losses). The most frequent aberrations were gains on chromosomes 2q (five patients), 13q (four patients), 10q (three patients), 21q (three patients), 1p (two patients), 1q (two patients), 3q (two patients), 5q (two patients), and 17q (two patients), as well as losses on chromosomes 1p (four patients), 17p (four patients), 12q (three patients), 13q (two patients), and 6q (one patient). On chromosome 13, the commonly gained region mapped to bands 13q21-q33 whereas bands 13q14-q31 were often underrepresented. A synopsis of all chromosomal imbalances is shown in Figure 2.

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Figure 1. A: Reverse DAPI profile in case 5. B: CGH images of the same metaphase. Note red region representing loss in chromosome 6q.

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Figure 2. Summary of gains and losses of DNA sequences identified by CGH. Gains are shown as vertical lines on the right side of the chromosome idiogram and losses on the left side. High-level amplification is marked as a thick black bar on the right side

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LOH at Chromosome 6q

The results by LOH analysis are shown in Table 2 and Figures 3 & 4. The LOH analysis for chromosome 6q was performed in the seven cases including five cases analyzed by CGH (cases 1–5) and two without CGH study (cases 8,9). Cases 6 and 7 were excluded because of lack of reference DNA. Of seven cases analyzed for LOH, six were informative for at least two 6q loci. Case 5 with 6q loss by CGH showed LOH at all informative loci including D6S300, D6S1639, D6S261, D6S407, and D6S292. No LOH of 6q was detected in cases that were normal by CGH.

Table 2. Results of CGH, LOH, and SSCP Analysis*
CaseDNA copy number changesLOH at 6qLOH at 1pLOH at 17p13P53 gene mutation
LossGain
  • *

    NI, noninformative; ND, not performed because of insufficient material.

11p31-pter,12q24,13q14-q31,3q,5q,10q22-q23,13q31-q34,15q23-qterNIND
24q21-q27,13q21-q31,17p10q, 13q31-q34, 21q21-qterNINDND
31p31-pter,12q243q,5q14-q34,18p,18q21-q23,21q21-qter+
48p12-p21,14q24,17p2q,11p12-p14NDND
51p34-pter,6q21-qter, 17p13,12q23-q24,1p21-p31,1q21-q31, 2q31-q32,11q14-q22, 17q21-q24,X+++
617p12-p132q12-q14,2q33-q36,10q23-q25,13q31-q32,18q22NDNDND
71p34-pter1p21-p31,1q21-q31,2q22-q32,6q22-q24,13q21-33,17q21-q24,22q12-q13NDNDNDExon 8
8ND
9NDNDND
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Figure 3. Summary of LOH involving chromosome 6q. Along the idiogram, each locus is indicated by a box. Case 5 reveals LOH at all the informative markers.

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LOH at Chromosomes 1p and 17p

Of four cases analyzed for LOH at 1p and 17p (cases 1,3,5,8), three (cases 3,5,8) were informative for at least two markers (Fig. 4). No LOH was identified in case 8. Cases 3 and 5 showed LOH at D1S214 and D1S503, which was compatible with the loss of chromosome 1p by CGH. The LOH at D17S559 was detected in case 5, which showed loss of 17p by CGH.

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Figure 4. Summary of LOH involving chromosome 1p and 17p. Along the idiogram, each locus is indicated by a box.

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P53 Protein Expression

Expression of p53 protein was assessed semiquantitatively. The case was regarded as negative when less than 10% of tumor cells were stained. Of six cases stained for p53 protein, 20–80% of tumor cells stained positively in four cases (2, 4, 6, 7).

P53 Gene Mutation

Of nine samples, one case (case 7) showed an abnormal band in exon 8 by SSCP analysis (Fig. 5).

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Figure 5. PCR-SSCP analysis of p53 mutations (exon 8) in nine nasal-type NK/T-cell lymphomas. Case 7 shows abnormal bands in exon 8. N, normal tissue; M, size marker.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Expression of CD56 is a common denominator of NK/T-cell lymphomas. Tumors arising in the nasal cavity are associated with EBV in more than 80% of cases. Although nasal NK/T-cell lymphomas are mostly of NK lineage, a few cases demonstrate TCR gene rearrangement (10, 13) and share similar histologic characteristics and high association with EBV (13, 14). NK/T-cell lymphomas arising in the extranasal sites are diagnosed as “nasal-type” when they are of NK lineage and positive for EBV (10).

The genetic information in nasal-type NK/T-cell lymphomas has been limited. Conventional cytogenetic study has been available in only 14 cases (4–6). An aberration involving chromosome 6q was reported in 5 of 14 cases. Other abnormalities including i(1q), del(7q), del(12q), del(17p), and 11q23 rearrangements were sporadically described. Although a limitation of our study is the number of cases (nine), the data show nonrandom alterations of specific chromosomal segments, some of them corresponding to the sites reported by conventional cytogenetic study.

Chromosomal losses and deletions are indicative of the involvement of tumor suppressor genes in tumorigenesis (15). The deletions in our study frequently involved chromosomes 1p, 17p, and 12q. Deletion of chromosome 1p, especially involving segment 1p36, is one of the common secondary cytogenetic changes detected in 12% of non-Hodgkin's lymphoma cases (16). Chromosomal segments spanning 1p31-p36 contain many candidate tumor suppressor genes including the MTS1 gene. Likewise, 17p is a chromosomal region containing several tumor suppressor genes including the p53 gene and one commonly deleted segment by conventional cytogenetic study in non-Hodgkin's lymphoma (17). Using conventional cytogenetics, alterations of chromosome 1p and 17p in nasal-type NK/T-cell lymphomas manifested as a deletion or as i(17)(q10) or i(1)(q10). The formation of an isochromosome results in loss of genetic material. Therefore, i(17)(q10) or i(1)(q10) represents one more way through which loss of one allele of p53 and other tumor suppressor genes is achieved. In nasal-type NK/T-cell lymphomas, alteration of the p53 gene is common. Abnormal expression of the p53 protein and mutation of the p53 gene were detected in 21.4–88.9% and in 22.2–60% of cases in studies conducted on Chinese and Japanese populations, respectively (7). In present study the mutation rate was lower than previous report, but high frequency of p53 protein expression and common loss of 17p by CGH suggested pathogenetic implication of tumor suppressor genes including p53 gene in nasal-type NK/T-cell lymphoma.

In our study, the low frequency of the 6q deletion by CGH is surprising. The 6q deletion is the most common alteration reported in 5 of 14 cases by conventional and molecular cytogenetic study (4–6, 18, 19). This discrepancy could be explained by the technical limitation of CGH. Although conventional cytogenetic study can detect changes in individual cells, these changes should be present in at least 50% of the cell samples used for CGH study. Because NK/T-cell lymphomas contain many inflammatory cells, the positive small fluorescence signal detected by CGH analysis could be blunted by admixed non-neoplastic cells (9). On the other hand, failure to detect more alterations of chromosome 6q by additional LOH analysis supports the result by CGH study.

The significance of the 6q deletion on the pathogenesis of nasal NK/T-cell lymphoma remains unclear. In non-Hodgkin's lymphoma, loss of 6q, particularly at the regions 6q21-23 and 6q25-27, has been well studied in B-lineage lymphomas. It is the most common secondary aberration. Loss of 6q is detected in up to 40% of cases after the tumor has been established, reflecting clonal evolution. Occasionally, it has been correlated with clinical features of non-Hodgkin's lymphoma such as tumor progression, transformation, and survival (20–22). In our study, most cases were primary tumors in stage I. Therefore, the low frequency of the 6q alteration might reflect the bias in case selection that included cases of early clinical stage before additional chromosomal alterations occurred.

Although DNA losses suggest secondary genetic alteration involving tumor suppressor genes, gene amplification including DNA gain is an essential mechanism of oncogene activation. The characterization of chromosomal amplicon areas will provide the means to discover mechanisms that activate several cellular oncogenes and other genes (23). Generally, conventional cytogenetic analysis detects more losses than gains of chromosomal material, whereas CGH is a more powerful method to detect gains over losses. In our series, DNA gains are more common than losses and involve chromosomal regions 2q, 13q, 10q, 21q, 3q, 5q, and 17q in the decreased order of frequency. Gains in 13q, 2q, 10q, and 21q are nonrandom, affecting three or more of cases.

Gains in 13q and 10q have not been described before in cytogenetic studies of NK/T-cell lymphoma. In our series, a gain of chromosome 13q mapped to bands 13q21 and 13q31-33, which contain several unidentified proto-oncogenes including the G-protein–coupled receptor 18 (24). Chromosome 2q also contains a few oncogenes including TCL4 rearranged in the human leukemic T-cell line (25). The long arm of chromosome 10 contains several known genes such as NFκB and RET, which have been implicated in tumorigenesis (26, 27).

In summary, nonrandom genetic alterations affecting specific chromosomal regions in nasal-type NK/T-cell lymphoma are detected by CGH. Despite the limitation of the CGH technique in detecting minor alterations, it provides information that can be used to discover the pathogenetic mechanism of nasal-type NK/T-cell lymphomas.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED
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