Cervical carcinoma in Southern Mexico: Human

papillomavirus and cofactors§

Berenice Illades-Aguiar PhDa,*, Enoc-Mariano Cortes-Malagon MSa,Veronica Antonio-Vejar MSa, Noelio Zamudio-Lopez MSa,

Luz del Carmen Alarcon-Romero PhDa, Gloria Fernandez-Tilapa PhDa,Daniel Hernandez-Sotelo MSa, Marco-Antonio Teran-Porcayo MDb,

Eugenia Flores-Alfaro MSa, Marco-Antonio Leyva-Vazquez PhDa

a Laboratorio de Biomedicina Molecular, Unidad Academica de Ciencias Quımico Biologicas,

Universidad Autonoma de Guerrero, Chilpancingo, Guerrero, Mexicob Instituto Estatal de Cancerologıa ‘‘Dr. Arturo Beltran Ortega’’, Acapulco, Guerrero, Mexico

Accepted 10 September 2008

Abstract

Background: This study was conducted to determine human papillomavirus (HPV) types in women with cervical cancer (CC) and normal

cervical cytology in the Southern region of Mexico, and to know the contribution of HPV types and cofactors in cervical cancer etiology.

Methods: A case–control study was performed in 133 women with CC and 256 controls. HPV detection was done by MY09/11 and GP5+/

GP6+ PCR systems and typing by restriction fragment length polymorphism or DNA sequencing. Results: HPV was found in 100% of CC and

35.5% of controls. The genotype distribution in CC was: HPV 16 (66.8%), 18 (9%), 31 (7.5%), 45 (4.5%), 58 (3.7%), 69 (3%), 52 (1.6%), 6,

11, 33, 56, and 67 (0.8% each). Among controls, HPV 33 followed by HPV 16 were the most frequent. Cervical cancer was associated with

HPV 16 (OR = 573.5), HPV 18 (OR = 804.4), and undetermined risk HPV (types 67 and 69) (OR = 434.3). Age at first intercourse<16 years

(OR = 9.6) and �3 births (OR = 16) were significant risk factors for CC. Conclusions: HPV 16, by far, is the most frequent type in CC, HPV

16 and 18 are responsible for 75.8% of the CC cases and high-risk HPV for 94.7%, which is useful data to take into account in vaccination

programs. HPV 33 is the most frequent type in controls and high-risk HPV are more common than low-risk HPV.

# 2008 Elsevier Ltd. All rights reserved.

Keywords: Human papillomavirus epidemiology in Mexico; Human papillomavirus genotypes in Mexico; Human papillomavirus and cervical cancer;

Cervical cancer in Mexico; Cervical cancer risk; Cervical cancer in Latin America; Cervical cancer co-factors; Human papillomavirus in normal cervix; HPV

typing; HPV PCR detection

www.elsevier.com/locate/cdp

Cancer Detection and Prevention 32 (2009) 300–307

1. Introduction

Cancer of the cervix uteri is the second most important

cancer among women worldwide with an estimated

§ Sources of support: This work was supported by Grants S-20 and

97SIBEJ-02-016 from the Sistema de Investigacion Benito Juarez-CON-

ACYT, Mexico.

* Corresponding author. Tel.: +52 747 4710901; fax: +52 747 4710901.

E-mail addresses: ibereni@yahoo.com.mx, ibereni@hotmail.com

(B. Illades-Aguiar).

0361-090X/$ – see front matter # 2008 Elsevier Ltd. All rights reserved.

doi:10.1016/j.cdp.2008.09.001

493,000 new cases and 274,000 deaths in 2002 [1]. More

than eighty percent of new cases are currently diagnosed in

developing countries [1], where there is a high incidence of

the disease because there are severe limitations in

screening programs as well as treatment facilities [2]. In

Latin America, a high incidence and mortality were

registered for cervical cancer (CC); by the year 2000, some

76,000 new cervical cancer cases and almost 30,000 deaths

were estimated for the whole region, which represent 16%

and 13% of the world burden, respectively. From Latin

B. Illades-Aguiar et al. / Cancer Detection and Prevention 32 (2009) 300–307 301

American countries, Mexico is seventh place in relative

incidence (age standardized rates – ASR – 40.5 per 100,000

women) and fifth in mortality (ASR 17.1 per 100,000

women [3].

In Mexico, cervical cancer is the most common type of

cancer among women [4]. Deaths due to cervical cancer

did not decrease during the 1990–2000 period; on the

contrary, there was a 0.76% yearly average increase.

Cervical cancer mortality shows heterogeneity in different

regions of the country. The Central region of Mexico,

where Mexico City is located, has the lowest mortality rate

and the lowest risk of death from cervical cancer, while the

Southern region has the highest mortality rate and the

highest mortality risk. The State of Guerrero, which is

located in the Southern region, is one of the states with the

highest rates of non-coverage by social security health

care services (47.7%), and one of the states with the

highest cervical cancer rates with the highest mortality

risk for this disease [5]. The State of Guerrero cervical

cancer mortality rate was 11 deaths per 100,000 women in

2005 [6].

Certain types of human papillomavirus (HPV) are the

central and necessary cause of cervical cancer worldwide

[7]. So far, nearly 100 HPV have been described [8] and

more than 50 different HPV types primarily infect the anal

and genital tracts. The IARC Monograph Working Group [9]

in 2005 concluded that human papillomavirus types 16, 18,

31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 66 are carcinogenic

for human beings and have been classified as high-risk or

oncogenic types. Munoz et al. consider that it is arbitrary to

classify HPV 66 as carcinogenic and they therefore propose

that HPV 66 as well as HPV 26, 53, 68, 73 and 82 should be

considered as probably carcinogenic [10]. HPV 6, 11, 13, 40,

42, 43, 44, 54, 61, 70, 72, 81, 89 are classified as low-risk

types. For HPV 2a, 3, 7, 10, 27, 28, 29, 30, 32, 34, 55, 57, 62,

67, 69, 71, 74, 77, 83, 84, 85, 86, 87, 90, 91 the risk of

cervical cancer is undetermined [10]. HPV 55 was

reclassified as a subtype of HPV 44 and is considered as

low-risk HPV type [11].

HPV 16 is the most prevalent type found in women with

cervical cancer worldwide and together with HPV 18, 31,

and 45 account for about 80% of invasive cervical cancer

collected from around the world [12].

Geographical variation in HPV type distribution is known

to occur in different regions of the world. Limited

information is available on the prevalence and distribution

of HPV types in cervical cancer in Southern Mexico. In

addition to this, there have been relatively few case–control

studies about invasive cervical carcinoma performed in

Mexico [13–17].

This study in the State of Guerrero, Mexico, was

undertaken to establish which HPV types are circulating in

women with cervical cancer and normal cervical cytology

from Southern Mexico, and to describe the contribution of

different HPV types and cofactors in cervical cancer

etiology.

2. Material and methods

2.1. Study population

The study was carried out in the Cancer Institute of the

State of Guerrero, a regional concentration Hospital located

in Acapulco, Guerrero, Mexico. The cases were 133

Mexican women with residence in Guerrero State, with

invasive cervical cancer recruited between 1997 and 2003.

Inclusion criteria for case subjects were that they had

histological confirmation of invasive cervical cancer

diagnosis, had not received any treatment, and that DNA

extracted from the biopsy samples was good quality. Of the

cases group, no exfoliated cells were obtained. Squamous

cell carcinoma (SCC) was diagnosed in 119 cases (89.5%)

and adeno/adenosquamous carcinoma (ADC/ADSC) in 14

(10.5%), by histological diagnosis, according to the

classification system of the International Federation of

Gynecology and Obstetrics (FIGO) [18]. The controls were

256 gynecological patients who attended the hospital

mentioned above for cytological screening with cytologi-

cally normal cervical epithelium according to the Bethesda

System [19]. Control women were selected to match the

expected age distribution of the case subjects and had to

fulfill the following inclusion criteria: negative diagnosis of

any kind of cancer, and to not have gone through cervical

conization or hysterectomy. The controls provide a cervical

scrape containing exfoliated cells from the cervix, from

which good quality DNA was confirmed to include them in

the study. Participants answered a standardized question-

naire on sexual behavior, reproductive history, smoking

habits, and socioeconomic background.

Informed consent was obtained from both cases and

control women. This study was approved by the ethical

committee of the Cancer Institute of the State of Guerrero.

2.2. Specimen collection

For cytological analysis and HPV detection, cervical

scrapes were collected by sampling the ectocervix with an

Ayre spatula and endocervix with a cytobrush, making sure

that tissue from the transformation zone was taken. Smears

were used for cytomorphological examination using

conventional Papanicolaou. For HPV detection, cytobrushes

with cervical scrapes were placed in lysis buffer (10 mM

Tris pH 8.0, 20 mM EDTA pH 8.0 and 0.5% sodium dodecyl

sulfate), and were removed after taking out cervical material

and stored at �20 8C until analysis. A tumor biopsy

specimen was also taken and was eluted in phosphate-

buffered saline (PBS), and stored at �70 8C until analysis.

2.3. HPV detection and typing

DNA was extracted according to the standard SDS–

proteinase K–phenol–chloroform method [20]. DNA

samples were tested by the MY09/11 PCR protocol

B. Illades-Aguiar et al. / Cancer Detection and Prevention 32 (2009) 300–307302

[21,22]. The consensus primers MY09 and MY11 target a

conserved 450-pb region of the HPV L1 gene. The reaction

mixtures (50 ml) contained 1 mg of target DNA, 0.8 mM of

each primer, 2 mM MgCl2, PCR buffer 1X, 150 mM of each

dNTP and 1.25 U of Ampli Taq GoldTM (Applied

Biosystems, Foster City, CA). DNA was amplified in a

GeneAmp PCR System 2400 (Applied Biosystems, Foster

City, CA) with the following steps: an initial step 10 min

denaturation at 95 8C, followed by 40 cycles of 95 8C for

1 min, 58 8C for 1 min, 72 8C for 1 min, and a final

extension at 72 8C for 10 min.

Integrity of DNA specimens was verified by amplifica-

tion of a 268-bp region of the human b-globin gene using

PC04 and GH20 primers [23]. HPV 16 plasmid, CaSki and

HeLa cells were used as positive controls, human DNA

without HPV DNA and water were used as negative

controls.

For HPV typing, amplified PCR products were digested

with restriction enzymes BamHI, DdeI, HaeIII, HinfI, PstI,

RsaI and Sau3AI (Invitrogen, Carlsbad, CA) and restriction

fragment length polymorphism (RFLP) analysis were

performed to identify more than 40 genital HPV types

[24]. Typing of HPVof randomly selected samples that were

positive for HPV 33 and HPV 16 was carried out by type-

specific PCR with primers designed for the E6 region of

these viral types [25,26] (see: Supplementary Data).

When samples analyzed with MY09/11 PCR protocol

were negative or the HPV could not be genotyped by

RFLP’s, the presence of HPV DNA was assessed using the

general GP5+/6+ PCR system [27]. PCR GP5+/GP6+

products were subjected to sequencing analysis. Briefly,

after the purification of PCR products using 75%

isopropanol and columns (Centri-Sep Spin Columns,

Applied Biosystems, Foster City, CA), these were sequenced

using Big Dye Terminator Chemistry Version 3 Cycle

Sequencing Kit (Applied Biosystems, Foster City, CA) in an

automated sequencer (310 ABI PRISM Genetic Analyzer,

Applied Biosystems, Foster City, CA). All the sequences

available for the HPV types were recovered from the NCBI

site [28]. HPV types were classified as high-risk versus low-

risk accordingly to Munoz et al. classification [10].

2.4. Statistical analysis

To estimate the relative risk of cervical cancer associated

with HPV types and other risk factors, odds ratios (ORs) and

95% confidence intervals (CIs) were calculated by using

logistic regression in non-adjusted models and adjusted

models for different risk factors. Statistical analysis was

performed using STATA software version 9.2.

3. Results

A total of 133 case patients with cervical cancer and 256

control subjects were included in this study. Of the cases,

119 (89.5%) were SCC and 14 (10.5%) were ADC/ADSC.

The average age of the cases was 52.2 years and of the

controls 50.5 ( p = 0.2). Fifty-three percent of the cervical

cancer cases occurred in women 50 years old or older, 41%

in women 35–49 years old, and 6% in women 34 years old or

younger. Table 1 summarizes the main sexual history and

reproductive factors characteristics of study subjects by case

and control status and the association of these characteristics

with cervical carcinoma after various adjustments. In the

fully adjusted model, start of sexual activity before age 16

(OR = 9.6, 95% CI, 3.5–26.5) and having 3 or more births

(OR = 16, 95% CI, 3–84) showed statistically significant

associations with cervical cancer. In the non-adjusted model

for age, menopause in women younger than 45 years showed

an apparent association with the risk of developing cervical

cancer, however, after adjusting for HPV infection, number

of sexual partners, age at menarche and smoking habits, the

association was found to be not significant (OR = 1.2, 95%

CI, 0.3–4.6). Other characteristics that were examined and

found not to be associated with cervical cancer included

number of sexual partners, age at menarche and smoking. Of

the women that participated in this study, 64.8% did not

provide the number of sexual partners they have had and

58.2% did not say if they smoked or not.

Among the 256 control women, specimens from 91

(35.5%) and all cases of SSC and ADC/ADSC were positive

for HPV DNA. Analysis of the HPV type-specific

distribution among cases and control women showed that

the most common HPV type in women with SCC was HPV

type 16 (65.5%) followed by HPV types 18 (9.3%), 31

(7.6%), 45 (5.1%), and 58 (4.2%). Two LR-HPV types (HPV

6 and 11) and two undetermined risk HPV types (HPV 67

and 69) were also detected. In women with ADC/ADSC,

HPV type 16 (78.7%) was the most frequent type followed

by HPV types 18 (7.1%), 31 (7.1%) and 69 (7.1%). In

control subjects, 76 (29.6%) were infected with high-risk

types and only 11 (4.3%) where infected with low-risk types.

It is important to note that in the control group HPV type 33

was the most recurrent (13.7%) followed by HPV types 16

(11.3%), 58 (1.6%) and 61 (1.6%). When we analyzed by

type-specific PCR, 5 samples with HPV 33 and 14 with HPV

16, they corresponded to the results obtained by MY-RFLPs.

Multiple infection was not found in either control or cases

(Table 2) (see: Supplementary Data).

HPV 16 phylogenetically related types found in this study

(a-9: HPV 31, 33, 52, 58, 67) were present in 14.4% of

cervical cancer cases and HPV 18 phylogenetically related

types (a-7: HPV 45) were present in 4.5% of cervical cancer.

The remainder 5.4% cases had a-5 (HPV 69), a-6 (HPV 56),

and a-10 (HPV 6 and 11) (Table 2).

The presence of HPV was associated with SCC (OR,

216.5; 95% CI, 56–+1) and with ADC/ADSC (OR, 27.1;

95% CI, 6.5–+1) (data not shown). Table 3 summarizes the

association of cervical cancer with HPV genotype infection

using five models and various adjustments. HPV infection

with any HPV type was associated with a 279-fold increase

B. Illades-Aguiar et al. / Cancer Detection and Prevention 32 (2009) 300–307 303

Table 1

Cervical cancer: associations with sexual history and reproductive factors.

Variable Cases (n = 133) Controls (n = 256) ORa (95% CI) ORb (95% CI) ORc (95% CI)

n (%) n (%)

Age at first sexual intercourse (years)

>20 17 (12.8) 95 (37.1) 1.0 1.0 1.0

16–20 59 (44.4) 121 (47.3) 2.7 (1.5–5.0) 2.3 (1.2–4.4) 2.6 (1.2–6.0)

<16 52 (39.1) 34 (13.3) 8.5 (4.4–16.8) 8.8 (4.1–18.8) 9.6 (3.5–26.5)

Did not answer 5 (3.75) 6 (2.3)

Parity

None 2 (1.5) 33 (12.9) 1.0 1.0 1.0

1–2 15 (11.3) 51 (19.9) 4.9 (1.0–22.6) 3.8 (0.8–19.0) 2.4 (0.4–14)

3 and more 114 (85.7) 172 (67.2) 10.9 (2.6–46.5) 12.5 (2.8–56.7) 16 (3.0–84)

Did not answer 2 (1.5) 0

Sexual partners

1 69 (51.9) 48 (18.8) 1.0 1.0 1.0

2–3 30 (22.5) 38 (14.8) 0.5 (0.3–1.0) 0.6 (0.3–1.0) 0.3 (0.1–1.0)

4 and more 5 (3.8) 4 (1.6) 0.9 (0.2–3.4) 0.9 (0.2–3.6) 1.0 (0.1–8.4)

Did not answer 29 (21.8) 166 (64.8)

Age at menarche (years)

14 and more 64 (48.1) 128 (50.0) 1.0 1.0 1.0

10–13 60 (45.1) 120 (46.9) 1.0 (0.6–1.5) 1.2 (0.7–1.9) 1.1 (0.6–2.0)

Did not answer 9 (6.8) 8 (3.1)

Age at menopause (years)

50 or more 16 (12.0) 51 (19.9) 1.0 1.0 1.0

45–49 25 (18.8) 53 (20.7) 1.5 (0.7–3.1) 1.5 (0.6–3.6) 2.0 (0.6–6.5)

<45 20 (15.0) 26 (10.2) 2.5 (1.1–5.5) 1.3 (0.5–3.5) 1.2 (0.3–4.6)

No menopause 36 (27.1) 111 (43.4) 1.0 (0.5–2.0) 0.5 (0.2–1.1) 0.6 (0.2–1.7)

Did not remember 36 (27.1) 15 (5.9)

Smoking

No 81 (60.9) 94 (36.7) 1.0 1.0

Yes 7 (5.3) 13 (5.1) 0.6 (0.2–1.6) 0.6 (0.2–1.6) 0.7 (0.2–2.9)

Did not answer 45 (33.8) 149(58.2)

OR = odds ratio; CI = confidence interval.a Odd ratio non-adjusted.b OR adjusted by sexual partners, age at menarche, smoking (except for the evaluated factor).c OR adjusted by factors of model c and HPV type by oncongenic risk (HPV negative, low-risk HPV, high-risk HPV and undetermined risk HPV).

in the risk of cervical cancer. Surprisingly, undetermined

risk HPV were associated with cervical cancer (OR = 434.3,

95% CI, 22.1–+1).

Analysis of the risk of developing cervical cancer with

HPV 16, HPV 18 and related types showed that the highest

risks were associated with HPV 18 (OR= 804.4, 95% CI,

71.1–+1) and HPV 16 (OR = 573.5, 95% CI, 74.4–+1).

The relative risk in women infected with HPV 18 related

types (OR = 312.2, 95% CI, 27.5–+1) was higher than that

among those infected with HPV 16 related types (OR = 86.1,

95% CI, 10.9–+1).

4. Discussion

This study provides information on the risk factors for

cervical cancer and the infection of HPV in the State of

Guerrero, Mexico. In this study, the presence of HPV DNA

was confirmed in 100% of cervical cancer cases (SCC and

ADC/ADSC) and in 35.5% of control women.

4.1. HPV genotype distribution

HR-HPVs were present in 94.7% of the cervical cancer

cases, similar to that found in other worldwide studies [7]

and in Mexico as well [17]. HPV 16 is by far the most

common HPV type identified in cervical carcinoma. The

HPV types identified in SCC, in order of decreasing

prevalence, were HPV 16, 18, 31, 45, 58, 69, 52 and 6, 11,

33, 56, 67; in ADC/ADSC they were HPV 16 and 18, 31, 69.

There was variation in HPV-specific prevalence between

different histological cancer types, HPV 16 was found more

often in ADC/ADSC (78.7%) than in SCC (65.5%); similar

to that found in North Africa and South America where HPV

16 was found in 72% of the ADC cases [29]; however,

worldwide HPV 16 was identified in 55.2% of SCC and in

31.3% of ADC [30], and in a study from Mexico City it was

found in 45.1% of the cervical cancer cases [17]. We found

that in Southern Mexico, HPV 18 was more frequent in SCC

(9.3%) than in ADC/ADSC (7.1%), which disagrees with

other studies in which it is found that throughout the world it

is present in 12.3% of SCC [30] and 37.7–39% in ADC/

B. Illades-Aguiar et al. / Cancer Detection and Prevention 32 (2009) 300–307304

Table 2

Distribution of HPV types in cases of cervical cancer and control women with normal cervical cytology.

HPV typesa Cervical cancer Controls

Squamous cell

carcinoma

Adeno/

adenosquamous

carcinoma

Total cases

n (%) n (%) n (%) n (%)

Total 119 (100) 14 (100) 133 (100) 256 (100)

Negative 0 (0) 0 (0) 0 (0) 165 (64.5

Positive 119 (100) 14 (100) 133 (100) 91 (35.5

HR-HPV 113 (95.0) 13 (92.9) 126 (94.7) 76 (29.6)

16 78 (65.5) 11 (78.7) 89 (66.8) 29 (11.3

18 11 (9.3) 1 (7.1) 12 (9.0) 3 (1.1)

31 9 (7.6) 1 (7.1) 10 (7.5) 3 (1.1)

33 1 (0.8) 0 (0) 1 (0.8) 35 (13.7

45 6 (5.1) 0 (0) 6 (4.5) 1 (0.4)

52 2 (1.7) 0 (0) 2 (1.6) 0 (0)

56 1 (0.8) 0 (0) 1 (0.8) 0 (0)

58 5 (4.2) 0 (0) 5 (3.7) 4 (1.6)

59 0 (0) 0 (0) 0 (0) 1 (0.4)

Probable HR-PV 0 (0) 0 (0) 0 (0) 3 (1.2)

53 0 (0) 0 (0) 0 (0) 3 (1.2)

LR-HPV 2 (1.6) 0 (0) 2 (1.6) 11 (4.3)

6 1 (0.8) 0 (0) 1 (0.8) 3 (1.1)

11 1 (0.8) 0 (0) 1 (0.8) 1 (0.4)

61 0 (0) 0 (0) 0 (0) 4 (1.6)

70 0 (0) 0 (0) 0 (0) 2 (0.8)

81 0 (0) 0 (0) 0 (0) 1 (0.4)

Undetermined risk-HPV 4 (3.4) 1 (7.1) 5 (3.8) 1 (0.4)

67 1 (0.8) 0 (0) 1 (0.8) 0 (0)

69 3 (2.6) 1 (7.1) 4 (3.0) 0 (0)

71 0 (0) 0 (0) 0 (0) 1 (0.4)

HPV, human papillomavirus; HR-HPV, high-risk human papillomavirus; LR-HPV, low-risk human papillomavirus.a Munoz et al. HPV classification [10].

Table 3

HPV infection and its association with cervical cancer.

HPV infection Cases (n = 133) Controls (n = 256) ORd (95% CI) ORe (95% CI)

HPV(1)

Negative 0 165 1.0* 1.0*

Positive (any type) 133 91 241.2 (33.2–+1) 279 (37.6–+1)

HPV types

LR-HPV(2) 2 11 30 (2.5–+1) 34.9 (2.8–+1)

HR-HPV(2) 126 76 273.6 (37.5–+1) 321 (43.1–+1)

Undetermined risk HPVa(2) 5 1 825 (44.9–+1) 434.3 (22.1–+1HPV 16(3) 89 29 506.4 (67.8–+1) 573.5(74.4–+1)

HPV 16 related typesb(3) 19 42 74.6 (9.7–+1) 86.1 (10.9–+1)

HPV 16 and related types(4) 108 71 251 (34.4–+1) 293.7 (39.3–+1HPV 18(3) 12 3 660 (63.7–+1) 804.4 (71.1–+1HPV 18 related typesc(3) 6 4 247.5 (23.9–+1) 312.2 (27.5–+1HPV 18 and related types(4) 18 7 424.3 (49.4–+1) 522.3 (57.5–+1HPV 16 or HPV 18(5) 101 32 520.8 (70.1–+1) 597.2 (78–+1)

HPV, human papillomavirus; OR, odds ratio; CI, confidence interval. (1)–(5)Models; *reference category for all models.a Undetermined risk HPV: 67, 69, 71 [10,24].b HPV 16 related types: 31, 33, 52, 58, 67 [10,24].c HPV 18 related types: 39, 45, 59, 70, 85 [10,24].d Odd ratio non-adjusted.e Odds ratio adjusted for age at first sexual intercourse and parity.

)

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B. Illades-Aguiar et al. / Cancer Detection and Prevention 32 (2009) 300–307 305

ADSC [29,30]; however, in North Africa the frequency of

HPV 18 in ADC/ADSC is 13.6% and in South America

19.6% [29]. The fraction of SCC attributable to HPV 16 and

18 was 74.8% while that for ADC/ADSC was 85.8%, almost

the same as reported worldwide [10]. In this study LR-HPV

were absent in ADC and were only found in SCC and control

women.

4.2. HPV and cervical cancer

The presence of HPV was associated with SCC and with

ADC/ADSC, as reported in many studies worldwide

[10,29,30]. Three-fourths of invasive cervical cancers in

this study were associated with HPV 16 and HPV 18.

However, ten more types were also associated with invasive

cervical cancer. The most frequent HPV species associated

with cervical cancer was a-9 HPV followed by a-7 HPV.

Odd ratios linked to HPV types demonstrated very strong

associations (OR > 100) for total HR-HPV, HPV 16, HPV

18, and HPV 18 related types, and strong associations

(OR > 18) for HPV 16 related types like in other worldwide

studies [10]. It is interesting to address that undetermined

risk HPV (HPV 67 and HPV 69) [10] were very strongly

associated with cervical carcinoma, so these findings could

contribute to determining the association of these HPV types

in the development of cervical carcinoma.

It is important to take into account that HPV status in the

control group is variable because transient infections could

not be differentiated from persistent infections, which could

influence in the estimation of the risk in the cases in which

infection is persistent. Epidemiologically, a basal risk is

present in both cases and controls, assuming that an effect of

the transitory infections could be present in the calculation

of the ORs. However, it would be difficult to evaluate in this

type of studies. Because this is not a follow up study, the

change in infection status throughout time cannot be

evaluated. In cases and control studies, a single measure

is done that allows us to know the current HPV infection

status, without letting us know if the infection is transitory of

persistent.

The geographic distribution and prevalence of HPV types

in cervical cancer varies worldwide. In this study we found

12 types of HPV including types 67 and 69, different from

that reported by Munoz et al. [31], who found 26 types in a

multicentric worldwide study in which types 67 and 69 are

not reported. The prevalence we found for HPV types 16, 45,

52 and 6 is similar to that found in Northern Africa and the

prevalence for types 18, 31, 58, 56 and 11 is similar to that in

Central/South America. Knowledge of geographic distribu-

tion of HPV types in cervical cancer has implications for

cervical cancer prevention and screening regarding the

composition of prophylactic vaccines and screening cock-

tails. At present, two HPV vaccines are available, a bivalent

vaccine (Cervarix) against HPV 16 and 18, and a tetravalent

vaccine against HPV 6, 11, 16, and 18 (Gardasil). In the

south of Mexico, the bivalent vaccine in theory could

prevent 75.8% of cervical cancer cases; the tetravalent

vaccine could prevent 77.4% of cases, leaving 22.6%

unprotected. On the other hand, an effective vaccine against

the 5 most common HPV types (16, 18, 31, 45 and 58) could

prevent 91.5% of the cases of cervical cancer.

The prevalence of infection by HPV in population-based

studies carried out in different regions of the world is

variable; in Sub-Saharian Africa it is 25.6%, in Asia 8.7%, in

South America 14.3%, and in Europe 5.2% [32]. The use of

hospital controls, like in our study, has advantages such as

high participation and disadvantages like potential for

selection bias. This partially explains the high frequency of

HPV in the controls (35.5%). The most common HPV type

in controls was HPV 33, followed by HPV 16, 58, 61, 6, 18,

31, 53, 70, 11, 45, 59, 71, and 81. It is important to mention

that HR-HPVs are present in 29.6% of the controls while

LR-HPVs are only present in 4.3%.

In comparison to the south of Mexico, worldwide

distribution of HPV types in cytologically normal women

reported by the International Agency for Research on

Cancer HPV in 2005 [32] showed that HPV 16 was the most

common HPV type, followed by HPV 42, 58, 31, 18, 56, 81,

35, 33 and 45. Our population is different from others

regarding predominance of HPV 33 in women with normal

cytology, which is higher than that of HPV 16. Type-specific

PCR for HPV 33 carried out in 11% of the samples of normal

cervical cytology with HPV 33 and in the only cervical

cancer sample with the this viral type, that were previously

typed by MY-RFLPs, confirmed that our findings were real.

In Mexican women with normal cytology, Torroella-Kouri

et al. [13] did not find HPV 33 in Mexico City and Lazcano-

Ponce et al. [4] found it in 1% in the State of Morelos. The

heterogeneity of the HPV genotypes distribution in Mexico

is evident in the study by Gonzalez-Losa et al. [33] in which

they found that in women from the State of Yucatan, HPV 58

was the most frequent type found in LSIL and HSIL, and in

cervical cancer cases its frequency was the same as HPV 16

(see: Supplementary Data).

In this study multiple infections were not found in cases

or controls. This could be due to the methods employed for

typing (RFLPs or sequencing) which are not suitable for the

detection of infections with multiple HPV types; these are

less sensitive than hybridization methods and, in case of

being positive, these will usually give an uninterpretable

mix-up of digestion/sequence patterns [34]. On the other

hand, in studies carried out in Mexican women, the presence

of multiple infections in women with normal cervical

cytology is low (0–2.4%) [4,13,35], although in women with

cervical cancer the prevalence is greater [13,33].

A total of 18 HPV types were detected in this study, 12

HPV types in cervical cancer and 14 HPV types in normal

cervical cytology. From those, 9 were HR-HPV, 1 probable

HR-HPV, 5 LR-HPV, and 3 undetermined risk-HPV. The

presence of these HPV types was detected using MY09/11

and GP5+/6+ PCR systems. Combination of two PCR

detection systems increased HPV DNA detection in cervical

B. Illades-Aguiar et al. / Cancer Detection and Prevention 32 (2009) 300–307306

cancer from 97.8% using MY09/11 PCR system to 100% by

analyzing MY-PCR negative samples with GP5+/6+ PCR

system. We found a higher prevalence than the worldwide

prevalence found in other studies (83–89% [30], >95% [31]

and 99.7% [7]), which reflects significant differences in

HPV DNA detection.

Our findings indicate that the commercially available

Hybrid Capture II (Digene) assay that detects 13 HR-HPVand

5 LR-HPV [36], would not detect genotypes 67 and 69 that

were found in 3.8% of cervical cancer cases in this study. It

would also not detect genotypes 53, 61, 70, 71 and 81 which

were found in 4.4% of the controls. It would be important that

the composition of screening ‘‘cocktails’’ for HR-HPV and

LR-HPV types be revised as proposed by other studies [37].

4.3. Cofactors for cervical cancer

Because infection by HR-HPV is a necessary, but not a

sufficient, cause for cervical cancer [7], it has been assumed

that other factors contribute to modulate the risk of transition

from cervical HPV infection to cervical cancer. Even though

there are many studies performed worldwide that analyze

these factors, in Mexico very few exist. In 1995, Lazcano-

Ponce et al. [16] found that the main risk factors for cervical

cancer in women from Mexico City (Central region) are

multiparity and a history of many sexual partners. The risk

factors found by Castaneda-Iniguez et al. [15] in 1998 in

women from the State of Zacatecas (Central-Northern

Mexico) are multiparity, early start of sexual activity and the

use of oral contraceptives. In 2005, Tirado-Gomez et al. [17]

found as risk factors in women from the Central region

(Mexico City and Veracruz) and Southern region (Morelos)

of Mexico low education levels, lack of access to health

services, unfavorable socioeconomic condition, multiparity,

presence of vaginal infection, and early start of sexual

activity.

In this study, we found that in the State of Guerrero,

located in the Southern region of Mexico, the risk factors of

invasive cervical cancer are having three or more births and

start of sexual activity before 16 years of age. High parity

may increase the risk of cervical cancer because it maintains

the transformation zone of the exocervix for many years,

facilitating the direct exposure to HPV and possibly other

cofactors. Hormonal changes induced by pregnancy may

also modulate the immune response to HPV and influence

risk of persistence or progression. It has also been proposed

that the developing cervix, around peri-menarchy, or the

healing cervix are high-risk situations for an HPV infection

to reach the basal layer and establish a persistent infection

[38]. Age, smoking habits, and number of sexual partners

were not risk factors in the development of cervical cancer,

as reported in other World populations. In the State of

Guerrero, polygamy is not a frequent practice among

women, in this study 60% of the women said that they were

monogamous and in a study that we carried out in Nahuatl

indigenous women in the year 2000 over 90% reported they

were monogamous (unpublished data). However, polygamy

is a common practice among men from the State of

Guerrero. A study shows that 98% of men have had more

than one sexual partner, of which 60% is infected with HPV

[39]. So, even though the number of the sexual partners the

women had is not a risk factor in the development of cervical

cancer in this study, the high promiscuity of the men could

explain the high frequency of infection by HPV and shows

why the sexual behavior of the men is an important factor in

the development of cervical cancer.

Acknowledgments

We thank all of the Instituto Estatal de Cancerologıa

‘‘Dr. Arturo Beltran Ortega’’ and Secretarıa de Salud

personnel who helped with this study at the clinic sites. We

thank Vıctor Hugo Garzon for management of the patients

and specimen collection, and Marco Antonio Jimenez for

histological evaluation of all biopsy material. We also

thank technicians of Laboratorio de Biomedicina Molecular

for their excellent laboratory assistance. And we would also

like to thank Dinorah Leyva-Illades (Texas A&M Health

Science Center) for revising the English style of the

manuscript.

Appendix A. Supplementary data

Supplementary data associated with this article can be

found, in the online version, at doi:10.1016/j.cdp.2008.

09.001.

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