Study On The Detection And Significance Of Bcl10 Novel Mutation In Ocular Adnexal Mucosa-associated Lymphoid Tissue Lymphoma

The mucosa-associated lymphoid tissue lymphoma of the ocular adnexal (MALT lymphoma ) is one of the most common anatomic sites occurred from extranodal marginal zone mucosa-associated lymphoid tissue lymphoma. Ocular adnexal MALT lymphoma accounts 10%~15% of orbital malignant tumors, may involve the orbital connective tissue , conjunctiva, lacrimal gland, eyelids. The mucosa-associated lymphoid tissue lymphoma is a distinct subgroup of B-cell non-Hodgkin Lymphomas(NHL). It is characterized by an indolent clinical behavior, tend to remain localized to the sites for long periods and to have a better prognosis. Up to now the diagnosis of mucosa-associated lymphoid tissue (MALT) lymphoma largely depends on the clinical and pathological findings due to absence of specific immunophenotype and molecular genetic markers. The histological typing of lymphoma is always complicated and is a dynamic process; besides, the symptoms of lymphoma is not well characterized, all making it difficult to differentiate lymphoma from inflammatory disorders such as inflammatory pseudotumor, reactive lymphatic hyperplasia, etc. It is difficult to follow the disease course and classify the histological types. The course of the disease is characterized by recurrence and progression. Pathological observation under microscope can not identify the borderline atypical lesions. The above factors directly affect the treatment outcomes and the prognosis of patients. Long term hormone treatment during recurrence often fails to obtain satisfactory outcome, only results in acute pain and systemic complications; severe and advanced cases may develop systemic stage IV lymphoma and die. Until now the mechanism of MALT lymphoma remains unknown. People in occidental countries are liable to have diffuse large B-cell lymphoma (DLBCL). Researchers have found that the mechanism of lymphoma was closely related to immunological and genetic factors. Increasing evidence showed that Bc110 participates in the activation NF-kappaB signal pathway, which results in apoptosis inhibition of tumor cells and plays an important role in the development of MALT lymphoma. This study is aimed to examine the expression of Bc110 gene in MALT lymphomas, atypical lymphatic hyperplasia, and reactive lymphatic hyperplasia involving the orbit, conjunctiva, lacrimal gland, and eyelid of Chinese patients. We identified novel mutations of Bc110 gene and investigated its relation with the activation of NF-kappaB, in an effort to assess the values of Bc110 gene novel mutation and the mutant Bc110 gene in diagnosis of MALT lymphoma, and to provide new evidence for the role of Bc110-activated NF-kappaB pathway in the development of MALT lymphoma.Objective1.To examine the expression of Bc110 gene in MALT lymphomas, atypical lymphatic hyperplasia, and reactive lymphatic hyperplasia involving the orbit, conjunctiva, lacrimal gland, and eyelid, and to discover novel mutations and their roles in clinical diagnosis of eye adnexal.2.To observe the expression of mutant Bc110 protein in MALT lymphomas, atypical lymphatic hyperplasia, and reactive lymphatic hyperplasia and its relation with the nuclear localization. To study the influence of mutant Bc110 protein on NF-kappaB pathway and to speculate the role Bc110 mutation-induced nuclear translocation of NF-kappaB in the development of MALT lymphoma.Methods1.Twenty-three specimens of ocular adnexal MALT lymphoma, 10 specimens of atypical lymphatic hyperplasia, and 10 specimens of reactive lymphatic hyperplasia were collected during 2005-2006 from the ophthalmology department of Shanghai Changzheng Hospital, Second Military Medical University; 12 normal peripheral lymphatic specimens were taken as control. The diagnosis of lymphatic lesions was based on the 2001 WHO diagnostic standard for lymphatic & hemopoietic neoplasma. The diagnosis in the present study was done by 2 experienced pathologists in a double-blind manner. The 23 patients with MALT lymphomas included 15 males and 8 females, with a mean age of 62.31±11.23 years (ranging 40-82 years). The 10 patients with atypical lymphatic hyperplasia included 6 males and 4 females, with a mean age of 66.92±7.5 years (ranging 55-76 years). The 10 patients with lymphatic reactive hyperplasia included 5 males and 5 females, with a mean age of 71.69±3.65 yeas (ranging 55-78 years). All the specimens were obtained during surgery and were preserved in liquid nitrogen immediately after harvesting.2.The expression of Bc110 gene in ocular adnexal MALT lymphomas, atypical lymphatic hyperplasia, and reactive lymphatic hyperplasia was examined by molecular methods. The DNA sequences were analyzed by Sanger method and the results were compared with those of the Genbank blast to identify novel mutations.3.The protein expression of mutant Bc110 and NF-kappaB in all the specimens were detected by immunohistological method (EnVision) and immunoflurescence (indirect method).4.Confocal microscope (Leica, TCS, SP2) was used to observe the colocalization of Bc110 and NF-kappaB expression. For the first channel of FITC, the maximal absorption wavelength was at 488nm, the maximal emission wavelength was at 520nm (second antibody labeling mouse anti-human Bc110 antibody, green fluorescence). For the second channel of Cyanine3, the maximal absorption wavelength was at 543nm, the maximal emission wavelength was at 570nm (second antibody labeling NF-kappaBp65 antibody, with red fluorescence). DAPI was used for nuclear staining and the UV emission was at 360mn.5.All the data were expressed as x±s . Bc110 expression and the mutation of Bc110 gene were compared between the experimental group and control group usingχ2 test and Fisher exact test. The relationship between nuclear expression of Bc110 with Bc110 mutation rate and NF-kappaB expression was analyzed byχ2 test, Fisher exact test, and Spearman analysis. All the data were processed by SPSS11.0 software; a P value less than 0.05 is considered statistically significant.Results1.Of the 43 specimens in experimental group, 21(48.84%) were positive of Bc110, of which 12(57.14%) had novel mutations of Bc110. There was a 129 bp deletion in the 299 site of exon, shortening the encoded protein from 233 amino acids to 99; we have registered this mutation in the GenBank (EF189176). Another 4 specimens had a same type of mutation: G→A transversion at position 638 in exon, leading to substitution of glycin by glutamate at 213. T→C transversion at position 308 in exon, leading to substitution of valine by alanine at 103. T→C transversion at position 368 in exon, leading to substitution of glutamate by glycin at 123. A→G transversion at position 374 in exon, leading to substitution of phenylalanine by glycine at 125. T→C transversion at position 5 in exon, leading to substitution of glutamate by glycine at 2. T→C transversion at position 412 in exon, leading to substitution of valine by alanine at 103. A transversion of A→G at position 145 in exon has been reported previsously. Four of the 10 atypical lymphatic hyperplasia specimens were positive of Bc110, including mutation a 33bp deletion at position 346 in exon, leading to changes of 116-126 amino acid, and they were the different products of mRNA editing; this mutation was also reported by others previously. Two of the 10 reactive lymphatic hyperplasia specimen were positive of Bc110: G→A transversion at position 638 in exon, which was the same as that of the 4 MALT specimens mentioned above. No Bc110 mutation was identified in the 12 normal lymphatic tissues. There was significant difference in Bc110 expression rates between the experimental group and the control group (P<0.05); The Bc110 mutation rates was significantly different in lesions with different degrees of malignancy (P<0.05). no significant difference was found in Bc110 mutation rates between different locations and different clinical stages (P>0.05).2.Twenty (86.96%) of the 23 MALT specimens were positive of Bc110, including 13 (65.0%) cases positive in both the nuclear and cytoplasm and 7(35.0%) cases positive only in the cytoplasm. Five (50%) of the 10 atypical lymphatic hyperplasia specimens had diffused expression of Bc110 in the cytoplasm. 1 case (10%) positive in both the nuclear and cytoplasm , three (30%) of the 10 reactive lymphatic hyperplasia specimens had expression of Bc110 in the cytoplasm. All specimens were negative of Bcl10 in the control group . The nuclear expression of Bc110 protein was significantly correlated with the mutation of Bc110 gene (P<0.05). The expression of Bc110 protein was no significant difference in lesions with different degrees of malignancy (P>0.05) . 18 (78.3 %) cases of the 23 MALT specimens were positive of NF-kappa B both in the nuclear and the cytoplasm, 5 cases(21.7%) positive only in the cytoplasm. Seven (70%) of the 10 atypical lymphatic hyperplasia specimens had diffused expression of NF-kappa B in the cytoplasm. 1 case (10%) was positive in both the nuclear and cytoplasm, five (50%) of the 10 lymphatic reactive hyperplasia specimens had NF-kappaB expression in the cytoplasm. In the control group, All specimens had diffused expression of NF-kappa B in the cytoplasm. The positive rates of NF-kappaB protein in ocular adnexal MALT lymphoma, atypical lymphatic hyperplasia, and reactive lymphatic hyperplasia, was significantly(P<0.05). The nuclear expression of Bc110 protein was significantly correlated with the expression of NF-kappaB (P<0.05).3. The signals of laser scanning confocal microscope was subjected to 3-D reconstruction using SFP option; FITC-labeled Bc110 protein was distributed in the nuclear and cytoplasm in a dotted manner or in clusters of green fluorescence granules. Cy3-labeled NF-kappaB protein was widely distributed in the cytoplasm. The reconstructed image showed co-localization of yellow fluorescence of Bc110 and NF-kappaB, including 13 cases of Bc110 and NF-kappaB co-expression. The expression of Bc110 was positively correlated with NF-kappaB co-expression (P<0.05).Conclusion1. We have identified 12 novel mutations of Bc110 gene in ocular adnexal MALT lymphoma, atypical lymphatic hyperplasia, and reactive lymphatic hyperplasia tissues in Chinese patients. Two mutations were also reported by others previously. The novel mutations are predominantly the substitution of glycine by glutamate (GGA→GAA) at 213, followed by deletion mutation: the CARD domain of Bc110 broke from the C terminal, shortening the amino acid from 233 to 99, which had been registered in the Genbank(EF189176).2. The discovery of novel Bc110 mutations in atypical lymphatic hyperplasia and lymphatic reactive hyperplasia tissues indicates that the examining Bc110 mutation is sensitive than pathological examination in diagnosis of ocular adnexal lesions, allows us to know the progression and nature of the lesions prior to any morphological changes and other differentiating parameters, and allows us to make accurate and timely diagnosis and obtain satisfactory outcome.3. The mutant Bc110 is associated with the malignancy of tumors; its protein is present in the nuclear and is positively correlated with the co-localization of NF-kappaB in the nuclear, which is consistent with the theory that Bc110 translocation activates NF-kappaB and subsequently induces inhibition apoptosis. Bc110 mutation can serve as a sensitive marker for diagnosis of MALT lymphoma. Besides, the detection of Bc110 mutation also provides new evidence for studying the role of Bc110-activated NF-κB pathway in pathogenesis of MALT lymphoma.