p63 is a member of the p53 tumor-suppressor gene family with structural homology to p53, but one that, from a functional point of view, shows both similarities and differences from p53 (1-5). In fact, the p63 gene transcript undergoes multiple alternative splicing and, in addition, can be transcribed from two different promoters, resulting in the generation of a variety of p63 isoforms, including three transactivating (TA-p63, α-β-γ) and three amino terminal-truncated proteins (ΔN-p63, α-β-γ) (1, 2, 5). Interestingly, these different molecular species can exert contrasting effects on the same molecular targets, interfering with p53 functions. TA-p63 molecules (especially TA-p63γ) have functions similar to p53 in inducing cell-cycle arrest and apoptosis. However, ΔN-p63 isoforms (especially theΔN-p63α isoform) act as a dominant-negative factor, inhibiting transcription activation by both p53 and TA-p63 isoforms (1, 2, 6). When abnormally over-expressed as a result of gene amplification, these latter p63 isoforms are thought to behave as oncogenic molecules (1, 5, 7-9).

Recently, p63 has been proposed to be a specific marker of precursor/stem cells in many epithelial tissues and to represent a candidate marker for exploring thymic stem cells (5, 10-12). The fine-tuning of different p63 isoforms in the basal and differentiating compartments is necessary to maintain the regenerative quality of epithelial stem cells, as well as the regulation of epidermal mesenchyme interactions, replicative senescence, and angiogenesis (13-15). Therefore p63 is essential during embryonic life for the correct development of limb craniofacial, epithelial differentiation and proliferation rather than tumor suppression (16). However, the final designation of p63 as an oncogene or a tumor suppressor gene is still under debate (6). In contrast, p53 is a well-known tumor suppressor gene located on chromosome 17p13.1 and encoding a nuclear phosphoprotein of 53 kDa (17). It is believed to induce cell cycle arrest in the presence of damaged DNA, finally leading cells to apoptosis. Regarding malignant lymphomas (MLs), p53 overexpression has been found in 33.3% (18) and 41.9% (19) of cases and has been related to disease-specific survival (18). Recently, alterations and loss of p63 expression in advanced stages of human bladder carcinoma have been described (20-22) but their clinical significance still has to be defined. Information regarding p63 expression in malignant lymphoma (ML) is scanty, and few data are available regarding ML. Starting from these grounds, we have investigated the expression of p63 in reactive hyperplasias (RHs) and in a series of MLs using immunohistochemistry.

To our knowledge, this is the first study to analyze the immunoreactivity of p63 and p53 in a large number of ML specimens and to correlate p63 and p53 immunoreactivity with clinical follow-up. More recently, MLs were described as p63 immunoreactive in a survey study on human tumors (23), but relationship between p63 and p53 immunostaining in respect to patient's prognosis with ML has not yet been reported. Di Como et al. (24) examined p63 expression profiles in a number of non-Hodgkin's B-cell lymphomas, including those classified as CLL/SLL, FL, DLBCL, anaplastic largecell lymphoma, MCL, and MZBCL. They observed diffuse to intense staining in DLBCL and FL (24). Furthermore, in FL, the strongest staining was observed in grade 3 cases in the larger neoplastic cells. Higher levels of p63 protein expression were found in tumor lymphocytes compared with normal lymphoid tissues by RT-PCR (24). Chilosi et al. (25) reported that T-cell precursor lymphomas did not express p63, whereas 7 of 8 cases of PMLBCL expressed TA-p63α. But details on ML subtypes and discussions on the significance of p63 overexpression in MLs were not provided (25). In humans, heterozygous germline p63 mutations have been described in EEC syndrome, in which a single case has been described with thymic hypoplasia (26) although no lymphoid or thymic abnormalities have been described in p63-null mice (16, 27). There is also another case study examined a sporadic incident of EEC syndrome (ectrodactyly ectodermal dysplasia-clefting syndrome) of complete form of the anomaly with late onset of non-Hodgkin's lymphoma (28). Interestingly, the non-Hodgkin's lymphoma was of the diffuse large-cell type, where we observed intense p63 immunoreactivity. Our results of study are consistent with those of previous studies (24, 28). In this study p63 was expressed in 38 cases of MLs studied (30.2%) including 32/61 cases (52.5%) of DLBCL, 1/8 cases (12.5%) of T-LBL, 4/14 cases (28.6%) of FL, 1/6 cases (16.7%) of T/NKL. Among p63 positive cases, p63 was diffusely and strongly expressed in 15/32 cases of DLBCL and 1/1 case of T-LBL. Remaining cases were focally and weakly expressed. p63 was not expressed in MCL, PTCL, MZBCL, plasmacytoma and HL. p63 and p53 were not expressed in RHs.

Recently, DLBCL tends to be divided according to CD10 immunoreactivity. So, we also analyzed p63 expression according to CD10 immunoreactivity in 52 DLBCL cases. In the present study, CD10 was expressed in 10/52 cases (19.2%) of DLBCL. Among 10 cases of CD10 expression, p63 was detected in only one case (1/10, 10%). In contrast, p63 was expressed in 12/42 cases (28.6%) of CD10 negative DLBCL. Although there is no statistical significance between p63 and CD10 expression, p63 showed a tendency to increase its expression in CD10 negative DLBCL comparing with in CD10 positive DLBCL.

There are no clear explanations why p63 is overexpressed in DLBCL. Yang et al. (1) speculated that because p63 lies close to the translocation break point found in some DLBCLs, genomic instability in this area may lead to the dysregulation of p63. However, this does not explain why TA isoforms are predominant in the small group of MLs by use of RT-PCR (24).

In contrast to p53, a protypical tumor suppressor gene, somatic mutations of the TP 63 gene are very rare, whereas germline mutations in TP 63 have been reported for patients with limb mammary syndrome, split-hand/split-foot malformation, ankyloblepharon-ectodermal dysplasia-clefting syndrome, and EEC syndrome (29-32). Therefore, it might be that alterations of TP 63 are epigenetic in nature. A number of studies have investigated the role of p63 in neoplastic transformation and tumor progression including squamous cell carcinomas (SCCs) of different organs (7, 8, 33, 34), invasive ductal carcinomas of the breast (35), transitional cell carcinomas (6, 36), and thymomas (25). SCCs from different organs express high levels of ΔN-p63 (7, 8, 34). The p63 gene may be the target of 3q27-29 gains common in SCC (1, 8).

In this context, we might speculate that when certain p63 isoforms are expressed, such as dominant-negative ΔN-p63, they could bind to and inhibit transactivation by p53 and TA-p63 (1). Alternatively, these ΔN-p63 isoforms, by binding to specific promoter elements, could block the transcription of otherwise critical genes, such as those involved in the apoptotic response. Rather than inactivating tumor suppressor activities (e.g., p53), altered patterns of p63 expression could be driving the transcription of oncogenic proteins, such as hMDM2, a target reported for TA-p63γ (37, 38). Further studies, integrating mechanistic approaches, are needed to elucidate the role of p63 in human cancer.

Regarding the prognostic importance of p63 expression in various types of human carcinomas, the results are controversible. Some authors reported an inverse correlation between p63 overexpression and advanced tumor stage and poor prognosis (6, 36). In thymomas, p63 expression was not correlated with clinical aggressiveness (24, 25) although others have reported a correlation between p53 expression and malignancy in thymomas. In this study, p63 expression appears to be related to poor survival in DLBCL. However, as shown in previous study (36), multivariate analysis failed to prove either p53 or p63 as an independent prognostic parameter in this study. Age was the only stastically significant independent prognostic factor.

In conclusion, p63 was overexpressed in specific histologic types of MLs such as T-LBL and DLBCL. In FL, p63 was weakly expressed in small numbers. The number of cells containing p63 and their distribution tend to depend on the degree of differentiation of lymphocytes. p63 was rarely coexpressed with p53. p63 expression appears to be related to poor survival although it was not stastically significant on multivariate analysis. Therefore, we could speculate that p63 could act indirectly as an oncogene by inhibiting p53 functions. Stage of differentiation of neoplastic lymphocytes appears to have correlation with p63 expression in MLs. Molecular study regarding different p63 isoform expression and p63 mutation could contribute to understanding the heterogeneity of DLBCL and progression of ML.