| Background and objectiveAathouhg craniopharyngioma(CP) is pathologically benign tumor, its treatment is a very challenging topic for study to all the neurosurgeons all the the time.With more understanding to it,and with the rapid development of modem medicine imaging techniques and microneurosurgery, more and more modern neurosurgery departments see total surgical resection of CP as its most important and prior management strategy.As to treanment results, the following two points are extremely troublesome,one is postoperative recurrence,and the other one is postoperative complications and qulity of life of patients.In recent years,in the point view of clinics,most researchers considered that surgical excision degree when operation on craniopharyngioma and its pathological subtype were two key points affecting postoperative recurrence of craniopharygioma patients. Pathologically, craniopharyngioma consists of two subtypes:adamantinomatous CP and squamous papillary CP.In recent years,the increasing number of researchers suggest that adamantinomatous- and squamous papillary-type CP are not only pathological structures,but also clinical manifestation and prognosis,distinctive variants. Adamantinomatous CP is reported to occur almost in childhood and early adolescent patients,the prognosis is bad and postoperative recurrence rate is high compared to squamous papillary CP sufferers.Because tumor cells proliferate by activity cycle,tumor cells' proliferation activity and proliferation speed is key internal agent that determines tumors biological behaviors.In recernt years,some domestic and abroad scholars have started to study the proliferating of craniopharyngiomas to understand whether different prognosis and postoperative recurrence rate of two subtypes of CP is also related to its internal proliferating.The proliferative markers they selected consisted mainly of PCNA, Ki-67,MIB-1,BrDU and Agnor, and so on.All the research conclusions are not coincident.p21 and hnRNP A2/B1 is two units that plays very important roles in the cell proliferative cycle.More and more studies showed that they were intimate relevant to some tumors' genesis and development.But as to its relation and significance to craniopharyngioma,no study can be discovered yet.Although the term of craniopharyngioma have been posed for more than one hundred years,its origin is not firmly established.It is generally thought to be derived from remnats of Rathke's pouch or squamous metaplasia, a number of studies suggested that both these two theories can't elucidate craniopharyngioma's origin perfectly.Recently,some oversea researchers have began to study craniopharyngioma's molecular and genetic structures to more deeply understand its genesis.Some investigations suggested that craniopharyngioma genesis is maybe relevant to its chromatosome certain gene change and then activation carcinogenic gene.And according to another study, adamantinomatous craniopharyngioma harboredβ-catenin exon 3 gene mutations.So far, there have been no reports on molecular or genetic study of craniopharyngioma genesis in domestic.Based on these findings,it is hypothesized that studies on the craniopharyngioma tumor cell's proliferativity and its genesis are attached great significance for further understanding of the internal cause of postoperative recurrence,which will provide theories for elucidating craniopharyngioma biological behaviors.The objectives of the research are:1)To undertake molecular pathological and molecular biological studies on p21 protein,hnRNP A2/B1 protein and hnRNP A2/B1 mRNA in adamantinomatous- and squamous papillary-type craniopharyngiomas;2)To study the difference of proliferativity in craniopharyngioma tissue of different pathological types as well as their clinical meanings;3)To study on the expression ofβ-catenin and its exon 3 genetic mutations in craniopharyngioma as well as the significance.Materials and Methods1 Study on p21 and hnRNPA2/B1 expression in craniopharyngioma1.1 Clinical dataSamples were from the 81 patients in department of neurosurgery NanFang hospital from January 2002 to November 2005,followed by postoperative pathological diagnosis to verify 62 cases of adamantinomatous craniopharyngioma and 19 cases of squamous-papillary craniopharyngioma.The patients included 49 males and 32 females,aged from 2 years old to 73 years old, with the mean age of 21.1 years old.All the sample were put into liquid nitrogen for cryopreservation.1.2 Empirical methodSP immunohistochemical method was used to detect the expression of p21 and hnRNP A2/B1 in the 81 samples of craniopharyngioma tissue,and then calculated the p21 Labeling Index(p21 LI) and hnRNP A2/B1 Labeling Index(hnRNP A2/B1 LI),to reflect the proliferation of different pathological subtypes of craniopharyngiom.P21 and hnRNP A2/B1 immunohistochehmical staining was then performed by the following procedures:the tumor samples were firstly fixed in 10% formalin,followed by routine paraffin imbedding and serial section in 4μm thick,which were then performed p21 and hnRNP A2/B1 immunohistochehmical staining.The immunohistochemistry was conducted in SP two-step method,which consisted mainly of microwave antigen repair, DAB-staining examinatin and. microscopic observation.The known positive sections were taken as the positive control and replace all samples with PBS as the negative control.The positively stained cells were characterized by buffy or even deep buffy cytoplasm or celluar nucleus. Five areas with high magnifying power (400×) and evenly distributed cells were selected as the observed area on each section to calculate the positive cell count and the total cell count within the visual field, followed by calculating the cell count mean of the five observed areas. The labelling index of cytoplasm or cellular nucleus was calculated with the following equation: LI=PositiveCellCount/TotalCellCount×100%.1.3 Statistical analysisThe statistical analysis was performed with SPSS10.0 software package and the data were presented with (Mean±Standard Deviation). The differences of p21 LI and hnRNP A2/B1 between different pathological types of CP were analyzed wit t test,and the correlationship between p21 LI and hnRNP A2/B1 LI in the two subtypes of CP and in all tissues of CP was analyzed with Pearson correlation analysis, being significant when P＜0.05.2 Study on hnRNPA2/B1 mRNA expression in craniopharyngioma2.1 Clinical data The same as 1.12.2 Empirical methodAs the operation instruction, the total RNA of the tumor tissue were extracted with Promega RNA extraction agent. 4μl RNA solution was added to the ingredient as the instruction of Promaega reverse transcription kit and reacted under the following conditions: 42℃for 60minutes, 95℃for 5 minutes, 4℃for 5 minutes. The hnRNP A2/B1 specific primers were:forward 5'-CTG TTT GTT GGC GGA ATT-3' and backward 5'-ACC CAT TAT AGC CAT CCC-3',with amplified fragment 226 base pair.Probe sequence was 5'-FAM-ATC ATC CAC TGT GGT AGC AGC CGC-TAMRA-3'.The reaction with 1μl TaqMan probe,1μl forward primer (10pmol/μl) ,1μl backward primer,2μl Cdna, 0.25μl Taq enzyme,1μl 10mMdNTP, 2μl 10×PCR Buffer and H2O 12.25μl was added to the PE7300 fluorescent quantitation PCR reaction system with the fluorescent quantitation RT—PCR device.The conditions for amplification were 93℃for 4 miniutes, 93℃for 30 seconds, 62℃for 30 seconds,and total 40 cycles. The result(Ct) from fluorescent quantitation system was analyzed and compared with PE7300 system SDS software.2.3 Statistical analysisThe statistical analysis was performed with SPSS10.0 software package and the data were presented with (Mean±Standard Deviation). The difference of hnRNP A2/BlmRNA expression between different pathological types of CP were analyzed with two independent sample t test,being significant when P＜0.05.3 Study onβ-catenin expression and genetic mutaions in craniopharyngioma3.1 Clinical dataSamples were from the 23 patients in department of neurosurgery NanFang hospital from January 2004 to January 2001,followed by postoperative pathological diagnosis to verify 20 cases of adamantinomatous craniopharyngioma and 3 cases of squamous-papillary craniopharyngioma.The patients included 15 males and 8females,aged from 3 years old to 58 years old, with the mean age of 21.6 years old.All the sample were put into liquid nitrogen for cryopreservation.3.2β-catenin protein expression in craniopharyngiomaPart of every tumor samples were firstly fixed in 10% formalin,followed by routine paraffin imbedding and serial section in 4μm thick,which were then performedβ-catenin immunohistochehmical staining.The concrete method could be seen from 1.2. The positively stained cells were characterized by buffy or even deep buffy in cells.t3.3 Detection ofβ-catenin exon 3 genetic mutations in craniopharyngiomaEvery sample DNA was extracted using TIANamp Genomicblood/cell/tissue genome DNA extraction kits from TIAGEN company.Samples were levigated in liquid nitrogen,followed next procedures according to instruction manuals.Extracted DNA samples were then refered to PCR experiment.The following primers, 5' -gatttgatggagttggacatgg-3' and 5'-tgttcttgagtgaaggactgag-3',were used to amplifyβ-catenin exon 3 sequence.PCR was performed for 3 minutes at 94℃for initial denaturing, followed by 26 cycles at 94℃degeneration for 30 seconds, 58℃renaturation for 30 seconds and 72℃elongation for 30 sections.PCR productions were electrophooressed in a 2% agarose electrophoresis,with the amplification fragments 228 base pair. Isolated PCR products were sequenced on an Applied Biosystems 310 Genetic Analyzer(Co Invitrogen.ShangHai).Sequence results were then analyzed with DNAstar software.Results1 Results of p21 and hnRNPA2/B1 protein expressionHnRNP A2/B1 was expressed diffusely in craniopharyngioma cells,mainly in cell nucleus and partly in cytolymph. HnRNP A2/B145 LI of adamantinous craniopharyngiomas (51.72%±8.31%) was significantly higher than that of squamous-papillary craniopharyngiomas (45.35%±7.51%) (P=0.004).P21 was also expressed dispersively in all layers of craniopharyngioma cells,but all in cell nucleus.Quantitative analysis showed that p21 LI of squamous-papillary craniopharyngiomas (34.05±6.77)was significantly higher than that of adamantinous type (28.24±4.60) (P=0.002). Through Spearman correiationship analysis,the expression of p21 and hnRNP A2/B1 in the two different pathological types of craniopharyngioma had no significant correlation(r=-0.197, P=0.078). The two LI was either not relative in squamous-papillary subtypes (r=0.442, P=0.058) .But in adamantinous craniopharyngioma,p21 LI and hnRNP A2/B1 LI was significantly negative correlation (r=-0. 271, P=0.033).2 Results of hnRNPA2/B1 mRNAThe melting curve of the hnRNP A2/B1 detected positive standard products and positive samples was smooth, each curve having obvious exponential amplification period. According to the cyclical threshold (Ct) value, the quantitative standard curve was drawn through logarithm transform, the initial template concentration having favorable linear relationship with Ct value. The regression coefficient of the standard curve from the melting curve of the hnRNP A2/B1 detected positive standard products was 0.997, showing favorable linear relationship, which suggested that the quantitative results were accurate and reliable.The expression quantity of hnRNP A2/B1 mRNA in admantinous CP (58.38±22.67)×103copies/μg was significantly higher than that in squamous-papillary CP(13.24±1.53)×103 copies/μg (t=6.738 P=0.010).3 Results ofβ-catenin expression and genetic mutations3.1 Immunohistochemical results ofβ-cateninImmunohistochemistry Ofβ-catenin showed quite different results between the two subtypes.All cases of the adamantinomatous type showed memberanous and cytoplasmic accumulation.Peripherally palisading cells showed somewhat stronger expression in both the cytoplasm and membrane.Clusters of palisading cells and cells along the epithelial cell whorls showed strong nuclear stainging.In contrast, squamous-papillary craniopharyngioma showed exclusively membranous expression. 3.2 Genetic mutations results ofβ-cateninOf all the 23 samples,there were no results in 5 samples could be detected perhaps duo to sample impurity.Sequence analysis discovered that no mutaions were presented in all the 3 cases of squamous-papillary craniopharyngioma.In contrast,of the next 15 cases of adamantinomatous type,6 samples(40%) could be detectedβ-catenin exon 3 genetic mutations.All of the mutations were missense mutations affecting the serine/threonine residues at GSK-3βphosphorylation sites.Three tumor samples showed a C to T transition(TCT to TIT) in codon 33,resulting in a substitution of serine by phenylalanine.One mutation affected codon 37(TCT to TGT, serine to aminothiopropionic acid).And in another two cases,the threonine residue 41 were affected(ACC to ATC, threonine to isoleucine).Conclusions:1 Based on the researches both in cell level and in molecular levels,we conclude that proliferativity between different pathological types of craniopharyngioma is significantly different,and that of adamantinomatous type is much higher than that of squamous-papillary subtype.The result helps to elucidate that ,on conditions of subtotal or part resection of craniopharyngioma, adamantinomatous craniopharyngioma is more apt to recur compared to squamous-papillary types.And this is one of the major internal causese that adamantinomatous craniopharyngioma poses bad clinical prognosis.2 Immunohistochemical analysis identified nuclearβ-catenin accumulation in adamantinomatous craniopharyngioma,but not in squamous-papillary type.This finding will be helpful in the neuropathological work-up of surgical specimens to discriminate adamantinomatous CP from squamous-pahpillary CP. 3 In adamantinomatous CP, nuclearβ-catenin expression was restricted to clusters of palisading cells and cells along the epithelial cell whorls.This unique celluar distribution patterns resemble that in pilomatricoma and calcifying odontogenic cysts.This suggest that not only morphological,but also maybe original similarities between adamantinomatous CP and odontogenic epithelium.4 Sequence analysis revealedβ-catenin exon 3 gene mutations in 40% of adamantinomatous CP and none of the squamous-pahpillary CP.The results suggest that adamantinomatous- and squamous-pahpillary craniopharyngioma are not only clinicopathologically, but also genetically, distinctive variants.Mutation of theβ-catenin gene therefore seems to play an important role in the tumorigenisis of adamantinomatous craniopharyngioma.
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