Human Papilloma Virus (HPV) Genotyping from Paraffin Block Archive of Patient with CIN Diagnose That Storage at Pathology Anatomy Department Faculty of Medicine Udayana University/ Sanglah Hospital Bali
Dwija, IBN.Putra1and I Gusti Ayu Sri Mahendra Dewi
1.Microbiology Department, Faculty of Medicine Udayana University. 2. Pathology Anatomy Department, Faculty of Medicine Udayana University
ABSTRACT
Human Papilloma Virus (HPV) is a DNA, doble stranded virus that infect cutaneous, feet and hand. Up to now more than a hundred type of HPV has been typing, and one third of its infected the anogenital tissue.
Cervical cancer is one of malignancy that caused by HPV. DNA of the HPV can be isolated from scraping of cervical epithelial and from tissue that embedded in paraffin block. The quality of specimen, method that used to embed, the method and time to keep the blocks in storage place will influence the quality of DNA of HPV. The aim of this research is to know the quality of the DNA and genotyping of the HPV from the archive of cervical tissue that has been embedded in paraffin block for more than 2 years in Pathology Anatomy Department Faculty of Medicine Udayana University/ Sanglah Hospital. Fifty blocks were slicing with 10 µm thickness, HPV’s DNA Isolated using PK-1 buffer method, PCR were done by set of general primer (GP5/6+), the PCR product was send to the Macrogen Company for sequencing and sequence result were analysis by Chromas 1.5 version. This research was conducted at Pathology Department Leids Universitair Medische Centrum (LUMC) Leiden, Netherland during April-May, 2012.
From the 50 block of tissue, DNA with best quality was isolated from 12 of 50 (24%) blocks and we failed to isolated DNA from 38 (76%) of paraffin blocks. From the 12 DNA isolated, PCR Product was detected from 9 specimen. This all of 9 PCR products was send to the Macrogen for sequencing. According to Chromas 1.5 version analysis, 3 of 9 (33,3%) is HPV 16, 1 (11,11%) is HPV 18, 1 (11,11%) is HPV 31, 1 (11,11%) is HPV 58, 1 (11,11%) is HPV 11 and 2 (22,22%) other has no sequencing result. From this result can we concluded that HPV 16 is the most HPV type isolated from the cervical tissue of paraffin block archive and the quality of the paraffin block is not good enough.
BACKGROUND
Gynecological cancer is still a major problem in Indonesia right now. Cervical Cancer is the most common gynecological cancer in Indonesia, based on Pathologic report, in 2002 as much as 2.532 cases of cervical cancer were registries with five years survival rate was poor in the higher stages of malignancies (Aziz. 2009). Human Papilloma Virus (HPV) is the most common agent of cervical cancer. HPV is a double stranded virus that infects coetaneous, feet and hand, with total genome is more than 7000 base pair (bp). Until now more than 100 genotypes has been established, and one third of them is categories as a high risk group.
Some kind of specimens can be use to isolation of DNA of HPV. Isolation of the HPV DNA from the Formalin Fixed, Paraffin Embedded (FFPE) tissue is one of the interests method in last few years to detect and genotyping of HPV from cervical specimen, head and neck cancer (Schlecht et al, 2011., Steinau, et al.,2011). Stored FFPE specimen has some of advantages, such as possible to be use as retrospective research and epidemiological study. HPV DNA that stored in a long time in FFPE still have a good quality, therefore, DNA fragmentation, DNA-protein cross linked because of the presence of formaldehyde, and the paraffin its self can be a source of negative effect in yield of DNA and PCR amplification (Steinau et al.,2011). Standard method that used to embedded the tissue and storage the FFPE block has been established, but different geographic condition such as temperature, humidity, and quality of the paraffin may be influence the quality of the FFPE block and DNA as well.
Detection of the high risk HPV (HR-HPV) is most important role to reduce the incidence of cervical cancer by active vaccination. Cervical cancer stills the most common cancer in developing countries, as found in Indonesia. Type specific HR-HPV is fluctuation in Indonesia during study (Vet et all, 2008; Boer et al.,2006).
MATERIAL AND METHODE
This Research is a cross sectional descriptive analysis using 50 archive of paraffin block from the patient with CIN diagnose that storage at the Pathology Anatomy Department Faculty of Medicine Udayana University Bali. Research was done at Pathology Department Leids Universitair Medische Centrum (LUMC) Leiden, Netherland, during April-May,2012.
DNA Extraction
As much as 1-2 slices of paraffin block of 10 µm thick was collected with new and sterile 1,5 ml micro tube. DNA extraction was done using PK-1 buffer method. Two hundred and fifty micro litter PK-1 buffers and 15 micro liter of Proteinase K were added to the microtube, the tube incubated at 560C overnight, until all the paraffin dissolved. If not all paraffin dissolved after overnight incubation add other 15 µl fresh Proteinase K and extended the incubation at 560C for 4 hour. After overnight incubation at 560C, followed by incubate the micro tube at 990C for 10 minutes to inactivate the proteinase K. Briefly centrifuge the micro tube at 13.000 rpm for 10 minutes at 40C. The liquid phase under the paraffin layer were collected and stored at -200C for further analysis.
HPV PCR
Polymerase Chain Reaction (PCR) was done using GP5/6+ primer pair(GP5+ : 5’-TTT GTT ACT GTG GTA GAT ACT AC-3’, GP6+ : 5’-GAA AAA TAA ACT GTA AAT CAT ATT-3’) (van den Brule et all.,2002), which is amplified the L1 genome of HPV. As much as 11,5 Biorad®-supermix, 1 ul of forward and reverse primer, and 6,5 ul of de-ionized of water and 3 micro liter of DNA template were added to the total 23 micro liter volume PCR reaction. For the quality of DNA β -globin PCR was also done using a specific primer pair.
Sequencing
Ten micro litter of PCR product was send to the Macrogen Company, Netherland for sequencing. Sequencing result was analysis using a free Chromas 5.1 program.
H&E Staining
RESULT AND DISCUSSION
Result
Among fifty slices of FFPE tissue were staining with H&E Staining, to determine the stage of the CIN. Among them 5(10%) is Non Keratinizing squamous cell carcinoma, 19 (38%) is CIN 1-Chronic cervicitis, 5 (10%) Chronic cervicitis, and 21 (42%) is CIN 1-2. With the range of age is 27-66 (mean 45 year old). The cell morphology of the cervical cell with atipia coilositic can seen in figure 1.
Figure 1
Normal ectocervical squamous epithelial layer (H&E, 450x). Cervical Intraepithelial Neoplasm (CIN) 1 with koilocytotic atypia,Figure 2
suspicious for HPV infection (H&E, 450x) H&E Staining from the paraffin block
Figur 1. H & E Staining from the normal cervical cell and Cervical Intraepithelial Neoplasia (CIN) I with Koilocytic Atipia suspected with HPV infection.
On conventional histopathological examination, the HPV infection in cervical cells characterized by atipia koilositik, characterized by core atipia in cervical squamous cells, the core size is varied and enlarged to three times its normal size, hypercromasia on core chromatin, irregular nuclear membrane, the cavitations or halo around the nucleus of the cell cytoplasm and cell membrane thickening
Based on the result of sequencing analysis of 9 specimen, 3 of 9 (33,3%) is HPV 16, 1 (11,11%) is HPV 18, 1 (11,11%) is HPV 31, 1 (11,11%) is HPV 58, 1 (11,11%) is HPV 11 and 2 (22,22%) other has no sequencing result.
Figur 2. HPV type (left), PCR with GP5/6+ which amplify 155 bp of L1 gene of HPV (right)
Some of un-specific band was also found during electrophoresis, for cross check the type of the HPV, the PCR product was send to the Macrogen Company for Sequence with result as bellow.
Figure : 3. Sequence result of the PCR product of HPV Typing.
Discussion
The recent finding of this research is extraction with PK-1 buffer has not good quality of the DNA. Some method and protocols of FFPE extraction has been published with a varying of success ( Gilbert et all.,2007; Man, et all., 2001). The quality of the DNA will determine the quality of the PCR, even PCR is needed a small amount of DNA. FFPE block can preserve the DNA for a long time period, extraction of DNA from FFPE specimens with applying the heat leaves melted paraffin and other some remain debris in the eluted DNA and influence the subsequent PCR reaction. DNA fragmentation, DNA-protein cross linked because of the presence of formaldehyde, and the paraffin its self can be a source of negative effect in yield of DNA (Stienau et all.,2011; Santa and Schneider.,1991).
It’s recognized that the quality and yield of DNA from FFPE specimen was low, even the extraction doing with perfect method, the best size of the DNA from FFPE for PCR amplifies is 450 bp (Hariri et al.,2012). Some of the primer pair for amplification of HPV L1 gene such as MY09/11, GP5+/6+, CPI/CPII which is amplifies different size of gene. We use the GP5+/6+ primer because this amplify the short fragment of DNA, about 155 bp, because of the possibility of fragmented DNA during storage, using primer with short fragment amplifies will improve the yield of PCR.
During this research HPV type 16 is the most prevalence type was found. Other research was also found the same result, de Boer et al (2006) and has found HPV 16 is the most prevalence in Jakarta. Multiple HPV type infection was found predominantly in adenosquamous carcinomas which is type 16 and 18 was dominant (Schellekens, et al., 2004). In multi centre study in Indonesia, Vet et al (2008) found HPV 52 was the
highest frequency, instead of HPV 16 and 18. Suggesting high risk HPV (HR-HPV)
(type 16,18 and 52) was same sirculating in Indonesia.
As a conclusion, the quality of the DNA that extracted from the paraffin block
archive is not good enough due to the quality of specimen during embedding or
storage situation. HPV 16 is the most prevalence HPV type found, instead
REFRENCE
Aziz , M. Farid. 2009. Gynecological cancer in Indonesia. J Gynecol Oncol Vol. 20, No. 1:8-10, March 2009.
Boer ,M.A. De , J.N.I. Vet, M.F. Aziz, S. Cornain, G. Purwoto, B.E.W.M. Van Den Akker, A. Dijkman, A.A.W. Peters & G.J. Fleuren. 2006. Human papillomavirus type 18 and other risk factors for cervical cancer in Jakarta, Indonesia. Int J Gynecol Cancer 2006, 16, 1809– 1814.
Gilbert MP, Haselkorn T, Bunce M, Sanchez JJ, Lucas SB, Jewell LD, Van Marck E, Worobey M: The isolation of nucleic acids from fixed, paraffin embedded tissues–which methods are useful when? PLoS One 2007, 6:e537
Hariri ,Susan, Martin Steinau, Allen Rinas, Julia W. Gargano, Christina Ludema, Elizabeth R. Unger, Alicia L. Carter, Kathy L. Grant, Melanie Bamberg, James E. McDermott, Lauri E. Markowitz, Noel T. Brewer, Jennifer S. Smith. 2012. HPV Genotypes in High Grade Cervical Lesions and Invasive Cervical Carcinoma as Detected by Two Commercial DNA Assays, North Carolina, 2001–2006. PloS One. Vol.7.March 2012.
Man YG, Moinfar F, Bratthauer GL, Kuhls EA, Tavassoli FA: An improved method for DNA extraction from paraffin sections. Pathol Res Pract 2001, 197:635–642
Schellekens Maaike C, Anneke Dijkman, Mohammad Farid Aziz, Budiningsih Siregar, Santoso Cornain, Sandra Kolkman-Uljee, Lex A.W. Peters, and Gert Jan Fleuren.2004. Prevalence of single and multiple HPV types in cervical carcinomas in Jakarta, Indonesia. Gynecologic Oncology 93 (2004) 49–53
Schlecht , Nicolas F, Margaret Brandwein-Gensler, Gerard J. Nuovo, Maomi Li, Anne
Dunne, Nicole Kawachi, Richard V. Smith, Robert D. Burk, and Michael B.Prystowsky. 2011. A comparison of clinically utilized human papillomavirus detection methods in head and neck cancer . Mod Pathol. 2011 October ; 24(10): 1295–1305.
Stanta G, Schneider C: RNA extracted from paraffin-embedded human tissues is amenable to analysis by PCR amplification. Biotechniques 1991, 11:304, 306, 308
Steinau , Martin. Sonya S. Patel,and Elizabeth R. Unger.2011. Efficient DNA Extraction for HPV Genotyping in Formalin-Fixed, Paraffin-Embedded Tissues. The Journal of Molecular Diagnostics, Vol. 13, No. 4, July 2011.
van den Brule AJ, Pol R, Fransen-Daalmeijer N, Schouls LM, Meijer CJ, Snijders PJ: GP5_/6_ PCR followed by reverse line blot analysis enables rapid and high-throughput identification of Human papillomavirus genotypes. J Clin Microbiol 2002, 40:779–787
