Detection of DNA by scraping the formalin-fixed paraffin-embedded tissue

Transcription

1 999 Detection of DNA by scraping the formalin-fixed paraffin-embedded tissue M H Bukhari 1 *, S Niazi 1, R Ghani 2, Z Rathore 1, R Basharat 1, N A Chaudhry 1, and S Naeem 1 1 Department of Pathology, King Edward Medical University, Lahore, Pakistan 2 Department of Biochemistry, Baqai Medical University, Karachi, Pakistan The manuscript was received on 16 December 2007 and was accepted after revision for publication on 7 May DOI: / JEIM385 Abstract: Background. Pakistan is a developing country and most of the research laboratories have limited required infrastructures for the diagnosis of cancer at molecular level. The polymerase chain reaction (PCR) on formalin-fixed paraffin-embedded tissues is becoming a popular procedure in the research centres. The study was conducted to introduce two new methods of DNA extraction for the PCR from formalin-fixed paraffin-embedded sections of surgical pathology specimens. Materials and methods. Two methods of DNA detection were used. In method A the formalin-fixed tissues were grossed, proper sections were taken, processed in an automatic tissue processor, embedded in paraffin blocks, and microtomic sections were made. In method B, the procedure was the same until embedding in the paraffin blocks, after which the selected paraffin blocks were set on a black card paper (4 cm64 cm) and 1 mg of pure scraped tissue was obtained using a scalpel, manually without using microtone. Results. DNA was successfully extracted but point mutation of p53 gene was not seen in SCP while it was determined in 96 and per cent cases of SCC by method A and method B respectively. There was no statistical difference in the results by both methods (P ). Conclusion. Scraping of the tissue for DNA detection was a simple method and may be performed in any laboratory. The reliability, reproducibility, and quality assurance of the present results are consistent with the already established techniques of DNA extraction for PCR. Keywords: DNA, formalin, paraffin-embedded tissue, p53, PCR, squamous cell carcinoma, squamous cell papilloma 1 INTRODUCTION The purpose of a polymerase chain reaction (PCR) is to make a huge number of copies of a gene. This is necessary to have enough starting template for sequencing. The polymerase chain reaction was invented in 1938 by Kary Mullis [1] as a simple and powerful biochemical technique that revolutionized biology by allowing extremely small amounts of DNA (even one molecule) to be amplified millions of times in a matter of hours. This DNA can then be visualized on a gel and further analysed [2, 3]. *Corresponding author: 26-MOF, GOR-3, Shadman, Lahore, 54000, Pakistan. drmhbukhari@yahoo.com Formalin-fixed paraffin-embedded tissue, stained with hematoxylin and eosin, has been routinely used for the diagnosis of many diseases for more than a hundred years. Detection of DNA from the fixed tissue specimens is important for both immunohistochemical (IHC) methods for the demonstration of protein antigens and PCR for molecular study [4, 5]. The tumour suppressor gene p53 is involved in predisposition to a wide variety of human cancers. Detection of its specific mutations were often hampered by a lack of available fresh or frozen tumour tissue samples for DNA extraction to confirm the suspected p53 mutation [6]. The purification of DNA from paraffin-embedded sections of human squamous cell carcinoma has been successfully used to detect the prevalence and

2 1000 M H Bukhari, S Niazi, R Ghani, Z Rathore, R Basharat, N A Chaudhry, and S Naeem localization of HPV using a modified in situ polymerase chain reaction (PCR) and in situ hybridization AT tailing (ISH-AT) [7, 8]. The Gentra system is an important method for genomic DNA purification and is known as the Puregene DNA purification system. The kit is easily available in developing countries [9]. A simple method of getting the tissue for the DNA extraction is introduced while keeping in mind the problems encountered in the laboratory infrastructure of developing countries. 2 MATERIALS AND METHODS The study included 100 cases, 20 cases of squamous cell papilloma (SCP) and 80 of squamous cell carcinoma (SCC) of various grades. These lesions were selected from all parts of skin, lips, and oral cavity. The data was collected from January 2006 to December Most of the samples were collected from the Department of Pathology, King Edward Medical University, while a few were obtained from other hospitals in Lahore. The data was collected with the consent of patients and a record was entered in a proposed proforma, designed for the study. All the ethics were followed before the collection of data. All the patients willingly deposited their biopsy specimens for histopathology in the Department of Pathology, King Edward Medical University, Lahore. Signatures of the patients or their relatives were taken for further study of p53 from these samples. The remaining samples were preserved for the use of patients in future. Basic histopathology reports were prepared in the Department of Pathology. DNA was extracted from these tissues for detection of p53 mutation by the PCR. Tissues were processed in the following way. 2.1 Detection of DNA from paraffin-embedded tissues Sections with microtome The tissues received were fixed in 10 per cent neutral buffered formalin to prevent autolysis and bacterial decomposition. Once the tissues were fixed, they were grossed, proper sections were taken, processed in an automatic tissue processor, and embedded in paraffin blocks for microtomic sections. Before cutting the tissue for DNA extraction for PCR, histopathological examinations of sections were carried out by hematoxylin and eosin (H&E) staining. This was done to confirm the targeted areas for detection of DNA. For purification of DNA from paraffin-embedded sections the following protocol was followed. 1. Immediately after removal from the patients, all samples were routinely fixed in 10 per cent neutral buffered formalin (the average period of fixation was 24 h at room temperature (RT)). 2. Fixed tissues were processed routinely through dehydration in graded ethanol, clearing in xylene, and embedded in paraffin blocks using an automatic processor. Additional paraffin was removed to expose the tissue and two sections were cut by microtome. These sections were stained with hematoxylin and eosin for the selection of paraffin blocks containing tumours to proceed for DNA extraction. Two competent surgical pathologists confirmed the histopathology of concerned tissues. 3. Two 10 mm thickness flaps were made from each tissue and collected in an autoclaved plastic microtube (1.5 ml) or the sections were kept on the glass slides if microtubes were not available. (Transfer the tissue from the slides to a suitable glass tube.) 4. A heating method based on the principles of the antigen retrieval technique was followed to yield better DNA quantity [6]. For this the sectioned tissues were incubated in an autoclave at 60 uc for one hour Simple alternative method of scraping without microtome 1. The selected paraffin block was set on a black card paper (4 cm64 cm) and the marked targeted area was sampled with the tip of a surgical blade on a scalpel. (Glass sheets can be used instead if card paper is not available.) 2. Additional wax on the covered surface of the tissue was scraped out and then the targeted tissue was carefully scraped further to a maximum depth of 1 mm, to avoid cutting through or mixing with underlying normal tissue. 3. The average surface area of tissue samples was approximately cm, although smaller fragments were also chosen successfully. 4. The scraped tissue, which consisted either of pieces or of a fine powder, was collected into a 1.5 ml microtube. 5. Multiple targeted areas scraped from different pathological lesions were chosen for DNA analysis from the same block.

3 Detection of DNA A new surgical blade and microtube were used for each case to avoid DNA contamination. 7. The tissues were scraped precisely with less inclusion of the wax component by assessing the density of the fixed tissue since the fixed and dehydrated tissues are harder than the wax. Using a scalpel and measuring with an electronic balance for DNA extraction, 1 mg of pure scraped tissue (hard grey brown) was collected from each block in a separate microtube. 8. The sections were incubated in an autoclave at 60 uc for one hour. 2.2 DNA extraction 1. 1 ml of xylene was added to the microtube containing tissue sections for 30 min for two changes and 75 per cent ethanol was added for 30 min with two changes. 3. It was then washed with phosphate buffered saline (PBS) for 15 min with two changes. 4. The 500 ml PBS and universal buffer solutions were mixed at ph 9 in a microtube containing the tissue sections. 5. It was heated at 120 uc using an autoclave for 20 min. 6. The tube was then allowed to cool for 5 min ml of lysis buffer was added (proteinase-k 20 mg/ml, 50 ml, 1M tris-hcl solution 10 ml, 0.5M EDTA 2 ml, 10 per cent SDS 100 ml, and distilled water 838 ml) and incubated at 52 uc overnight until all tissue fragments were dissolved completely ml phenol, chloroform, and isopropanol alcohol were added in a ratio of 25:24:1 to the de-waxed tissue, mixed by vortex and centrifuged at RT and g for 10 min. 9. Supernatant fluid was transferred to an autoclaved microtube using a 100 ml pipette and one volume of chloroform was added to the supernatant mixed by vortexing and centrifuged at g for 5 min. 10. The upper aqueous supernatant was carefully removed to another fresh microtube. Then 0.1 volume of 3M sodium acetate was added to the new tube and mixed by vortexing; 1 volume of isopropanol was added and incubated at 220 uc overnight. 11. The precipitated DNA was centrifuged at g at 4 uc. The supernatant fluid was discarded and the sediment washed once with 75 per cent ethanol. The extracted DNA was collected after further centrifugation and the final yield of DNA was dissolved in 50 ml of distilled water after drying completely in a hood. 2.3 Procedure of the polymerase chain reaction [9, 10] PCR reactions Standard PCR reactions were performed using Taq DNA polymerase and primers were designed for aberrant p53 at axon 7 with sequence 59-GACT- CCTACCGGAAACAGGT-39 and 59-CTGTACTGATGGA- TGTCTTC-39 for the 287 bp fragment, to amplify a fragment of Exon-7 of the p53 gene. Thirty-five cycles of amplification were performed on DNA extracted from the sample of interest using a denaturing temperature of 95 uc for 25 min, annealing at 54 uc for 1.50 min, and extension at 72 ucfor1.30min[9, 10] Electrophoresis condition Fifteen microlitres of the PCR products were removed and mixed with 3 ml of a loading buffer and then loaded on a 2 per cent agarose gel. The TBE buffer was used for the electrophoresis. The gel was set at 100 V for 1 h and then stained with ethidium bromide. After staining, the bands were seen under ultraviolet (UV) light. The point mutations were characterized with a 100 bp (base pair) DNA ladder for the p53 gene at 287 bp. 2.4 Data analysis Data were collected and entered in the tables and computerized. Fisher s exact test was used to compare the results of both methods A and B [8]. 3 RESULTS The cases were considered positive when white bands appeared on the strips of gel against 287 bp. Ladder (M), positive control (+ve), and negative control (2ve) were used to analyse the results of the conventional PCR. In this study 20 cases were of benign papilloma and 80 cases were of squamous cell carcinoma. Mutation of p53 was not detected in any papilloma by the PCR performed by both methods of DNA extraction.

4 1002 M H Bukhari, S Niazi, R Ghani, Z Rathore, R Basharat, N A Chaudhry, and S Naeem Mutation of p53 was successfully detected from 77 (96 per cent) cases of squamous cell carcinomas from the tissues obtained by microtomy (method A) and 75 (93.75 per cent) cases from the tissues obtained by scraped tissue (method B). There is no statistical difference between both methods (P ) (Table 1 and Fig. 1). 4 DISCUSSION PCR detection of specific mutations of any gene on DNA has become a vital factor in oncology and this requires fresh or frozen tumour samples. This facility is not available in most of the centres in developing countries like Pakistan. These research studies were often impeded by a lack of availability of required samples for DNA extraction to confirm the suspected mutation [1, 2, 11]. When work was started to detect p53 mutations in SCC at Lahore in 2002, no facility was available to initiate the work. A search of the literature revealed that no simple technique was available to extract DNA from paraffin-embedded tissue that could be used at the research laboratory in Lahore. Keeping in mind this problem, other scientists have also adopted different techniques to overcome this for DNA isolation to permit mutational analysis of p53 from minimal amounts of paraffin-embedded archival tissue samples. The authors followed Chen and Clejan [12], Schubert et al. [7], and Koyama et al. [8], who introduced very simple methods for DNA detection from formalin-fixed paraffin-embedded tissues but these techniques are still cumbersome to perform in an area with limited facilities and for the research students in universities of Pakistan. The authors modified the procedures to make them easier for local use. In the present study the methods of Chen and Clejan [12] and the Gentra system [6] were further simplified. In the present study a specialized microtome was not used to take the sections; the routine microtome used for histopathology was used to perform sectioning. Simple tubes were used to collect samples Table 1 Comparison of PCR of DNA extracted by methods A and B Method of DNA extraction PCR +ve PCR 2ve Total A B Total P is non-significant (P ). Fig. 1 p53 point mutation is shown as wide white transverse lines by conventional PCR methods against ladder M of 100 bp at 287 bp (base pair) from the patients of squamous cell carcinoma 47 53, 55, and 57, while no point mutation is seen from the patients of carcinoma 54, 56 and 58 (method A) (C stands for carcinoma and +ve and 2ve control bars are shown on either side of ladder M) used for purification of DNA (method A) and the results of tissue collected by simply scraping with knives from paraffin blocks without using a microtome were also compared. These tissues measured cm and were then used for DNA purification (method B). The p53 mutation was successfully detected in the laboratory from the tissues taken by both methods (A and B). No p53 mutation was detected in any SCPs. These findings are consistent with Lin et al. [13], who did not find p53 mutation in HPV affected SCP. Using method A, p53 mutation was detected in 77 (96 per cent) samples of SCC as compared to 75 (93.75 per cent) samples when method B was used. The results of DNA extraction by PCR detection were compared for both techniques. There was no significant difference in these results (P ). On comparing results from both methods with available international data the reliability of the present results was ensured. The reliability was tested by positive detection of mutated DNA from all the tissues obtained by both methods and the percentage of p53 mutation in SCC but no mutation in SCP. These results are consistent with international data of p53 mutation in SCC detected by the PCR that is in a range per cent [14 20]. The percentage of p53 mutation in the present malignant cases is higher as compared to other studies due to the reliable methods of DNA extraction used. These findings of p53 mutations are also consistent with the findings of Maurer et al. [20] who found 92 per cent mutated p53 protein expression in SCC by the PCR, while, in the present study, this frequency of p53 mutation in SCC was 96 and 93 per cent detected by methods A and B respectively.

5 Detection of DNA CONCLUSION The present method of DNA extraction is very simple and can be used in research laboratories in developing countries with little available infrastructure. The reproducibility and quality assurance of these results is satisfactory and comparable with already established but cumbersome and costly techniques. ACKNOWLEDGEMENTS The authors are grateful to Professor Dr Nikhat Siddique (Karachi University, Karachi, Pakistan) who helped in designing this PCR program and gave encouragement in developing new methods. They are also grateful to laboratory staff at King Edward Medical University and Shaukat Khanum Memorial Cancer Hospital, Lahore, Pakistan for their cooperation during the research work. REFERENCES 1 Murtaza, I., Mushtaq, D., Margoob, M. A., Dutt, A., Wani, N. A., Ahmad, I., and Bhat, M. L. A study on p53 gene alterations in esophageal squamous cell carcinoma and their correlation to common dietary risk factors among population of the Kashmir valley. World J. Gastroenterol., 2006, 12, Mullis, K. B. Target amplification for DNA analysis by the polymerase chain reaction. Ann. Biol. Clin. (Paris), 1990, 48, Vierstraete, A. University of Ghent. URL Updated , available from avierstr/principles/pcr.html, accessed 21 May Taylor, C. R. Paraffin section immunocytochemistry for estrogen receptor. The time has come. Cancer, 1996, 77, Shi, S. R., Imam, S. A., Young, L., Cote, R. J., and Taylor, C. R. Antigen retrieval immunohistochemistry under the influence of ph using monoclonal antibodies. J. Histochem. Cytochem., 1995, 43, Shi, S. R., Cote, R. J., and Taylor, C. R. Antigen retrieval techniques: current perspectives. J. Histochem. Cytochem., 2001, 49, Schubert, E. L., Bischoff, F. Z., Whitaker, L. L., Pleasants, L. M., and Hansen, M. F. A method to isolate DNA from small archival tissue samples for p53 gene analysis. Human Mutation, 2005, 2, Koyama, K., Uobe, K., and Tanaka, A. Highly sensitive detection of HPV-DNA in paraffin sections of human oral carcinomas. J. Oral Pathol. Med., 2007, 36, Gentra system. Pure gene DNA purification system. Genomic DNA purification kit, instruction. Minnesota, USA, 2006, pp Shi, S. R., Cote, R. J., Wu, L., Liu, C., Datar, R., Shi, Y., et al. DNA extraction from archival formalinfixed, paraffin-embedded tissue sections based on the antigen retrieval principle: heating under the influence of ph. J. Histochem. Cytochem., 2002, 50, Agresti, A. A survey of exact inference for contegency tables. Statist. Sci., 1992, 7, Chen, B. F. and Clejan, S. Rapid preparation of tissue DNA from paraffin-embedded blocks and analysis by polymerase chain reaction. J. Histochem. Cytochem., 1993, 41, Lin, K. Y., Westra, W. H., Kashima, H. K., Mounts, P., and Wu, T. C. Co-infection of HPV-11 and HPV- 16 in a case of laryngeal squamous papilloma with severe dysplasia. Laryngoscope, 1997, 107, Jonason, A. S., Kunala, S., Price, G. J., Restifo, R. J., Spinelli, H. M., Persing, J. A., et al. Frequent clones of p53-mutated keratinocytes in normal human skin. Proc. Natl Acad. Sci. USA, 1996, 93, Ren, Z.-P., Ahmadian, A., Pontén, F., Nistér, M., Berg, C., Lundeberg, J., et al. Benign clonal keratinocyte patches with p53 mutations show no genetic link to synchronous squamous cell precancer or cancer in human skin. Am. J. Pathol., 1997, 150, Ziegler, A., Jonason, A., Simon, J., Leffell, D., and Brash, D. E. Tumor suppressor gene mutations and photocarcinogenesis. Photochem. Photobiol., 1996, 63, Ziegler, A., Jonason, A. S., Leffell, D. J., Simon, J. A., Sharma, H. W., Kimmelman, J., et al. Sunburn and p53 in the onset of skin cancer. Nature, 1994, 372, Ziegler, A., Leffell, D. J., Kunala, S., Sharma, H. W., Gailani, M., Simon, J. A., et al. Mutation hotspots due to sunlight in the p53 gene of nonmelanoma skin cancers. Proc. Natl Acad. Sci. USA, 1993, 90, O Connor, D. P., Kay, E. W., Leader, M., Murphy, G. M., Atkins, G. J., and Mabruk, M. J. Altered p53 expression in benign and malignant skin lesions from renal transplant recipients and immunocompetent patients with skin cancer: correlation with human papillomaviruses? Diag. Molec. Pathol., 2001, 10, Maurer,T.A.,Christian,K.V.,Kerschmann,R.L., Berzin,B.,Palefsky,J.M., Payne,D.,et al. Cutaneous squamous cell carcinoma in human immunodeficiency virus-infected patients. A study of epidemiologic risk factors, human papillomavirus, and p53 expression. Arch. Dermatol., 1997, 133,