In: Bladder Cancer: Etymology, Diagnosis, and Treatments ISBN: 978-1-61728-002-3

Editor: William E. Nilsson © 2009 Nova Science Publishers, Inc.

Chapter 8

DETECTION AND GENOTYPING BY MICROARRAY OF

HUMAN PAPILLOMAVIRUS IN TISSUE OF BLADDER

AND BLADDER CARCINOMA BIOPSY

Antonio Sorlózanoa, Ana Isabel Moraga

b, Manuel Pareja

c,

Enrique Villegasa, María Luisa Villahermosa

b,

José Antonio Carrilloa and Jose Gutiérrez

a+*

aDepartment of Microbiology, School of Medicine, University of Granada,

and +Service of Microbiology, University Hospital Virgen de las Nieves, Granada, Spain

bGENOMICA, ZELTIA Group, Coslada, Spain

cDepartment of Urology, University Hospital San Cecilio, Granada, Spain

ABSTRACT

Background

Several studies have analyzed the association between bladder cancer (BC) and

Human Papillomavirus (HPV) infection using various direct and indirect diagnostic

techniques. However, even though there may be evidence of such association, the debate

on whether it is sheer coincidence or on whether a relationship of causality exists remains

open due, amongst other factors, to the lack of a high sensitivity standardized procedure

that can detect the virus. Thus, the aim of this work was to develop a technique with

appropriate sensitivity that may allow the detection of HPV DNA both in tumour tissue

biopsies as well as in healthy vesical tissue biopsies, while, at the same time, allowing the

detection of the genotype in question.

* Corresponding author: Departamento de Microbiologia, Facultad de Medicina, Avda de Madrid 11, E-18012

Granada, Spain. E-mail: josegf@ugr.es

Antonio Sorlózano, Ana Isabel Moraga, Manuel Pareja et al. 2

Material and Methods

Eighty-five biopsies of vesical tissue were obtained from patients operated upon at

the Urology Unit of Hospital San Cecilio in Granada, Spain: 34 bladder cancer and 51

healthy vesical tissue biopsies. Initially, the CLART®HPV (GENOMICA, ZELTIA

Group) system was used in all samples. This technique amplifies a fragment of the L1

region of HPV and identifies the genotype through microarray. With the aim of

increasing the sensitivity in HPV DNA detection, a nested-PCR specific for each of the

genotypes with greater oncogenic risk were designed.

Results

HPV DNA was detected in 5.9% of the biopsies (2 samples with genotype 16 and

3 with genotype 82) through CLART®HPV detection. HPV DNA was however detected

in 17.6% of the samples analyzed (1 with genotype 35, 2 with 51, 3 with 52, 2 with 58, 5

with 82 and 2 with two concomitant genotypes) through nested-PCR. Nested-PCR

increased the sensitivity in the detection of genotypes 18, 33, 51 and 82 by two 10-fold

serial diluted in relation with the CLART®HPV system, and in the detection of

genotypes 52 and 58, by one 10-fold serial diluted, although the same sensitivity was

maintained in both techniques in the detection of genotypes 35 and 39.

Conclusion

The development of an ultrasensitive technique in the future for detecting HPV DNA

in bladder cancer and in vesical healthy tissue biopsies will allow the determination of a

causal relationship between HPV infection and bladder carcinoma.

INTRODUCTION

Bladder carcinoma (BC), often considered as only one type of neoplasia, comprises a

heterogeneous group of processes which encompasses lesions of very different

biopathological behaviour. Various clinical observations have shown that, notwithstanding

(high grade) in situ carcinoma, BC comprises of two main clinicopathological groups:

superficial and deep or musculo-invasive. Each of them has a different natural history and,

maybe also different risk factors in their development [1]. The mechanisms associated to the

appearance and progression of these tumours, as well as the possible risk factors involved,

continue being subject to study to date [1-7].

On the other hand the oncogenic potential of the human papillomavirus (HPV) has been

clearly proven [8] and, although most studies have centred on tumours of the female genital

tract, specially those of the neck of the uterus, the study of its participation in other urogenital

tumours, amongst them, BC, has also been promoted [9-11]. In this sense, various authors

have tried to establish the relationship between HPV infection and BC, mainly with

transitional cell tumours [12], a possible relationship based on the virus epithelial tropism and

on the anatomic proximity of the urogenital area.

A meta-analysis carried out by our team shows how the results obtained from the

44 studies analyzing said relations (all published before June 2005) are contradictory [13]. In

Detection and Genotyping by Microarray of Human Papillomavirus in Tissue… 3

39 studies, the researchers determined the presence of HPV DNA and found a prevalence of

between 0% [14-22] and 100% [23]; pooled estimation of the presence of the infection was

16.0% (95% CI 12.8 to 19.1) and pooled Odds Ratio (OR) estimation was 2.3 (95% CI 1.3 to

4.1). Seven studies analyzed HPV infection by detecting the antigen or antibodies and the

prevalence found was between 14% [24] and 60% [22,25]. Pooled estimation of the

prevalence of infection was 32.4% (95% CI 17.0 to 47.8) while pooled OR estimation was 2.9

(95% CI 1.7 to 5.3).

Globally considered, then, although most of the studies show the presence of the

infection, however there is a significant dispersion of the results, it may be because the

methodologies employed in such studies varied, were badly standardized (in some cases

control groups were absent) and/or scarcely explicit, thus making it difficult for totally valid

conclusions to be obtained. In spite of all the research work, therefore, it is impossible to

obtain definite conclusions that may indicate the participation of HPV in the development of

vesical neoplasia [13].

Several other articles were published after said meta-analysis, and until September 2009;

again, however, while some state that HPV does not seem to play a role in the pathogenesis of

either type of bladder cancer [26,27], others state that HPV might play a causative role in BC

[11,12,28]. Notwithstanding, these analyses, like the previous ones, are lacking in sufficient

methodological information, so that they do not allow us to establish a definite conclusion.

In the last few years the microbiological tests used to detect HPV infection have

undergone changes. Today it is most common to use PCR but this should not detract from

other techniques that rely on host response or that detect the virus in situ, the sensitivity and

reliability of which have been compared. In support of this fact is the satisfactory relationship

between the results obtained by Ludwig et al [29], who used direct and indirect results to

detect infection.

Our retrospective study of the published results of HPV involvement in bladder cancer

[13] leads to some interesting conclusions. One of the conclusions is that comparisons among

studies can be complicated by various factors: 1) the different populations studied may have

different risk factors, such as genetics, geography and life-style, of which any might affect

carcinoma development in the bladder epithelium; 2) different researchers often use different

techniques with varying specificity and sensitivity; 3) technical mistakes made by the

laboratory researcher or even the choice of the right primer can affect the experimental

results. Thus, current diagnostic methods such as biopsy specimens may well provide low

numbers of HPV genomes and they are often inconclusive for identifying the specific HPV

strain, encouraging researchers to further examine universal detection methods using DNA.

The less valid results of our study could be due to a lack of HPV test sensitivity. For this

reason, it is the intention of HPV experts and the WHO to establish international biological

standard materials for the diagnosis and therapy of human disease. The availability of

international HPV DNA standards would contribute to the field of HPV prevention, diagnosis

and treatment [30,31].

Thus, the aim of our study was to develop a technique for detecting HPV DNA in

biopsies of BC and of healthy vesical tissues, with adequate sensitivity and which may also

allow the detection of the genotype in question.

Antonio Sorlózano, Ana Isabel Moraga, Manuel Pareja et al. 4

MATERIAL AND METHOD

Study Design and Tissue Samples

Eighty-five patients were selected from those who underwent surgery at the Urology Unit

of University Hospital San Cecilio in Granada, Spain, between September 2003 and April

2007. Two fragments of approximately 25 mm2

of vesical tissue were obtained from all of

them through biopsy. All biopsies were stored at -80 ºC until their subsequent processing.

Table 1. Characteristics of patients and bladder tumour lesions

Patient/Sample Sex Age Smoking Category tumour Histological grade Stage

1 Male 55 No Recurrence G1 T1

2 Male 55 Yes Recurrence G2 T1

3 Male 59 Yes Recurrence G1 T1

4 Male 63 Yes Primary G2 Tx

5 Male 86 Yes Primary G2 T2

6 Male 51 Yes Primary G2 T1

7 Male 39 Yes Primary G3 Tx

8 Male 65 Yes Primary G3 T1

9 Male 78 Yes Recurrence G2 T1

10 Male 61 Yes Recurrence Gx Tx

11 Male 61 Yes Primary G3 T1

13 Male 64 Yes Primary G3 T2

15 Male 77 Yes Primary G2 T1

16 Male 76 No Primary G3 T2

17 Male 74 Yes Primary G3 T2

18 Male 61 Yes Primary G2 T1

19 Male 72 Yes Primary G2 T1

20 Male 36 No Recurrence G1 Ta

21 Male 78 Yes Primary G2 T1

22 Male 59 Yes Primary G3 T2

23 Male 61 Yes Primary G2 T1

24 Male 51 Yes Primary G2 Ta

25 Male 68 Yes Recurrence G2 T1

26 Male 53 Yes Primary G2 T1

27 Male 67 Yes Primary G1 Ta

28 Male 68 Yes Recurrence G2 Ta

30 Male 76 Yes Primary G3 T2

31 Male 74 Yes Recurrence G2 Ta

33 Male 82 No Recurrence G2 T1

34 Male 82 Yes Primary G3 T1

35 Male 69 Yes Recurrence G1 T1

36 Male 78 Yes Recurrence G2 T1

37 Male 72 Yes Recurrence G2 T1

38 Male 78 Yes Primary G3 T2

Detection and Genotyping by Microarray of Human Papillomavirus in Tissue… 5

In 34 patients (Table 1) the biopsy was obtained from the vesical tumour lesion after

undergoing transurethral resection (TUR). All of them were male. The mean age of the group

was 66 years, with a range of 36 to 86 years. 88.2% were, or had been, smokers. Of the

vesicle lesions resected, 61.8% were primary tumours while the rest were recurrent. 9.5% of

the primary tumours were Ta, 47.6% were T1 and 33.3% were T2. Assessing the histological

grade, we discovered that within the primary tumours 4.8% were G1, while 47.6% were G2

and also G3. Of the recurrent, 30.8% were G1 and 61.6% were G2; in said cases, 23.1% were

Ta and 69.2% were T1.

Healthy vesicle mucosa was biopsied from the 51 remaining patients (Table 2). The

majority of these patients underwent prostate adenomectomy surgery using Freyer's technique

(46 patients), two patients underwent TUR surgery of prostate carcinoma; one, inflammatory

vesical lesion surgery and two (both women), TUR surgery of infiltrating endometrial

carcinoma. The mean age of the group was 70 years, with a range of 53 to 82 years. 54.9%

were, or had been, smokers.

Table 2. Characteristics of patients who underwent biopsy of healthy bladder tissue

Patient/

Sample Sex Age Smoking

Patient/

Sample Sex Age Smoking

12 Male 82 Yes 61 Male 69 Yes

14 Female 74 No 62 Male 65 Yes

29 Male 62 Yes 63 Male 77 No

32 Female 73 No 64 Male 68 Yes

39 Male 78 Yes 65 Male 71 No

40 Male 75 No 66 Male 69 Yes

41 Male 78 No 67 Male 75 Yes

42 Male 77 No 68 Male 65 Yes

43 Male 75 No 69 Male 68 Yes

44 Male 65 Yes 70 Male 70 No

45 Male 72 No 71 Male 71 No

46 Male 64 No 72 Male 73 Yes

47 Male 70 No 73 Male 80 Yes

48 Male 66 Yes 74 Male 74 No

49 Male 74 No 75 Male 65 No

50 Male 63 Yes 76 Male 77 Yes

51 Male 81 Yes 77 Male 76 Yes

52 Male 64 Yes 78 Male 67 Yes

53 Male 65 Yes 79 Male 60 No

54 Male 68 No 80 Male 73 No

55 Male 70 Yes 81 Male 75 No

56 Male 81 Yes 82 Male 73 Yes

57 Male 80 Yes 83 Male 60 Yes

58 Male 53 Yes 84 Male 63 No

59 Male 64 No 85 Male 67 Yes

60 Male 63 No

Antonio Sorlózano, Ana Isabel Moraga, Manuel Pareja et al. 6

Preliminary Detection of HPV DNA in Bladder Biopsies through

CLART®HPV

Genotyping of eighty-five bladder biopsies was performed with CLART®HPV

(GENOMICA, ZELTIA Group, Coslada, Spain) [32], This CE labelled kit comprises a

multiplex-amplification of L1 conserved region which is performed using biotin-labeled

primers, and a hybridization of the amplicon (450 bp) on a low-density tube array containing

HPV type-specific probes. HPV types genotyped with the kit included high-risk (HR)

genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82; low-risk (LR)

genotypes 6, 11, 40, 42, 43, 44, 54, 61, 66, 70, 72, 81 and 89 and genotypes with probable

high-risk (PHR) 53 and 66. The amplified fragment contain highly conserved regions used as

PCR targets for the amplification of different types of HPV virus, but shows enough

variations as to allow the differentiation of each type of virus using specific probes.

The amplified product is visualized through a tube microarray (ArrayTube, AT), a system

that basically consists of the inclusion of a microarray in the lower part of an Eppendorf-type

test tube. This type of technology allows the simultaneous detection of multiple molecular

markers of diagnostic utility, in this case, as stated, a fragment of the L1 region of 35 different

types of HPV, and the controls necessary to guarantee the reliability of the results obtained.

The amplified fragments obtained through PCR were hybridized with their respective

specific probes immobilized in specific and known areas of the AT tube; then they were

incubated with a conjugate of streptavidin-peroxidase. This binds to the biotin present in the

amplified products through streptavidin and the peroxidase activity provokes the appearance

of a product that is insoluble with 3,3',5,5'-tetramethylbenzidine (TMB), and thus its

precipitation takes place in the areas of the AT tube where hybridization occurs.

Two internal controls were also added to the CLART®HPV. On the one hand, a fragment

of 892-bp of human CFTR (Cystic Fibrosis Transmembrane Regulator) gene is amplified and

serves as control of the human DNA present in the sample and, on the other, all the tubes also

contain a modified plasmid that serves as inhibition control for the amplification reaction.

This internal control is amplified with the same primers as the human CFTR gene, but the

size of the amplified part is different (1202-bp). Both controls are amplified simultaneously

with HPV in a single reaction tube.

The DNA was extracted from all biopsied vesical tissue samples and purified following

the manufacturer instructions. Five microliters of DNA were amplified in a final volume of

50 l. Amplification conditions consisted of 95 °C for 9 min; 45 cycles of consecutive

denaturation, annealing, and DNA extension (94 °C for 30 sec, 55 °C for 1 min, and 72 °C for

90 sec, respectively) and final extension for 8 min at 72 °C. All PCR tubes for CLART®HPV

have the reagents necessary for amplifying a fragment of the L1 region of the 35 HPV

genotypes (buffer, MgCl2, dNTPs, taq polymerase, primers specific for amplifying HPV

DNA and primers for both controls, at the appropriate levels).

Amplification products were also subsequently hybridized using the AT system with the

aim of determining the genotype. The image obtained in the visualization of the microarray

corresponds to the probe imprint pattern observed in Figure 1 (each genotype is detected three

times).

Detection and Genotyping by Microarray of Human Papillomavirus in Tissue… 7

R 70 71 72 73 81 82 83 84 85 89 R

51 52 53 54 56 58 59 61 62 66 68

R 18 26 31 33 35 39 40 42 43 44 45

81 82 83 84 85 89 CI DNA 6 11 16

54 56 58 59 61 62 66 68 70 71 72 73

31 33 35 39 40 42 43 44 45 51 52 53

82 83 84 85 89 CI DNA 6 11 16 18 26

56 58 59 61 62 66 68 70 71 72 73 81

39 40 42 43 44 45 51 52 53 54

R CI DNA 6 11 16 18 26 31 33 35 R

R: Developing control. Biotin probes are inserted at these positions, such that, as long as the developing

is correctly executed, these localizations are revealed. This appropriately shows how the array is

situated in the reading. No PCR product is detected.

CI: Probes for detecting the internal control of the PCR (1202-pb fragment)

DNA: Probes for detecting a fragment of 892-bp of human CFTR gene

Blank: No probe printed.

As observed, all genotypes go in triplicate

Figure 1. Probe impression pattern on the AT for the detection of the different genotypes

Development of a Nested-PCR Method for Detecting HPV DNA in Bladder

Biopsies

A nested-PCR was designed to increase the sensitivity in HPV DNA detection. Its first

amplification is that described in the section above (CLART®HPV) and, for the second, new

primer pairs were designed specifically for each of the genotypes of higher oncogenic risk

(16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82) (data not shown), as well as new

amplification conditions. For oligonucleotides design we used sequences available in the

database http://www.ncbi.nlm.nih.gov/pubmed/. Specifically we selected primers set, using

the Oligo 6 Software (Molecular Biology Insights, Inc., Cascade, CO, USA).

Five microliters of the first amplification product were then used as the template for the

second reaction in a final volume of 50 l. In all assays, amplification conditions consisted of

95 °C for 9 min; 45 cycles of consecutive denaturation, annealing, and DNA extension (94 °C

for 30 sec, 50 °C or 55 ºC -depending of the specific nested- for 1 min and 72 °C for 90 sec,

respectively) and final extension for 8 min at 72 °C. Amplification products were

electrophoresed through a 2% agarose gel and visualized with a UV transilluminator after

ethidium bromide staining. Different size or patterns of bands were obtained with each

nested-PCR specific of HPV. To confirm the detected genotypes through the nested-PCR in

those samples, purified PCR products were sequenced on ABI Prism 3100 Genetic Analyzer

Antonio Sorlózano, Ana Isabel Moraga, Manuel Pareja et al. 8

(Applied Biosystems, Foster City, CA, USA). The sequences were submitted to

the

comparative sequence program BLAST (www.ncbi.nlm.nih.gov/blast/)

for searching

sequences with the highest degree of similarity to identify the genotype.

Transformation, Cloning and Sequencing of HPV DNA

In order to determine the sensitivity of detection of higher oncogenic risk HPV

genotypes, both through the CLART®HPV as well as through new nested-PCR, HPV DNA

samples with known genotypes were subjected to transformation, cloning and sequencing

procedures (Protocols and Plasmid Bank of GENOMICA).

To begin with, HPV DNA was amplified from the genotheque samples using primers

specific for each genotype. Amplified products were separated by agarose gel electrophoresis

and purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA).

Purified DNA was cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA),

transformed into Escherichia coli novablue (Novagen Inc., Madison, WI, USA) competent

cells through the heat shock method and incubated overnight at 37°C with shaking.

Transformation was detected on LB agar plates supplemented with ampicillin / IPTG

(isopropyl-D-1-thiogalactopyranoside) / X-Gal (5-bromo-4-chloro-3-indolyl- -D-

galactopyranoside) by screening the blue-white colonies. A recombinant clone was identified

as the white colony on an X-Gal and IPTG plate.

The recombinant colonies were then screened by PCR with primers specific for the

cloning site of the vector, and those containing inserts of the expected size were grown

overnight in 10 ml of LB media supplemented with ampicillin. The plasmids of these colonies

were then extracted using the PureYield™ Plasmid Midiprep System (Promega) kit.

After purification, the insert DNA fragments were identified through sequencing using

automatic DNA sequencer (ABI Prism 3100 Genetic Analyzer). The DNA fragments were

spectrophotometrically quantified (SmartSpecTM Plus Spectrophotometer, Bio-Rad, Spain). A

theoretical number of plasmid copies and reaction efficiency were calculated considering the

molecular weight. The plasmid was 10-fold serial diluted (from 104 to 10

-1 plasmid copies/ l)

and stored at -20°C. Five microlitres of those dilutions were used to determine the sensitivity

of the CLART®HPV and for nested-PCR.

RESULTS

Detection of HPV DNA in Bladder Biopsies with CLART®HPV

After DNA amplification of all samples and, subsequently, the visualization through

agarose gel electrophoresis, the results shown in Figure 2 were obtained. All samples showed

a fragment of 892-bp corresponding to the human CFTR gene, except for sample 14 where,

notwithstanding, the internal control was correctly amplified (1202-bp fragment). A fragment

of 450-bp was also observed in samples 6 and 9 (tumour tissue samples), 14, 53 and 60

(healthy vesical tissue samples). HPV DNA was thus detected in 5 of the 85 samples studied

(5.9%).

Detection and Genotyping by Microarray of Human Papillomavirus in Tissue… 9

From left to right :

Molecular weight marker VIII (Roche Diagnostics,

Mannheim, Germany)

Samples 1, 2, 3, 4, 5, 6, 7, 8, 9, 10

Negative control

Molecular weight marker VIII

From left to right :

Samples 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23

Molecular weight marker VIII

From left to right :

Molecular weight marker VIII

Samples 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37

Two negative control

Samples 38, 39

Molecular weight marker VIII

From left to right :

Molecular weight marker VIII

Negative control

Samples 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,

53, 54, 55, 56

Molecular weight marker VIII

From left to right :

Molecular weight marker VIII

Samples 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,

70, 71, 72

Molecular weight marker VIII

From left to right :

Molecular weight marker VIII

Samples 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85

Two negative control

Molecular weight marker VIII

Figure 2. Visualization by agarose gel electrophoresis (CLART®HPV)

The amplified products of all the samples were subsequently hybridized to detect their

genotype using the AT technology. As Table 3 and Figure 3 show, sample 6 was positive for

genotype 16, sample 9 for genotypes 61 and 82, sample 14 for genotype 82, sample 53 for

genotype 16 and sample 60 for genotype 82. The sensitivity of the AT technology was not

higher than the detection by agarose gel electrophoresis. The microarray only allowed the

determination of the genotype in the positive samples.

Antonio Sorlózano, Ana Isabel Moraga, Manuel Pareja et al. 10

Table 3. Genotype detected by CLART®HPV or nested-PCR and sequencing

No.

Sample Biopsied tissue

Genotype

detected by

CLART®HPV

Genotype detected by nested-PCR and

sequencing

35 39 51 52 58 82

5 Tumour tissue +

6 Tumour tissue 16

8 Tumour tissue +

9 Tumour tissue 61+82 +

10 Tumour tissue +

14 Healthy bladder tissue 82 +

17 Tumour tissue +

21 Tumour tissue +

22 Tumour tissue +

29 Healthy bladder tissue +

37 Tumour tissue +

53 Healthy bladder tissue 16 +

56 Healthy bladder tissue +

60 Healthy bladder tissue 82 + +

70 Healthy bladder tissue + +

75 Healthy bladder tissue +

Sample 6: genotype 16 Sample 9: genotypes 61 and 82 Sample 14: genotype 82

Sample 53: genotype 16 Sample 60: genotype 82

Figure 3. Genotype detected by CLART®HPV

Detection and Genotyping by Microarray of Human Papillomavirus in Tissue… 11

Nested-PCR Method for Detection of HPV DNA in Bladder Biopsies

To date, our research group has been able to effectively develop nested-PCR for the

detection of genotypes 18 (obtaining an amplification fragment of 266 pb), 33 (276 pb), 35

(288 pb), 39 (224 pb), 51 (251 pb), 52 (205 pb), 58 (151 pb) and 82 (155 pb). We are still

developing nested-PCR for the detection of genotypes 16, 31, 45, 56, 59, 68 and 73.

The results in Figure 4 were obtained after DNA amplification of all the samples using

the nested-PCR designed for the 8 genotypes and after the visualization through agarose gel

electrophoresis. The subsequent sequencing of the amplified fragments confirmed the

positivity of the samples, as observed in Table 3: the virus was detected in 8 BC biopsy

samples (23.5% of this group) (2 with genotype 52, 2 with 58 and 4 with 82); and 7 samples

of healthy vesical tissue were also positive (13.7%) (1 with genotype 35, 2 with 51, 1 with 52,

1 with 82, 1 with genotypes 35 and 82, and 1 with 39 and 82). HPV DNA was therefore

detected in 17.6% of the samples studied.

Quantification of the Sensitivity of Both Methods

As occurred with the development of nested-PCR, our research group has currently

developed recombinant clones for genotypes 18, 33, 35, 39, 51, 52, 58 and 82, used to

determine the sensitivity of the CLART®HPV system and of the nested-PCR for each

genotype. The results are outlined in Table 4.

Figure 4. Visualization by agarose gel electrophoresis (nested-PCR of different genotypes)

Antonio Sorlózano, Ana Isabel Moraga, Manuel Pareja et al. 12

Table 4. Sensitivity (in plasmid copies/reaction) of CLART®HPV

and nested-PCR for detection of eight HPV genotypes

Genotype CLART®HPV Nested- PCR

5×104 5×103 5×102 5×101 5×100 5×10-1 5×104 5×103 5×102 5×101 5×100 5×10-1

18 + - - - - - + + + - - -

33 + + + - - - + + + + + -

35 + + + + - - + + + + - -

39 + + + + - - + + + + - -

51 + + + - - - + + + + + -

52 + + + - - - + + + + - -

58 + + + + - - + + + + + -

82 + + + + - - + + + + + +

As can be observed, nested-PCR increased the sensitivity in the detection of genotypes

18, 33, 51 and 82 by two 10-fold serial diluted; in the detection of genotypes 52 and 58 by

one 10-fold serial diluted; but the same sensitivity was maintained in both techniques in the

detection of genotypes 35 and 39. Specifically the nested-PCR is able to detect 500 copies of

genotype 18, 50 copies of genotypes 35, 39 and 52, 5 copies of genotypes 33, 51 and 58 and

only 1 copie of genotype 82.

DISCUSSION

HPV may not be cultured in vitro and serology studies have limited accuracy because of

their failure to distinguish between current and past infections [34]. That is why, if there is

need for a study on the relationship between the virus and any neoplasic process, whether

uterine neck carcinoma or, as in our case, bladder cancer, it is more appropriate to use HPV

DNA detection techniques [35]. In this sense, PCR is the technique with highest sensitivity,

confirmed in several publications [35-37]. However, considering that there are over a hundred

genotypes of the virus [35], of which 15 show high oncogenic risk [38], the use of a PCR

with primers specific for each genotype demands a lot of time and work and, in addition,

requires validation for each of them [36].

The originality of the CLART®HPV system lies in it being a single procedure, in two

phases, the first being the amplification of an region of the HPV genome which is conserved

in most of the genotypes, the L1 region, and the second to typify the virus present in the

sample through microarrays, with which, in addition to obtaining faster and objective results,

it allows for the concurrent detection of mixed infections in one sample.

However, to carry out a pathogenicity study in which we can establish the possibility of

causal relationship, in addition to establishing the existence (or lack thereof) of an

epidemiologic relationship between the viral infection and cancer, it would be necessary to

create a technique with the highest possible sensitivity, such as a nested-PCR. And that is

what happened in our research. Currently the nested-PCRs developed increased their

sensitivity in comparison with the CLART®HPV in up to 100-fold, thus allowing an increase

in the percentage of samples in which the virus was detected, 5.9% to 17.6%.

Nevertheless, our work is not yet finished. We are developing nested-PCR for genotypes

16, 31, 45, 56, 59, 68 and 73, to be later applied to the 85 samples of the assay. Our final

Detection and Genotyping by Microarray of Human Papillomavirus in Tissue… 13

objective would be to develop an ultra-sensitive technique that may allow the detection of

higher oncogenic risk genotypes in different tissue samples, in three steps. The first step

would still entail the amplification of the L1 region in the same conditions as presently done

by the CLART®HPV system; the second step would be a multiplex-nested-PCR in which the

amplification conditions would allow amplification of the DNA sequences specific for each

genotype, and a third step of microarray typification.

Finally, although we can not as yet reach any conclusions on the relationship between

bladder carcinoma and HPV infection, we believe that in a near future the current

development of this new technique will allow us to definitely determine whether or not said

relationship exists. Probably the development of multiple PCR techniques for the detection of

a high number of serotypes will improve the HPV detection and association to bladder cancer.

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