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 B.
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.Putra1 and 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.
Keyword: HPV typing, Paraffin Block, CIN, Sanglah Hospital
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).
As we know there is no data about the isolation of the HPV DNA and Genotyping.
from the FFPE block archive in Pathology Anatomy Department Faculty of Medicine
Udayana University/ Sanglah Hospital. The aim of this study is to determine the genotyping
of HPV that isolated from the archive of the paraffin block that stored at the Pathology
Anatomy Department, Faculty of Medicine Udayana University.
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 ATT3’) (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
To analysis the abnormalities of the cervical squamosa cell, H&E staining were done with
the standard method. The pathologist will read the staining slide to determine the stages of
malignancies.
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).
Figure 2
Cervical Intraepithelial Neoplasm (CIN) 1 with koilocytotic atypia,
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
DNA extraction with PK-1 buffer among fifty specimens has not good quality of DNA,
that shown in the qPCR there is no band in the electrophoresis gel, to get good quality of
DNA, we decide to purified the DNA with a robotic system, after purification, among 12
(24%) specimen has a good quality of DNA and still no DNA detected was in 38 (78%)
specimen. PCR analysis we did from the 12 specimens but in electrophoresis the PCR
product detected in only 9 specimens.
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.
GP5/6+
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 HPV-18,31 and 52.
REFRENCE
Aziz , M. Farid. 2009. Gynecological cancer in Indonesia. J Gynecol Oncol Vol. 20, No. 1:810, 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
Vet ,JNI. MA de Boer, BEWM van den Akker, B Siregar, Lisnawati, S Budiningsih, D
Tyasmorowati, Moestikaningsih, S Cornain, AAW Peters and GJ Fleuren. 2008. Prevalence
of human papillomavirus in Indonesia: a population-based study in three regions. British
Journal of Cancer (2008) 99, 214 – 218.
with CIN Diagnose That Storage at Pathology Anatomy Department Faculty of
Medicine Udayana University/ Sanglah Hospital Bali
Dwija, IBN.Putra1 and 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.
Keyword: HPV typing, Paraffin Block, CIN, Sanglah Hospital
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).
As we know there is no data about the isolation of the HPV DNA and Genotyping.
from the FFPE block archive in Pathology Anatomy Department Faculty of Medicine
Udayana University/ Sanglah Hospital. The aim of this study is to determine the genotyping
of HPV that isolated from the archive of the paraffin block that stored at the Pathology
Anatomy Department, Faculty of Medicine Udayana University.
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 ATT3’) (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
To analysis the abnormalities of the cervical squamosa cell, H&E staining were done with
the standard method. The pathologist will read the staining slide to determine the stages of
malignancies.
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).
Figure 2
Cervical Intraepithelial Neoplasm (CIN) 1 with koilocytotic atypia,
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
DNA extraction with PK-1 buffer among fifty specimens has not good quality of DNA,
that shown in the qPCR there is no band in the electrophoresis gel, to get good quality of
DNA, we decide to purified the DNA with a robotic system, after purification, among 12
(24%) specimen has a good quality of DNA and still no DNA detected was in 38 (78%)
specimen. PCR analysis we did from the 12 specimens but in electrophoresis the PCR
product detected in only 9 specimens.
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.
GP5/6+
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 HPV-18,31 and 52.
REFRENCE
Aziz , M. Farid. 2009. Gynecological cancer in Indonesia. J Gynecol Oncol Vol. 20, No. 1:810, 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
Vet ,JNI. MA de Boer, BEWM van den Akker, B Siregar, Lisnawati, S Budiningsih, D
Tyasmorowati, Moestikaningsih, S Cornain, AAW Peters and GJ Fleuren. 2008. Prevalence
of human papillomavirus in Indonesia: a population-based study in three regions. British
Journal of Cancer (2008) 99, 214 – 218.