Role of Human Papillomavirus Types 16, 18, and 52 in Recurrent Cystitis and Urinary Bladder Cancer Among Egyptian Patients

Abstract

Background

Bladder cancer is a common malignancy in Egypt. Human papillomavirus (HPV) could have a possible etiologic role in bladder carcinogenesis. We aimed to estimate the prevalence of HPV-16, -18, and -52 in Egyptian patients with bladder cancer or recurrent cystitis, and to study the correlation of type-specific HPV-immunoglobulin (Ig)G with polymerase chain reaction (PCR) results and different clinicopathologic parameters.

Methods

This study was conducted on 60 inpatients of the Urosurgery Department at the Theodor Bilharz Research Institute (TBRI), who were identified histopathologically and clinically as cancer bladder (group I, 20 patients), cystitis (group II, 24 patients), and cancer bladder with cystitis (group III, 16 patients), and a fourth group of 20 healthy control subjects (for serologic testing). Patients were subjected to detection of HPV-16 and -18 DNA by PCR on bladder tissue biopsies (BTB) and buffy coat cells (BCC) and serum IgG antibodies to L1 capsids of HPV-16 and -52 IgG by enzyme-linked immunosorbent assay (ELISA).

Results

HPV-16 and -18 DNA were detected in BTB (30% and 10%, respectively) with significantly higher rates (44.4%) in bladder cancer than cystitis cases (11.11%), with significant association with schistosomal affection (78.6% and 25%, respectively) and recurrence (48%, HPV-16). There was a significant association of transitional cell carcinoma (TCC) with HPV-16 in 69.2% and 61.1% of BCC and BTB, respectively. Multiple HPV types 16, 18, and 52 were significantly higher than single types (79.2% and 20.8%, respectively). The observed absolute association between seropositivity of HPV-52 (11.7%) and HPV-16 (26.7%) was significantly associated with TCC in patient groups only.

Conclusion

Our study confirmed the significant association of HPV-16, -18 and -52 with bladder cancer in Egyptian patients, with the suggestion of viral synergistic action in bladder carcinogenesis. Such HPV types were significantly associated with TCC tumors of low grade and high stage, with schistosomal affection and recurrence tendency. HPV serology would pave the way for better management and follow-up of patients and for optimal design and evaluation of HPV vaccination.

Introduction

Bladder cancer is a common malignancy in Egypt.[1,2] The increasing incidence of bladder carcinoma observed over the past 3 decades has stimulated research into the identification of possible etiologic agents. The significant association between human papillomavirus (HPV) infection and cancer bladder has been reported.[3,4]

More than 100 HPV genomes have been characterized on the basis of nucleotide sequence homology of viral DNA, and at least 42 types have an established etiologic role in tumors of the urogenital tract and anal region. Molecular and epidemiologic studies have distinguished HPV types into oncogenic types, eg, HPV-16, -18, -31, -33, -35, -45, -52, and -58. HPV-16 is the most prevalent oncogenic HPV genotype, with regional variation being highest in Europe and lowest in Africa.[5] The prevalence of other HPV types varies across geographic locations: HPV-35, -45, -52, -56, and -58 are more common in HPV-positive women in sub-Saharan Africa than in Europe, whereas HPV-52 and -58 are more prevalent in Japan and China.[6]

The role of HPV in the pathogenesis of carcinoma of the urinary bladder has been extensively investigated with contradictory results in non-Egyptian reports, ranging between 0%[7] and 80%.[8] Egyptian reports from the National Cancer Institute of Cairo University, Cairo, Egypt, revealed that the association between HPV and human cancer bladder ranged from 23%[9] to 49%.[10] The choice of specimen and the different techniques performed seem to influence the number of false-positive and false-negative results, eg, the use of single or multiple tumor fragments, fresh or fixed and paraffin-embedded material, the number of virus genotypes, contamination by plasmid DNA or polymerase chain reaction (PCR) products, and the use of high- or low-sensitivity techniques (Southern blot, PCR, or in situ hybridization).[11]

In addition, serology with HPVL capsids, viruslike particles (VLPs), as antigens has also been used to elucidate the association of oncogenic types with cervical neoplasia.[12] Moreover, antibody response to HPV-16 L capsids was significantly associated with the development of HPV-16-positive cervical intraepithelial neoplasia in a cohort study of university women.[13]

HPV-16 and/or -18 genomic sequences were identified in the urinary tract of female patients with recurrent and persistent urethritis and cystitis, and in transitional tumors of the bladder.[3,4]

However, the question of whether prior infection with a given type of HPV can reduce the risk for infection with a different HPV type and subsequent intraepithelial neoplasia development is unresolved. Similarly, it is not clear whether vaccination against a given HPV strain can protect against infections with other types, especially phylogenetically related types. Recently, some prospective studies that were based on viral DNA detection have reported that prior infection may not reduce the risk for acquisition of other HPV types.[14,15] To address this issue, the antibody response (immunoglobulin [Ig]G) to L1 capsids can be used as a marker of cumulative HPV exposures because serum antibodies are known to persist on long-term follow-up even after the clearance of viral DNA.[13]

The current study was designed to estimate the prevalence of HPV-16, -18, and -52 in Egyptian patients with bladder cancer or recurrent cystitis by PCR. Moreover, we aimed to investigate the magnitude and prevalence of HPV-VLP-specific IgG and its correlation with HPV-DNA and different clinicopathologic parameters.

Patients and Methods

This study was conducted from January 2001 to December 2006 on 60 patients (47 males and 13 females) who were admitted to the Urosurgery Department of the Theodor Bilharz Research Institute (TBRI); their ages ranged from 16 to 75 years (mean, 49.37 ± 13.61). They were divided into 3 groups of patients. Patients' groups were identified as cancer bladder (group I, 20 patients), recurrent cystitis (group II, 24 patients), and cancer bladder with cystitis (group III, 16 patients). A fourth group of 20 healthy control subjects was included for serologic testing. They were matched one-to-one with cases on age (within 5 years) and were attending the Urosurgery Department at TBRI. They were proved to be free clinically, radiologically, and microbiologically from chronic cystitis and bladder cancer. The Declaration of Helsinki[16] was followed for each case and control. Patients were diagnosed clinically by different urologic symptoms and/or signs, transurethral cystoscopic examination followed by histopathology of biopsies, or resected bladder masses. Schistosomal affection was diagnosed upon the presence of schistosomal eggs in urine sediment, demonstration of hematoxylin-eosin-stained bladder tissue sections, or association of serologic evidence. All tumors were graded pathologically from G1 to G3, according to the extent to which the tumor cells resemble the normal cells, and tumor staging, according to the degree of infiltration of the bladder wall, perivesical fat, and adjacent structures.[17]

Sample Collection

The bladder tissue biopsies (BTB), blood sample collection, and clinical data collection were conducted in a retrospective manner as the cases were selected. Blood samples (10 mL) were collected from all subjects. One part (5 mL) of blood was collected in EDTA tubes for separation of buffy coat rich in leukocytes for detection of HPV-16 and -18 DNA by PCR. The second part (5 mL) was centrifuged and stored at −85°C until it was used for performing serologic markers for diagnosis of HPV-16 and -52 for cases and control groups. Bladder biopsies were divided into 2 pieces: The first was kept in formalin for pathologic examination, whereas the other piece was directly stored in liquid nitrogen at −70°C to be further examined by PCR assay for detection of HPV-16 and -18 DNA.

Laboratory Investigations

Detection of HPV-16 and -18 DNA in BTB and buffy coat cells by PCR technique. Total cellular DNA was extracted from BTB according to Ausubel and colleagues[18] and from buffy coat cells (BCC) according to Higuchi.[19] HPV DNA was amplified by the PCR method with 2 sets of primers specific for E6 regions of HPV-16 or -18 DNAs that would amplify and detect a specific band of the expected size (140 bp), supplied at a concentration of 100 pmol/microliters (mcL) (Biometra, Göttingen, Germany). Their sequences, according to previously published data,[20] were as follows:

• p16F: (5′-AAGGGCGTAACCGAAATCGGT-3′);

• p16R: (5′-GTTTGCAGCTCTGTGCATA-3′);

• p18F: (5′-AAGGGAGTAACCGAAAACGGT-3′); and

• p18R: (5′-GTGTTCAGTTCCGTGCACA-3′).

Serologic detection of HPV-16 and -52 IgG by enzyme-linked immunosorbent assay with baculovirus recombinant HPV-16 VPLs.

• Detection of IgG antibodies to HPV-16 and -52 was performed with purified L1 capsids as antigens. The production of HPV-16 and -52 L1 capsids was described by Seedorf and colleagues.[21] DNA fragments containing the entire L1 open reading frame nucleotides 5637-7154 and 5565-7154 for HPV-16 and -52, respectively, were prepared by PCR with synthetic primers for HPV-16 and -52: 5' ends of sense primers contained Sma I recognition sequences, and 5' ends of antisense primers contained XbaI. HPV genome DNA were then cloned as templates.

• Cloning into pVL1393 (baculovirus-transfer vector, PharMingen, San Diego, California) as described by Matsumoto and colleagues.[22]

• Purification of VLPs according to Kirnbauer and colleagues.[23]

• Enzyme-linked immunosorbent assay (ELISA) for detection of IgG antibodies against HPV-16 and -52 in human sera. The cutoff values were determined statistically. The mean and SD were calculated for all 80 samples, and those specific absorbances higher than the mean +2 SD were considered seropositive. The mean and SD were calculated for those with specific absorbances lower than the initial mean +2 SD, and those higher than the second mean +2 SD were considered seropositive. The calculation was repeated until there were none to be excluded for the subsequent calculation, and the last mean +2 SD was chosen as the cutoff value.[24,25]

Statistical Evaluation

Statistical evaluation was done with Epi Info software for data management and analysis. Sensitivity, specificity, and predictive values of different assays were calculated against the results of HPV-16 and -18 DNA in bladder tissue by PCR. Chi-square and Fisher's exact test were used for significance testing of proportional data. The level of significance was established at P < .05. A histogram was used to illustrate ELISA representative results.

Results

Histopathologic Data

Among the 36 patients with cancer in groups I and III, 25 cases (69.4%) were diagnosed histopathologically as transitional cell carcinoma (TCC), 7 cases (19.4%) as dysplasia, and 4 cases (11.1%) as squamous cell carcinoma (SCC). Low-grade tumor (1 and 2) was detected in 27 tumors (75%), and muscle invasion (T2 and T3) was observed in 14 tumors (38.9%). Among the 40 patients with cystitis (groups II and III), 30 cases (75%) were diagnosed histopathologically as chronic nonspecific cystitis 8 cases (20%) as chronic schistosomal cystitis, 1 case (2.5%) as acute nonspecific cystitis, and 1 case (2.5%) as cystitis follicularis. Twenty-six of 60 enrolled patients (43.3%) were recurrent cases after transurethral resection or resection.

Detection of HPV-16 and -18 DNA by PCR

PCR analysis for the detection of HPV-16-specific (Figure 1) and HPV-18-specific DNA in BTB revealed the following: Among 60 patients, type-specific primers analyzed positive HPV-16 and -18 DNA in 30% and 10% of patients, respectively. Additionally, PCR analysis of BCC of the same patients revealed rates of positivity of 21.7% and 5% for HPV-16 and -18, respectively.

Figure 1.

Electrophoretic analysis of human papillomavirus (HPV)-16 amplification products. The 140-bp product was obtained. Lanes 1, 10: DNA standard size marker. Lanes 2, 3: positive and negative controls, respectively. Lanes 4, 5, 6, 8, and 9: bladder biopsy specimens positive. Lane 7: bladder biopsy specimen negative.

The concordance of HPV detection type results with clinicopathologic parameters is revealed in Table 1. Schistosomiasis was associated with 14 BTB, and 92.9% of such schistosomal patients were associated with HPV. Significant association of the overall virally infected patients with grade 2, stage III TCC tumors was found (P < .05; Table 1).

Table 1.

Clinicopathologic Data for HPV Positive Subjects

Clinicopathologic Data	HPV-16 DNA		HPV-16 IgG	HPV-18 DNA		HPV-52 IgG
	Bladder Tissue (N = 18)	Buffy Coat (N = 13)	(N = 16)	Bladder Tissue (N = 6)	Buffy Coat (N = 3)	(N = 7)
Age, mean	53	45	45	52	33	50
Range	19-70	19-66	19-60	32-65	19-45	19-70
Sex, Male	16	11	13	6	2	6
Female	2	2	3	0	1	1
Schistosomal affection
Present	11‡	5	7*	5†	2	4†
Absent	7	8	9	1	1	3
Recurrence
Yes	12*	11†	12†	3	2	3
No	6	2	4	3	1	4
Tumor type
TCC	11*	9*	11*	3	1	5
SCC	2	2*	2	1	0	1
Dysplasia	3	1	1	1	1	1
Cystitis type
CSC	3	2	3	3*	2*	3*
CNSC	5	6	6	1	1	0*
ANSC	0	0	0	0	0	0
CF	1	0	0	0	0	0
GradeX
1	2	2	3	0	0	2
2	10*	6	7	2*	1*	4*
3	2	2	2	3	2	1
StageXX
1	5	3	5	1	0	4*
2	3	2	2	1	0	1
3	4*	4*	4*	2*	1	2

HPV = human papillomavirus; IgG = immunoglobulinG; TCC = transitional cell carcinoma; SCC = squamous cell carcinoma; CSC = chronic schistosomal cystitis; CF = cystitis follicularis; CNSC = chronic nonspecific cystitis; ANSC = acute nonspecific cystitis

X: Grades 1, 2, and 3 refer to microscopic classification of well, moderately, and poorly differentiated tumors, respectively.

XX: Clinical stages: T1 = invasion of lamina propria; T2 = invasion of superficial muscle; T3 = invasion of deep muscle.

^*P < .05

^†P < .001

^‡P < .0001

The distribution of HPV-16 and -18 among the studied groups is summarized in Table 2. HPV-16 DNA yielded positivity rates of 46.7%, 42.9%, and 8.3% of BTB among groups I, II, and III, respectively. Among the 36 patients with cancer (groups I and III), HPV-16 DNA were significantly detected in 16 (44.4%) BTB compared with patients with cystitis (group II) (2 of 24, 8.3%; P < .01).

Table 2.

Distribution of HPV Detection Results Among the Studied Groups

Studied Groups	Number	HPV-16 Positivity in:			HPV-18 Positivity in:		HPV-52 Positivity in Serum N (%)
		Bladder Tissue N (%)	Buffy Coat N (%)	Serum N (%)	Bladder Tissue N (%)	Buffy Coat N (%)
Group I	20	9 (45.0)	5 (25.0)	7 (35.0)	2 (20.0)	0 (0.0)	0 (0.0)
Group II	24	2 (8.3)	3 (12.5)	3 (8.3)	1 (4.7)	1 (4.7)	3 (12.5)
Group III	16	7 (43.8)	5 (31.3)	6 (37.5)	3 (18.8)	2 (12.5)	4 (25.0)
Total	60	18 (30.0)	13 (21.7)	16 (26.7)	6 (10.0)	3 (5.0)	7 (11.7)
Group IV	20	NA	0 (0.0)	0 (0.0)	NA	0 (0.0)	0 (0.0)

HPV = human papillomavirus; NA = not analyzed

Detection of HPV-16 and -52 IgG by ELISA

Representative results of HPV-16 and -52 IgG serum antibodies are summarized in Figure 2. The frequency distributions of the majority of the samples from the healthy donors appeared to be normal and to peak near a specific absorbance of 0.2. The samples with specific absorbance values higher than the statistically determined cutoff value (0.35) were considered to be seropositive. Of the 60 studied subjects, 16 (26.7%) were positive for HPV-16 IgG and 7 (11.7%) were positive for HPV-52 IgG.

Figure 2.

Detection of immunoglobulin (Ig)G antibodies to viruslike particles (VLPs) of human papillomavirus (HPV)-16 (A) and HPV-52 (B) by enzyme-linked immunosorbent assay (ELISA) with VLPs as antigens. The number of serum samples is plotted against intervals of specific absorbance. The arrow indicates the cutoff value determined statistically, 0.35.

The distribution of HPV-16 and -52 IgG among the studied groups is illustrated in Table 3. None of the healthy donors were positive for HPV-16 and -52 IgG. Therefore, the prevalence rates for HPV-16 and -52 IgG in cancer bladder cases with or without cystitis (groups I and III) and the cystitis cases (group II) were significantly higher than the controls (seronegative) (P < .05). Moreover, prevalence rates for HPV-16 and -52 IgG in groups I and III were significantly higher than the group II cases (P < .05).

Table 3.

Seroreactivity to HPV-16 and 52

HPV DNA	NO = 60	HPV-16 IgG		HPV-52 IgG
		Positive (N = 16) N (%)	P Value	Positive (N = 7) N (%)	P Value
HPV-16 BTB
Present	18	12 (66.7)	< .00001	5 (27.8)	< .01
Absent	42	4 (9.5)		2 (4.8)
HPV-16 BCC
Present	13	13 (100)	< .0000001	6 (46.2)	< .0001
Absent	47	3 (6.4)		1 (2.1)
HPV-18 BTB
Present	6	3 (50)	NS	1 (16.7)	NS
Absent	54	13 (24.1)		6 (11.1)
HPV-18 BCC
Present	3	2 (66.7)	NS	1 (33.3)	NS

HPV = human papillomavirus; IgG = immunoglobulinG; BTB = bladder tissue biopsy; BCC = buffy coat cells; NS = nonsignificant

Significant differences (P < .05)

Seroreactivity to HPV-16 and -52

We analyzed the association between seropositivity to L1 capsids and the presence of viral DNA (Table 3). HPV-16 seropositivity was significantly higher in the HPV-16 DNA BTB-positive cases (66.7%) than the negative cases (9.5%) (P < .00001). Similar results were recorded for HPV-52 IgG (P < .01). In regard to HPV-16 DNA in BCC, there was absolute association with seropositivity to HPV-16 (P < .0000001) and strong association with seropositivity to HPV-52 (P < .0001).

Association of Single and Multiple HPV Types With Oncogenesis

Of the 60 studied patients, 24 (48.3%) were positive for HPV-16, -18, and -52 that were detected by any of the assessed methods. Multiple infections, 19 (79.2%), with different HPV types were statistically more significant than those of a single infection, 5 (20.8%) (P < .0001).

Sixteen of the 60 patients under study (26.7%) were seropositive for HPV-16 and/or -52. Multiple infections were recorded in 7 cases (43.75%) and a single infection in 9 cases (56.25%) with no significant difference. However, the association of multiple-type infections with oncogenic HPV (TCC) (6 of 7, 85.7%) was found to be statistically significant (P < .02) compared with a nonsignificant association with single-type infection (3 of 9). Of interest, all HPV-52-seropositive cases were absolutely associated with HPV-16 IgG positivity.

On the other hand, HPV DNA types 16 and/or 18 were detected by PCR in BTB and/or BCC in 23 of 60 studied patients (38.3%). Relying on DNA-dependent assays, infection with a single HPV type was significantly higher than with multiple types (P < .02). Multiple infections with both types were found in 7 of 23 (30.4%), and in the remaining 16 (69.6%) single infection was recorded. The association of either single or multiple infections with oncogenic HPV (TCC) (7 of 23, 30.4% and 16 of 23, 69.6%, respectively) was found to be statistically insignificant.

Discussion

HPV has been shown to play a role in the development of anogenital and bladder cancers.[26,27] We examined the possible etiologic role of HPV-16 and -18 in bladder carcinogenesis or recurrent cystitis with PCR on BTB and BCC. Also, we evaluated the association of type-specific IgG antibodies against HPV-16 and -52 L1 capsids with HPV DNA types and clinicopathologic parameters.

We relied on tumor tissue and BCC for case subjects as a source of HPV DNA. HPV-16 and -18 genomes were detected in BTB with higher significant rates of 44.4% and 13.9% in bladder cancer cases (groups I and III) than in cystitis cases (group II), 8.3% and 4.7%, respectively, with similar results in BCC. These findings were nearly similar to those obtained from Egypt[10,28] and Italy.[27] The high prevalence of HPV DNA in bladder tumor tissue and peripheral leukocytes in the present study and others emphasizes the role in bladder carcinogenesis. Carcinoma development may be triggered by HPV infection through inactivation of the tumor suppressor pRB by the HPV-16 oncoproteins E6 and E7 leading to genomic instability, which is thought to be an essential part of the conversion of a normal cell to a cancer cell.[26]

However, several studies have reported an incidence of HPV among patients with bladder cancer with widely differing results. Some studies have reported an incidence of 0% to 10%[27]; other studies have reported an incidence of 10% to 50%[29]; and some studies have even reported an incidence of ≥ 50%.[29] Several explanations of this variability have been proposed, including sampling problems, contamination, sensitivity of the detection systems, and geographic variation.[30] None of the healthy controls were positive for HPV-16 and -18 DNA in BCC, which was in accordance with Aglianò and colleagues.[27] In contrast, Barghi and colleagues[31] had an HPV positivity rate of one of the largest control groups (5%) of bladder tissue, with PCR on paraffin-embedded bladder tissue specimens.

Many investigations have reported a low prevalence of HPV in TCC.[32] For example, Sur and colleagues,[33] from South Africa, detected HPV DNA in only 1 of 64 screened paraffin wax-embedded TCCs. Youshya and colleagues,[34] from the United Kingdom, suggested that HPV is unlikely to play an etiologic role in the development of bladder TCC. On the other hand, 2 Japanese studies have reported higher incidences of HPV-18 DNA among bladder TCC (81% and 31%, respectively).[35,36] In regard to tumor type, our results revealed that the prevalence of HPV-16 in bladder TCC was significantly higher than that of SCC. Moreover, there was no significant difference between the prevalence of HPV-18 and the 2 types of carcinomas. Such dissimilarity suggested that the association of HPV with TCC may vary with geographic locations.[32]

It was reported that in 40% to 80% of patients, bladder tumors recur within 12 months after standard treatment by transurethral resection.[37] Recent epidemiologic studies suggested that the risk for urologic malignancies may be related to exposure to infectious agents, especially in those patients whose tumors show high recurrence rates.[38] In our study, there was a significant association between recurrent cases and the positivity rate of HPV-16. Moreover, the assessed BTB yielded a significant association between low grade (1, 2) and stage III (muscle invasion) tumors and the prevalence rate of HPV-16. Youshya and colleagues[34] suggested that HPV may play a significant role in the progression of TCCs toward higher stages and/or grades by inactivation of the tumor suppressors or other unknown mechanisms.

The high frequency (78.6%) of HPV positivity in schistosomiasis-associated bladder carcinomas among our patients and 13 of 14 (92.9%) of patients with schistosomiasis was positive for HPV infection. Such results agreed with Khaled and colleagues,[10] who reported HPV infection in 53% among Egyptian schistosomal cases with bladder cancer. HPV-associated bladder cancer has been reported in immunosuppressed patients. This may explain the significant rate of HPV bladder infection in our immunodeficient patients with schistosomiasis, as reported by several investigators.[39,40] Four (8.7%) of the 46 patients without schistosomiasis were positive for HPV infection. Evidence with schistosomiasis in the study groups was correlated to HPV positivity. Schistosoma could be a confounding variable.

An antibody response to HPV-16 was observed in 66.7% patients positive for the corresponding HPV DNA type, which is consistent with previous studies of HPV capsid serology.[26] De Gaetani and colleagues[11] detected IgG antibodies to HPV-16 VLPs in 70.5% of patients with corresponding HPV DNA. Le Cann and colleagues[41] suggested that anti-HPV antibodies detected by enzyme immunoassay with L1 capsids must be considered a marker of virus replication. On the other hand, 33.3% of the assessed HPV-16 DNA-positive patients were seronegative for HPV-16 IgG. This finding also agrees with De Gaetani and colleagues,[11] who reported that 25% of HPV-16 DNA-positive patients were serum antibody-negative. The long lag time from HPV infection to antibody seroconversion with the transient nature of the disease could explain the possibility of undetectable levels of antibodies.[42]

Of the HPV-16 DNA-negative patients, 9.5% were positive for the corresponding antibody. However, other researchers[11] reported that 57.7% of the HPV-16 DNA-negative patients were serum antibody-positive. Such a discrepancy in results may be attributed to the presence of the infection at a site that was not sampled. Therefore, they emphasized the importance of using multiple biopsies from the same case to make a positive identification of the presence of HPV in cases that otherwise might be considered negative. This suggests that the virus does not infect neoplastic tissue uniformly, but that it has a focal distribution; therefore, HPV DNA is found only when the biopsy is taken from the right place in the tumor. Another possible explanation is HPV-16 nonbladder cancer or infection involvement.[43]

Several serologic studies with mainly HPV-16 VLPs have demonstrated that infection with HPV is followed by a serologic immune response to viral capsid proteins. However, the titer of detectable serum antibodies to HPV VLPs was low. This immune response is largely HPV type-specific and directed against conformational epitopes. Follow-up studies have demonstrated that seroconversion most frequently occurs between 6 and 18 months after DNA detection. Anti-VLP antibodies are rarely observed in patients with transient HPV DNA, but are associated with the persistence of HPV DNA detection.[44]

It is evident from the present study that there was a significant association between the presence of anti-HPV-16 VLPs in serum with the tendency of tumor or infection to recur. Cohort studies indicated that HPV infection with oncogenic types is mostly transient and that only a small proportion of those infected become carriers and then develop intraepithelial neoplasia.[44]

Infection with multiple HPV types – 16, 18, and 52 – was significantly higher than single-type HPV among the assessed cases. These results were comparable to those of other researchers who reported that such mixed infections raise the possibility of synergism between viruses in bladder carcinogenesis. Such synergism already has been suggested in other human tumors, as some epithelial malignancies of the anogenital tract.[45] Therefore, further investigations are required in order to establish whether the finding of such mixed infections in bladder tumors just represents an occasional event or alternatively may play some role in the development and progression of some urologic malignancies in humans.[11]

The observed absolute association between seropositivity of HPV-52 and -16 was consistent with Matsumoto and colleagues,[22] who found that seropositivity to multiple HPV-16, -18, and -52 L1 capsids could indicate past or current exposures to different strains rather than cross-reactivity, because of the largely HPV type-specific assay. Moreover, seropositivity to the assessed multiple HPV types was significantly associated with TCC of the bladder. Ho and colleagues[46] suggested that specific association between seropositivity in proportion to the number of oncogenic HPV types increases the risk for cervical intraepithelial neoplasia development. Immunologic cofactors may also be considered because seropositivity to multiple L1 capsids is most frequently observed in immunosuppressed individuals.[47,48]

In the present study, HPV-16 seropositivity was still associated with TCC of bladder tissue even after acquiring seropositivity to HPV-52. Höpfl and colleagues[49] declared that the combined immunologic responses elicited by different HPV types may not influence cervical intraepithelial neoplasia development, but rather lead to its persistence and progression to invasive cervical carcinoma. Although the HPV type specificity of the cellular immune response is still unknown, cellular immunity is suggested to play an important role in cervical intraepithelial neoplasia regression.

In the present work, single HPV DNA-type infection in BTB was significantly higher than coinfection with multiple HPV types. However, Matsumoto and colleagues[22] could not detect multiple HPV types among either cases or control groups. In fact, a prospective, viral DNA study in college women reported that concurrent or sequential detection of different HPV types confers an increased risk for intraepithelial neoplasia development.[46]

None of the healthy donors were positive for anti-HPV-16 or -52 L1 capsids in serum. In one study, IgG against HPV-16 capsids was demonstrated among 55% of anal cancer patients but only among 4% of the control subjects, suggesting that HPV-16 capsid antibodies are serologic markers for anal cancer and that the exposure to HPV-16 appears to be a major risk factor in the majority of anal cancers.[48] The reason why patients with dysplasia or cancer may be infected with HPV-16 more frequently than healthy subjects is not clear.[26] Matsumoto and colleagues[22] observed high seropositivity to HPV-16, -18, and -52 in controls, which showed that infections with oncogenic types were relatively common in Japan.

Prior infection with a given HPV strain that elicits antibody response may not prevent infections by other strains. Thus, a prophylactic vaccine to target a broad spectrum of oncogenic HPV types would be necessary.[22] The preliminary data suggest that bivalent HPV-16 and HPV-18 vaccine may also protect against HPV-16-related types (-31, -33, -35, -52, and -58) and HPV-18-related types (-39, -45, -59, -68, and -85).[50] A human papilloma vaccine is available; it prevents infection with high-risk types of HPV and has the potential to greatly reduce the occurrence of cervical cancer as well as other HPV-related cancers. Currently, commercially available HPV vaccines either target HPV-6, -11 for genital warts and HPV-16, -18 for cancer. The vaccine is approved for use in girls and women between the ages of 9 and 26 years.[51]

In conclusion, our study confirms the significant association of HPV-16, -18, and -52 with bladder cancer in Egyptian patients. Therefore, the interesting hypothesis of a viral synergistic action in bladder carcinogenesis is strongly suggested. In addition, such HPV types were significantly associated with TCC tumors of grade 2 and stage III, with schistosomal affection and recurrence tendency. HPV serology can pave the way for better management and follow-up of patients and for optimal design and evaluation of HPV vaccination. The availability of such reliable serologic markers would enable conclusive epidemiologic studies for such infectious causes of human cancer to explain regional variations in cancer incidence, especially in developing countries where the logistics for systematic screening by cytology are difficult to establish with immediate implications for the fight against cancer.