Protein Expression And Genetic Aberrations Of FGR And TP73 In Peripheral T Cell Lymphoma, Not Otherwise Specified

Background and Objective Peripheral T cell lymphoma, not otherwise specified (PTCL-NOS), represents a heterogeneous group of mature T cell malignancies characterized by an aggresive behavior and a dismal prognosis, with five-year survival rate ranged from 20% to 30%. There are significant differences in geographical and racial incidence of PTCL-NOS, which most commonly occurs in China and other Asian countries as compared with that in Western countries. Its pathogenesis is not clear now, and may be associated with the infection of Epstein-Barr virus (EBV) and human T-cell leukemia virus-1 (HTLV-1). Our group previously investigated the genomic aberrations in 31 cases of PTCL-NOS by using array-based comparative genomic hybridization (a-CGH), with the findings that 9 cases changed significantly in 1p36.32~36.11, of which 8 cases showed recurrent gains, and one showed deletion. FGR and TP73, located in 1p36.11 and 1p36.32, respectively, are tumor-associated genes, and participate in the pathogenesis of haematopoietic and lymphoid tumors. Since the role of FGR and TP73 on PTCL-NOS have not been addressed so far, the aim of this study was to detect the protein expression and genetic changes of both genes, to validate our previous findings, and to analyze their possible involvement in PTCL-NOS pathogenesis, providing scientific basis for its pathologic diagnosis and prognostic evaluation.Methods Immunohistochemistry (IHC) and interphase fluorescence in situ hybridization (FISH) were used in this study to detect the expression and genetic alterations of FGR and TP73 in PTCL-NOS tissues. The procedures were as follows:1. Cases studiedFormalin-fixed and paraffin-embedded (FFPE) tissues of 34 cases of PTCL-NOS (from Oct. 2001 to Oct. 2010) were enrolled from the Departments of Pathology, Shanxi Cancer Hospital, 304 Hospital, 307 Hospital and 306 Hospital of PLA, of which 19 cases were investigated by 1Mb resolution array-based comparative genomic hybridization (a-CGH). 10 cases of tonsil and reactive lymphoid hyperplasia were used as control.2. ImmunohistochemistryThe protein expression of FGR and TP73 was detected by EnVision two-step immunohistochemical staining technique according to the technical introductions of the kit. Positive control and blank control were set up in each experiment.3. Probes preparation(1) BAC DNAsThe BAC DNAs of FGR and TP73 are contained in RP1-159A19 (1p36.13) and RP5-1092A11 (1p36.2~36.33), respectively, and both are a geneous gift of Professor Hongxiang Liu from Cambridge University, UK. The brief procedures were that the plasmids were extracted from cultured BAC clone by using Qiagen's Qiaprep Miniprep kit, then purified, and finally checked with a spectrophotometer. The DNA concentrations were 15 ng/μl more or less.(2) TempliPhi amplification of circular BAC DNASince 1μg DNA was needed for probe labeling with the method of nick translation, the BAC DNAs were amplified by using TempliPhi Amplification Kit, which could amplify circular DNA preferentially, efficiently and specifically, in order to get a concentration between 200 and 800 ng/μl.(3) Probe labelingNick Translation Kit (Abbott molecular-Vysis, American) was utilized for BAC DNA labeling with green fluorescence fluorescein isothiocyanate (FITC), for that chromosome enumeration probe 1 (CEP1) we bought was labeled with orange fluorescence rodamin.(4) Probe precipitationBecause of containing substrates unreacted as well as organic and inorganic ions, the labeled probes should be purified, and also because BAC DNA contain many DNA repetitive sequences, it is needed to add human COT-1 DNA to the probes so as to block nonspecific hybridization. The procedures were as follows: human COT-1 DNA, 3 M NaAc and 100% enthanol were added to the nick translation reaction mixture to coprecipitation in -80℃, washed with 70% enthanol after centrifuging at low temperature, precipitated in -80℃again and then centrifuged, and finally resuspended in LSI/WCP hybridization buffer (Abbott Molecular-Vysis, USA) and stored at 4℃. 4. Interphase fluorescence in situ hybridizationThe genetic aberrations of FGR and TP73 were investigated in PTCL-NOS as well as tonsil tissue with homemade LSI probes and commercial probe CEP1 (served as control).5. Statistical analysisAll calculations were done by using the SPSS 17.0 statistical software package. Survival analysis of the follow-up data was performed using the Kaplan-Meier method, and log-rank test was used to compare the difference of survival rates between two groups. Statistical comparisons involving binary variables were performed using two way tables for the Fisher's exact test. P<0.05 was considered significance in all statistical calculations.Results1. Clinical characteristicsAmong the 34 cases of PTCL-NOS, 23 cases were male, and 11 cases were female, with the ratio of 2.1:1, median age was 52 years (range, 6 to 83 years). There were 20 cases occurred in lymph node, 12 cases in extranodal sites, and the sites in other two cases were unclear. When the diagnosis was established, staging information was available in 12 cases, of which 2 cases were in stageⅠ, 2 cases in stageⅡ, 5 cases in stageⅢ, 3 cases in stageⅣ, and the others unclear. The follow-up data were obtained only in 18 out of 34 cases, in which 15 cases died and 3 cases were still alive. Histologically, the tumors showed effacement of the normal architecture with diffuse infiltration of polymorphous lymphoid cells. Most cases consisted of numerous medium-sized and/or large cells with irregular, hyperchromatic or vesicular nuclei, prominent nucleoli and many mitotic figures. Clear cells and Reed-Sternberg (RS)-like cells were also noted. Rare cases had a predominance of small lymphoid cells with atypical, irregular nuclei. High endothelial venures may be increased.2. Expression of FGR and TP73 proteinsSome tumor cells expressed FGR, and the protein located in cytoplasm/nucleus. Among 34 cases, 11 cases expressed FGR, with the positive rate of 32.4%. Besides that, inflammatory cells such as granulocytes and histiocytes also expressed FGR in the tumor and tonsil tissues. Only 5 cases out of 34 cases expressed TP73, and showed low level expression in cytoplasm, and the positive rate was 14.7%; Tonsil and reactive lymphoid tissues did not express TP73.3. Genetic alterations of FGR and TP73In general, 7 of 34 (20.6%)cases of PTCL-NOS showed genetic aberrations, of which 4 cases had changes on both of the loci of FGR and TP73, including 3 cases of amplification and 1 loss of heterozygosity (LOH), 1 case of FGR amplification and other 2 TP73 amplification only. CEP1 amplification was detected in 4 cases (11.8%), simultaneously associated with FGR/TP73 gene amplification. No alteration of both genes was detected in the tonsil and reactive lymphoid tissues.4. Clinical significance of the genetic changesKaplan-Meier survival analysis showed that TP73 positive group of patients with PTCL-NOS had poor prognosis (P<0.05), as compared with TP73 negative group, but we did not find this phenomenon between FGR positive group and FGR negative group (P>0.05); Both FGR/TP73/CEP1 alteration group vs. group without FGR/TP73/CEP1 alteration and TP73 alteration vs. group without TP73 alteration had a trend of poor prognosis, however, there was no statistical significance by log-rank test (P>0.05). The overall survival time between FGR genetic alteration group and group without FGR genetic alteration, between FGR amplification group and group without FGR amplification, between TP73 amplification group and group without TP73 amplification, and between CEP1 copy number increase group and group without CEP1 copy number increase, all had no statistical significance. Fisher's exact test showed that protein expression of TP73 was significantly related with the FGR/TP73/CEP1 genetic alterations, TP73 genetic alteration, and TP73 amplification, P value were 0.048, 0.029 and 0.015, respectively. But there were no correlations between sex, age, occurrence site and all kinds of genetic alterations, as well as protein expression of FGR and TP73.Conclusion1. The methods utilized to prepare LSI probes, such as amplifying extracted BAC DNA using TempliPhi Amplification Kit, labeling fluorescence with nick translation method, and then applying FISH on paraffin-embedded tissue with the prepared probes, could detect the changes of specific genes associated with lymphomas, and thus FISH play a significant role in the pathogenetic study, pathologic diagnosis and prognostic evaluation of lymphoma.2. FGR,a member of SRC family, is an EBV-associated gene, may have a role in the development and progresson of PTCL-NOS. In PTCL-NOS, protein overexpresson of TP73, a member of TP53 tumor suppressor gene family, means to have a poor prognosis, and DeltaNp73, one isoform of TP73, may have a role on it. TP73 amplification may be an important prognostic factor in PTCL-NOS, and further studies are needed to clarify the pathogenetic implications of TP73 in PTCL-NOS.3. Aberration of 1p36.11-36.32 may be the specific genomic changes in PTCL-NOS, and it may play a role in its diagnosis and prognosis, and so as polyploid of chromosome 1.