Gubitak heterozigotnosti gena p53 u kongenitalnim sakrokokcigealnim teratomima

Kongenitalni sakrokokcigealni teratom je najčešći tumor zametnih stanica u dječjoj dobi. Tumor je dobroćudan i sastoji se od diferenciranih zrelih tkiva podrijetla svih triju zametnih listića, ali može sadržavati i nezrela tkiva. Obično se dijagnosticira pri porođaju. Mutacije gena p53 utvrđene su u više od 50% zloćudnih tumora ljudi, ali postoje oskudni podatci o njihovoj učestalosti u kongenitalnim sakrokokcigealnim teratomima. U ovom je radu analiziran gubitak heterozigotnosti (LOH) gena p53 u 9 kongenitalnih sakrokokcigealnih teratoma pomoću lančane reakcije polimeraze (PCR) i polimorfizma dužine restrikcijskih fragmenata (RFLP) na temelju dvaju polimorfnih mjesta (Bst U1 i Msp 1). Kongenitalni sakrokokcigealni teratomi su dijagnosticirani u 8 ženske i 1 muškog djeteta u dobi od 3-30 dana. Tumori su bili od 5,5 do 17 cm u najvećem promjeru. LOH je utvrđen u jednom tumoru. Šest je tumora bilo homozigotno za oba analizirana markera (neinformativno), dok su dva bila informativna za Bst U1, ali bez LOH-a u tumorskom tkivu. Imunohistokemijski je utvrđena pozitivna reakcija na p53 u 4 tumora. Na temelju ovakvih rezultata zaključujemo da je LOH za p53 rijetka pojava u kongenitalnom sakrokokcigealnom teratomu te bi valjalo analizirati veći broj tumora radi bolje procjene uloge p53 u nastanku ovih tumora.
Congenital sacrococcygeal teratoma (CSCT) is the most common germ cell tumor of infancy and childhood. The tumor is benign, consists of fully differentiated mature tissues and usually is diagnosed at birth. CSCTs may also contain immature tissues, most commonly of neural origin. However, in most instances immature teratomas are associated with a favorable prognosis (1, 2). CSCT may recur within a few years after initial surgery, probably due to incomplete resection. The p53 tumor suppressor gene, located on chromosome band 17p13, encodes a 53-KDa nuclear phosphoprotein that functions as a transcriptional regulator of the cell cycle (3). The p53 acts at the G1/S checkpoint to induce growth arrest or apoptosis of a cell with damaged DNA. With homozygous loss of p53, DNA damage goes unrepaired, mutations become fixed in dividing cells leading the cell to malignant transformation. Mutations of the p53 gene or its expressed protein have been found in more than 50% of human cancers, however, there are few data regarding the status of p53 in CSCT (4, 5, 6, 7).

In this study we analyzed the loss of heterozygosity (LOH) of p53 in nine CSCT by polymerase chain reaction/ restriction fragment length polymorphism (PCR/RFLP) based on two polymorphous sites (Bst U1 and Msp 1). MATERIAL AND METHODS Patients' charts and surgical pathology reports of all CSCTs diagnosed in neonates and infants (up to 1 month of age) between 1985 and 2001 at the Children's Hospital Zagreb were reviewed. Routine histopathologic analysis and immunohistochemistry of tumor tissues was performed at the Department of Pathology, School of Medicine, University of Zagreb. Genetic analysis was performed at the Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia. For histologic, immunohistochemical and genetic analysis, the original paraffin blocks were utilized. Tissue specimens and genomic DNA extraction: Eight 5 _m sections were cut from each paraffin block. Two slides (the first and the eighth) were stained with hematoxylin-eosin in order to determine the border between the tumor and the corresponding non-tumorous tissue. Genomic DNA was extracted separately from these separated parts from unstained slides marked 2-7. A single pathologist reviewed all sections.

DNA extraction and PCR: Genomic DNA was extracted as previously described (8): Sections were deparaffinized with two washes in xylene (2x2hours), xylene was rinsed with ethanol and dried, deparaffinized samples were incubated in digestion buffer (50 mM TRIS-HCl pH 8.5, 1 mM EDTA, 0.5% Tween 20) containing 500 _g/ml of Proteinase K and incubated for 48 hours at 37_ C. Proteinase K was inactivated at 95_C for 8 minutes. Amplicons were obtained from 5 _l of the boiled supernatant in 25 _L reaction volume containing 2.5 _L of Buffer 1 (Eppendorf), 1.5mMMgCl2, 10 mMdNTP’s (2.5mMeach), 5 pmol of each primer and 1.0 U of Taq polymerase (Eppendorf). The samples were predenaturated (94_C for 2 minutes), and subsequently processed in 40 cycles consisting of 40 sec at 95_C, 35 sec at 58_C and 35 sec at 72_C, and final extension at 72_C for an additional 10 minutes. The presence of the bands was verified by agarose gel electrophoresis. The primer pairs used in these reactions were previously described (9, 10). LOH analysis: Loss of heterozygosity in the p53 gene was analysed by PCR/RFLP method based on two polymorphic sites: in exon 4 (Bst U1) and in intron 6 (Msp 1). For RFLP analysis, 10-15 _L of the PCR products were digested overnight with 10 U of Bst U1 (exon 4) or Msp 1 (intron 6) (Roche, Germany) in a total volume of 25 _L. The digested PCR products were separated on PAGE and silver stained. Loss of the upper band or two lower bands in the tumor tissue was considered as LOH when compared with the presence of three bands in corresponding nontumorous tissue (one cut, and one uncut allele). Immunohistochemistry was performed on paraffin embedded archival material using the primary antibody to p53 (1:25 dilution) (DAKO, Glostrup, Denmark). The peroxidase/antiperoxidase method was used on deparaffinized sections with 3.3'-diamino-benzidine-tetrahydrochloride as a chromogene. Briefly, tissue sections were cut at 5 _m thickness, dewaxed and incubated in 10 _mol/L sodium citrate in a pressure cooler. The pressure cooler was placed in a microwave oven and heated for 30 minutes. The slides were than cooled at room temperature for 30 minutes. Endogenous peroxidase activity of the tissue samples was quenched with an aqueous solution of 3% hydrogen peroxide for 3 minutes, followed by 3 rinses with Tris-buffered saline. As a positive control we used a known positive section of mammary carcinoma. Omission of the primary antibody served as the negative control for all antibodies. Microwave pretreatment was performed to improve immunostaining. The intensity of staining was graded semiquantitatively as (-) for no immunostaining, (+) for weak staining (up to 10% of positive cells), (++) for moderate staining (10-25% of positive cells), and (+++) for intense staining (more than 25% of positive cells). At least 500 cells were counted for each case. All slides were examined independently by two pathologists.
Tumors were found in 10 female and one male patient. There were 9 mature and 2 immature SCTs. Mature teratomas were composed of different, well-differentiated mature tissues including skin and skin adnexa, mature glial tissue, seromucous glands, and cystic structures lined by columnar epithelium (Figure 1). The tissue of 9 CSCT was available for this study (Table 1). They were diagnosed in 8 female and 1 male children aged from 3 days to one month. The tumors measured from 5.5 to 17 cm at the largest diameter. All nine tumors were located in the postsacral area with no other associated anomalies. LOH was detected in one tumor sample. Six samples were non-informative (homozygous) for both analyzed markers, while two samples were informative for BstUI, but without LOH in tumor tissue (Table 1 and 2). The p53 positive immunostaining was found in 4 tumors.
At present, eight patients with mature SCTs are alive without recurrences in the follow-up interval ranging from 1-12 years, and one died due to bilateral pneumonia 3 days after surgery (case 1).
There are few small series and case reports dealing with genetic changes, mostly the oncogenes expression in CSCT (4, 5, 6, 11, 12, 13). The p53 reactivity was determined by immunohistochemistry in the CSCT with a yolk sac tumor in the microcystic areas, the neuroectodermal rosettes and glandular area (4). The authors used an anti-p53 antibody that recognizes the N-terminus of wild type and mutated p53. p53 nuclear positivity was found in the teratoma with a yolk sac tumor component in the microcystic and glandular types (4). In our previous study, slightly to moderately p53 positive immunostaining was found in two immature and three mature teratoma (5). Of all cases analyzed in this study, four showed positive immunostaining for p53. The case with LOH for p53 immunohistochemically demonstrated a slightly positive reaction for p53. However, it is a case of a female child aged 6 days, who presented with mature teratoma and was well and without signs of recurrence during the period of follow- up. On the contrary, p53 mutations were not detected in the tissues of both immature ovarian teratoma of the pregnant mother and intracranial teratoma of her fetus using polymerase chain reaction and electrophoresis. (5). Charoenkwan e t a l (7) have recently published results obtained on 29 immature teratomas, among them five immature CSCTs (four of them in female patients). They reported occasional, focal p53 positivity in three and a continuously negative reaction in the remaining two sacrococcygeal tumors. They observed an association between p53 immunopositivity with mutations in the p53 gene in only 1 of 29 cases. The p53 diffuse immunostaining was demonstrated in about 60% of their cases (18/29) and was associated with recurrence or the presence of malignant elements in over 50% of the cases (7). p53 immunostaining was also reported in sporadic cases of different congenital tumors, including oligodendroglioma and neurofibroma (14, 15, 16). N a r i t a e t a l (14) observed p53 accumulation in the majority of cells of congenital oligodendroglioma and they suggested that this tumor should be considered as malignant regardless of its benign features observed at histological examination. It seems that LOH of p53 is a rare genetic event in congenital CSCT. However, it is obvious that more studies on larger groups of patients are needed.
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Kategorija: Klinička zapažanja
Broj: Vol. 49, No 1, siječanj - ožujak 2005
Autori: B. Krušlin, K. Gall-Trošelj, A. Čizmić, H. Čupić, M. Turčić, M. Belicza
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