p53 Mutations in nasal natural killer/T-cell lymphoma from Mexico: association with large cell morphology and advanced disease

Abstract

Nasal NK/T-cell lymphoma is a unique form of lymphoma highly associated with Epstein-Barr virus, and with a characteristic geographic distribution. Recently, we showed that p53 is overexpressed in a high percentage of nasal NK/T-cell lymphomas. The aim of this study was to analyze the status of the p53 gene, and correlate it with the expression of p53 protein and its downstream target, the cyclin-dependent kinase inhibitor p21, in a series of 25 cases of well-characterized nasal NK/T-cell lymphoma from Mexico. The highly conserved exons 5 to 8 of the p53 gene were amplified by polymerase chain reaction and screened for mutations by denaturing high-pressure liquid chromatography. Abnormal polymerase chain reaction products detected by denaturing high-pressure liquid chromatography and additional selected cases were sequenced. In addition, the incidence of loss of heterozygosity at the p53 locus was analyzed in 12 cases. Of the 25 patients, 17 were male and 8 female (M:F ratio, 2.1:1), with a median age of 43 years (range, 21 to 93 years). Morphologically, most of the cases were composed of a mixture of medium-sized cells and large transformed cells (21 cases), and four cases were composed exclusively of large transformed cells. Three different groups determined by p53 gene status and expression of p53 protein were identified: group 1 was p53 +/p53 mutated (five cases, all with p53 missense mutations). Morphologically, three of the five cases were composed of large cells. All five cases revealed overexpression of p53 in the majority of the tumor cells with a mean of 86%. Unexpectedly, three of these cases also showed overexpression of p21. Four of the five patients presented with clinical stage IVB and died with disease. Group 2 was p53+/p53 wild-type (10 cases). Histologically, nine cases were of the mixed type, and one of the large cell type. The percentage of p53 overexpressing cells was lower than in the previous group with a mean of 23%. p21 was positive in 7 of the 10 cases. Six patients in this group presented with clinical stages I to II and four patients with advanced disease (stage III and IV). Five patients are alive 12 to 120 months later (mean, 24 months), three with no evidence of disease. Group 3 was p53-/p53 wild-type (10 cases). All cases showed mixed cell morphology. p21 was positive in 5 of 10 cases. Four patients presented with clinical stage I to II and six patients with advanced disease. Four patients are alive with no evidence of disease 9 to 60 months later (mean, 10 months). Overall, p53 mutations were present in 24% (5 of 21) of the evaluable cases, all of them overexpressing p53 in the majority of tumor cells. Cases with p53 mutations were associated with large cell morphology (P = 0.0162) and presented more often with advanced stage disease. Loss of heterozygosity at chromosome 17p was found only in 2 of the 12 (17%) cases investigated, both cases showed p53 mutations of the remaining allele. P21 overexpression (60% of cases) is frequent in nasal NK/T-cell lymphoma and seems to be independent of p53 gene status. The overexpression of p53 and p21, independent of p53 mutations, although as yet not clear, might be the result of Epstein-Barr virus infection, and warrants further investigation.

Figure 1.

A: Nasal lymphoma with angioinvasion. The neoplastic cells infiltrate the wall of a medium-sized vessel. The neoplastic infiltration is composed of a mixture of medium-sized cells and large transformed cells intermingled with rare eosinophils and plasma cells. B: In contrast to the previous example, the neoplastic population is composed of large transformed cells with cytological atypia, nuclear irregularities. and clumped chromatin. Note the presence of rare small lymphocytes (arrows). H&E; original magnifications, ×400.

Figure 2.

Mutational analysis of p53 in nasal NK/T-cell lymphomas from Mexico by DHPLC. A: A normal DHPLC melting curve of any given exon, suggestive of wild-type p53. B: A questionable DHPLC melting curve, suspicious in this case of a p53 mutation in exon 6. The sequence analysis was normal. The red arrow highlights the area where an equivocal second peak is seen. C: Abnormal DHPLC pattern strongly suggestive of a p53 mutation (red arrow). The sequence analysis confirmed a mutation in exon 7, codon 242 (case 3).

Figure 3.

Immunohistochemical study of p53 and p21 in nasal NK/T-cell lymphomas from Mexico with p53 missense mutations (A and B) and wild-type p53 gene (C and D). A: Nasal lymphoma with strong nuclear overexpression of p53 in the majority of tumor cells and negative p21 expression (case 3, group I). B: In contrast to the previous example, note the positive nuclear expression of p21 in some tumor cells (case 5, group I). C: Nasal lymphoma positive for p53 with variable nuclear intensity and p21 expression (case 11, group 2). D: Nasal lymphoma with p53 expression in <5% of tumor cells and high expression of p21 (case 17, group 3). Immunoperoxidase staining, original magnifications, ×400.

Figure 4.

LOH analysis. Electropherogram of normal/tumor pair in cases 1 and 3 (left and right side, respectively) The p53 informative locus is shown. The y axis represents the peak height in fluorescence units. Top: Amplification from normal tissue. The green peaks represent the heterozygous alleles. Bottom: Amplification from tumor tissue, the red arrows mark the lost allele.