**4. Clinical utility of molecular HPV diagnosis**

The incidence of cervical cancer in the decades to come might in fact increase in developing countries due to the aging of the population and the persistent absence of adequate screening programs [95]. In countries with screening, the target of preventive efforts has shifted from cervical cancer detection to the diagnosis and treatment of cancer precursor lesions. However, the morphologic basis of the screening test cannot be substantially improved, inherently diminishing the accuracy for precursor lesion diagnosis. In these countries, a false negative Pap test occurs in 30% of all cervical cancers diagnosed and another 10% is attributable to errors in following up abnormal cytology reports [95,96].

with both oncogenic HPV vaccine types. Women of all ages should be able to make a well-

Screening protocols are likely to be modified taking advantage of the higher validity of HPV tests as compared to the conventional Pap smear. Many clinical trials have compared HPV DNA testing and cytology in screening scenarios and concluded that HPV test offers a greater sensitivity (in the range of 30%) and a reduced specificity (in the range of 8%) as compared to cytology. Moreover HPV tests are less demanding in terms of manpower and quality control and automated equipments are available for high throughput performance [100]. Other biomarkers are under evaluation to increase the specificity of screening programs and for the triage of HPV positive women with normal cytology. These include HPV typing, p16 INK

p16INK4a has been successfully deployed for the classification of HPV-related disease for several reasons: the expression of p16INK4a is directly linked to the HPV oncogenic action, since continuous expression of E7 is necessary to maintain the malignant phenotype, the expression of p16INK4a is independent of the HPV type, and therefore, genotyping does not need to be performed, and the expression of p16INK4a by cycling cells is a specific marker of HPV-E7 overexpression or other events that inactivate Rb by immunochemistry. Additionally, improving diagnostic accuracy and reproducibility, the use of p16INK4a immunohistochem‐ istry may help in identifying CIN1 lesions that are associated with HR-HPV types; these lesions are at an increased risk for progression to high-grade dysplasia or carcinoma [98]. Thus, the clinical assessment of HPV infection uses a combination of diagnostic cytologies, such as the

The new guideline regarding screening for the early detection of cervical precancerous lesions and cancer was published by the ACS and American Society for Colposcopy and Cervical Pathology (ASCC) in American Journal of Clinical Pathology [102]. The new guideline includes a review of molecular screening tests and strategies, it suggest that perhaps the largest immediate gain in reducing the burden of cervical cancer incidence and mortality could be attained by increasing access to screening (regardless of the test used) among women who are currently unscreened or screened infrequently. Incorporation of HPV testing may offer advantages over what is already a successful screening strategy if utilized (ie, cytology) [103]. Incorporation of HPV testing into cervical cancer screening strategies has the potential to allow both increased disease detection and increased length of screening intervals. The recommen‐

Pap test in association with complementary DNA test and hostp16INK4a [101].

**Population Screening Method\* Management of Screen Results Comments**

guidelines2.

Aged <21 years (y) No screening HPV testing should not be used for

HPV-positive ASC-US† or cytology of LSIL or more severe: Refer to ASCCP

screening or management of ASC-

HPV testing should not be used for screening in this age group

US in this age group

Molecular Tools for Detection Human Papillomavirus

http://dx.doi.org/10.5772/55710

43

informed decision when considering HPV vaccination [99].

immunostaining and others [101].

dations are described in the Table 3.

3 y

Cytology alone every

Aged 21-29 years

(y)

Despite this limitation, a significant reduction in cervical cancer has been achieved with cytology-based technology and screening strategies [95]. HR-HPV DNA testing is currently recommended for triage of cytological diagnoses of ASCUS, as a cotest with the Pap smear in the general screening of women ≥30 years of age, and for follow-up of women after colono‐ scopy and treatment [97].

Histological examination of colposcopy-guided biopsies is still considered the "gold standard" in the assessment of cervical lesions; however, the histologic assessment of these lesions is limited to the interpretation of the morphology, with little to no information regarding the risk of persistence, progression, or regression. In addition, histologic assessment of cervical lesions is complicated by interobserver variability. The main interpretive categories include distin‐ guishing normal from dysplasia (CIN) of any grade and low-grade (CIN1) lesions from highgrade (CIN2/3) lesions. Errors in histologic diagnosis lead to either overtreatment of patients who will not benefit from intervention or, conversely, undertreatment of patients with clinically significant high-grade lesions that received false negative diagnoses [98].

Contemporaneous cervical cancer screening guidelines from the American Cancer Society (ACS) and the American College of Obstetricians and Gynecologists (ACOG) in effect acknowledged the extremely high sensitivity of FDA-approved Pap and HPV co-testing by specifically accepting lengthened screening intervals for women who test negative on both cytology and HPV tests [103]. There is ample evidence that the detection of HPV DNA in cervical samples has a higher sensitivity for cervical cancer and precancerous lesions than the Pap test and high-quality HPV tests are routinely used in prevention programs in some developed countries [99].

Although the recent introduction of a highly effective prophylactic HPV vaccine has great promise for the prevention of persistent infections and precancerous lesions, cervical cancer screening will still be required because the current vaccines do not protect against all carcino‐ genic HPV types and do not treat preexisting HPV infections and related disease [97]. Since persistent infection with HR-HPV is a risk factor for progression to cervical cancer and with the advent of HPV vaccines, it is increasingly relevant to perform HPV genotyping to identify oncogenic HPV vaccine types. HPV genotyping is of clinical interest, since the risk of devel‐ oping a precancerous lesion is between 10%, and 15% with HPV types 16 and 18, and below 3% for all other high-risk types combined. Genotyping information could provide more information regarding risk-stratification as well as persistence of infection [98].

While current guidelines and recommendations consistently advise on vaccinating young girls before their sexual debut, natural history studies indicate that all sexually active women are at risk of new oncogenic HPV infections and of development of cervical lesions and cancer throughout their lives. The reviewed data suggest that most sexually active women have the potential to benefit from HPV vaccination, with the exception of those with current infections with both oncogenic HPV vaccine types. Women of all ages should be able to make a wellinformed decision when considering HPV vaccination [99].

the morphologic basis of the screening test cannot be substantially improved, inherently diminishing the accuracy for precursor lesion diagnosis. In these countries, a false negative Pap test occurs in 30% of all cervical cancers diagnosed and another 10% is attributable to errors

42 Human Papillomavirus and Related Diseases – From Bench to Bedside A Diagnostic and Preventive Perspective

Despite this limitation, a significant reduction in cervical cancer has been achieved with cytology-based technology and screening strategies [95]. HR-HPV DNA testing is currently recommended for triage of cytological diagnoses of ASCUS, as a cotest with the Pap smear in the general screening of women ≥30 years of age, and for follow-up of women after colono‐

Histological examination of colposcopy-guided biopsies is still considered the "gold standard" in the assessment of cervical lesions; however, the histologic assessment of these lesions is limited to the interpretation of the morphology, with little to no information regarding the risk of persistence, progression, or regression. In addition, histologic assessment of cervical lesions is complicated by interobserver variability. The main interpretive categories include distin‐ guishing normal from dysplasia (CIN) of any grade and low-grade (CIN1) lesions from highgrade (CIN2/3) lesions. Errors in histologic diagnosis lead to either overtreatment of patients who will not benefit from intervention or, conversely, undertreatment of patients with

Contemporaneous cervical cancer screening guidelines from the American Cancer Society (ACS) and the American College of Obstetricians and Gynecologists (ACOG) in effect acknowledged the extremely high sensitivity of FDA-approved Pap and HPV co-testing by specifically accepting lengthened screening intervals for women who test negative on both cytology and HPV tests [103]. There is ample evidence that the detection of HPV DNA in cervical samples has a higher sensitivity for cervical cancer and precancerous lesions than the Pap test and high-quality HPV tests are routinely used in prevention programs in some

Although the recent introduction of a highly effective prophylactic HPV vaccine has great promise for the prevention of persistent infections and precancerous lesions, cervical cancer screening will still be required because the current vaccines do not protect against all carcino‐ genic HPV types and do not treat preexisting HPV infections and related disease [97]. Since persistent infection with HR-HPV is a risk factor for progression to cervical cancer and with the advent of HPV vaccines, it is increasingly relevant to perform HPV genotyping to identify oncogenic HPV vaccine types. HPV genotyping is of clinical interest, since the risk of devel‐ oping a precancerous lesion is between 10%, and 15% with HPV types 16 and 18, and below 3% for all other high-risk types combined. Genotyping information could provide more

While current guidelines and recommendations consistently advise on vaccinating young girls before their sexual debut, natural history studies indicate that all sexually active women are at risk of new oncogenic HPV infections and of development of cervical lesions and cancer throughout their lives. The reviewed data suggest that most sexually active women have the potential to benefit from HPV vaccination, with the exception of those with current infections

information regarding risk-stratification as well as persistence of infection [98].

clinically significant high-grade lesions that received false negative diagnoses [98].

in following up abnormal cytology reports [95,96].

scopy and treatment [97].

developed countries [99].

Screening protocols are likely to be modified taking advantage of the higher validity of HPV tests as compared to the conventional Pap smear. Many clinical trials have compared HPV DNA testing and cytology in screening scenarios and concluded that HPV test offers a greater sensitivity (in the range of 30%) and a reduced specificity (in the range of 8%) as compared to cytology. Moreover HPV tests are less demanding in terms of manpower and quality control and automated equipments are available for high throughput performance [100]. Other biomarkers are under evaluation to increase the specificity of screening programs and for the triage of HPV positive women with normal cytology. These include HPV typing, p16 INK immunostaining and others [101].

p16INK4a has been successfully deployed for the classification of HPV-related disease for several reasons: the expression of p16INK4a is directly linked to the HPV oncogenic action, since continuous expression of E7 is necessary to maintain the malignant phenotype, the expression of p16INK4a is independent of the HPV type, and therefore, genotyping does not need to be performed, and the expression of p16INK4a by cycling cells is a specific marker of HPV-E7 overexpression or other events that inactivate Rb by immunochemistry. Additionally, improving diagnostic accuracy and reproducibility, the use of p16INK4a immunohistochem‐ istry may help in identifying CIN1 lesions that are associated with HR-HPV types; these lesions are at an increased risk for progression to high-grade dysplasia or carcinoma [98]. Thus, the clinical assessment of HPV infection uses a combination of diagnostic cytologies, such as the Pap test in association with complementary DNA test and hostp16INK4a [101].

The new guideline regarding screening for the early detection of cervical precancerous lesions and cancer was published by the ACS and American Society for Colposcopy and Cervical Pathology (ASCC) in American Journal of Clinical Pathology [102]. The new guideline includes a review of molecular screening tests and strategies, it suggest that perhaps the largest immediate gain in reducing the burden of cervical cancer incidence and mortality could be attained by increasing access to screening (regardless of the test used) among women who are currently unscreened or screened infrequently. Incorporation of HPV testing may offer advantages over what is already a successful screening strategy if utilized (ie, cytology) [103]. Incorporation of HPV testing into cervical cancer screening strategies has the potential to allow both increased disease detection and increased length of screening intervals. The recommen‐ dations are described in the Table 3.


On the other hand, educative action to prevent cervical and non cervical cancers, which are part of basic health actions, should be considered a professional commitment to the popula‐ tion's quality of life and a care quality commitment, emphasizing patients' autonomy in selfcare. In the study in Brazil aimed to evaluate the applicability of an educational booklet that contained information for the general population about promotion and prevention of infec‐ tions and neoplasic process caused by the HPV. The authors enphatized it is necessary to promote and improve campaigns to the population about the HPV and its relations with the

Molecular Tools for Detection Human Papillomavirus

http://dx.doi.org/10.5772/55710

45

Proper condom use as a primary prevention measure should remain a top priority for health officials. Campaigns with a primary aim to increase sexually transmitted infection (STI) knowledge and awareness with the intention of influencing risk perceptions amongst those sexually active, may not effectively translate into an increase in prevention behaviors. To reach the public health goal of reducing STI prevalence, barriers to engaging in STI prevention need

Education should not only be considered an extra activity, but an action that redirects practices at health promotion as a whole. Suggests that preventive knowledge about the natural history of cervical and non cervical cancer and, such as use of HPV vaccination in both sex will decrease the incidence of HPV associated cancers and has the potential to be of great significance to high-risk female and male populations, the largest group to suffer from HPV-associated

In addition, molecular tools are a relatively new division of laboratory medicine that detects, characterizes, and/or quantifies nucleic acids to assist in the diagnosis of human disease. Molecular assays augment classical areas of laboratory medicine by providing additional diagnostic data either in a more expeditious manner or by providing results that would not be obtainable using standard methodologies. These methods are used for detecting infectious agents have several advantages when compared to classical approaches, because these methods generally do not require growth in culture media, like HPV, which cannot be grown

In this context, researchers benefit from having a variety of molecular diagnostic tests at their fingertips; however, the clinical laboratories in the United States have a more limited selection of FDA-approved tests for HPV. Many of the HPV diagnostic kits available in regions such as Europe and Canada have not been approved for clinical use in the United States. Meanwhile, laboratories may choose to use non-approved tests as analyte specific reagents (ASRs) or home

Regardless of the technical method used, careful consideration is necessary in the evaluation of diagnostic techniques for HPV screening. Most infectious disease tests strive for the highest possible analytical sensitivity, and PCR is typically the optimal method to achieve that standard. However, the more important standard for HPV screening is not analytical sensi‐ tivity but clinical sensitivity and specificity. Clinical utility of HPV screening is based on the prediction of cervical cancer, not simply the presence of the virus. Especially in young adult populations, detecting the HPV virus has little clinical use because the vast majority of these

brews, although more extensive validation is required in these cases [59].

neoplasic process to preventive strategies [104].

to be addressed, including preventive strategies [105].

cancers within this greater population [105].

in conventional cell cultures [106].


ASCCP, American Society for Colposcopy and Cervical Pathology; ASC-US, atypical squamous cells of undetermined significance; CIN2, cervical intraepithelial neoplasia grade 2; HPV, human papillomavirus; LSIL, low-grade squamous intraepithelial lesion.

\* Women should not be screened annually at any age by any method.

† ASC-US cytology with secondary HPV testing for management decisions. \* Modified from [102].

**Table 3.** Summary of Recommendations of The American Society for Colposcopy and Cervical Pathology

On the other hand, educative action to prevent cervical and non cervical cancers, which are part of basic health actions, should be considered a professional commitment to the popula‐ tion's quality of life and a care quality commitment, emphasizing patients' autonomy in selfcare. In the study in Brazil aimed to evaluate the applicability of an educational booklet that contained information for the general population about promotion and prevention of infec‐ tions and neoplasic process caused by the HPV. The authors enphatized it is necessary to promote and improve campaigns to the population about the HPV and its relations with the neoplasic process to preventive strategies [104].

**Population Screening Method\* Management of Screen Results Comments**

44 Human Papillomavirus and Related Diseases – From Bench to Bedside A Diagnostic and Preventive Perspective

guidelines2

contesting

genotypes

to colposcopy

Cytology alone every HPV-positive ASC-US† or cytology of

guidelines2

Aged 30-65 years

Aged "/>65 years

intraepithelial lesion.

(y)

HPV and cytology "cotesting" every 5 y

(preferred)

No screening following adequate

recommendations

as unvaccinated women)

\* Women should not be screened annually at any age by any method.

negative prior screening

HPV vaccinated Follow age-specific

(same

(y)

Cytology negative or HPV-negative ASC-US†: Rescreen with cytology in 3 y

HPV-positive ASC-US or cytology of LSIL or more severe: Refer to ASCCP Screening by HPV testing alone is not recommended for most clinical

Women with a history of CIN2 or a more severe diagnosis should continue routine screening for at

and without a history of CIN2 or a more severe diagnosis in the past 20

y or cervical cancer ever

least 20 y

settingst.

HPV positive, cytology negative: Option 1: 12-mo follow-up with

Option 2: Test for HPV16 or HPV16/18

• If HPV16 or HPV16/18 positive: refer

• If HPV16 or HPV16/18 negative: 12 mo follow-up with cotesting

Cotest negative or HPV-negative ASC-US: Rescreen with cotesting in 5 y

LSIL or more severe: Refer to ASCCP

Cytology negative or HPV-negative ASC-US†: Rescreen with cytology in 3 y

After hysterectomy No screening Applies to women without a cervix

ASCCP, American Society for Colposcopy and Cervical Pathology; ASC-US, atypical squamous cells of undetermined significance; CIN2, cervical intraepithelial neoplasia grade 2; HPV, human papillomavirus; LSIL, low-grade squamous

† ASC-US cytology with secondary HPV testing for management decisions. \* Modified from [102].

**Table 3.** Summary of Recommendations of The American Society for Colposcopy and Cervical Pathology

Proper condom use as a primary prevention measure should remain a top priority for health officials. Campaigns with a primary aim to increase sexually transmitted infection (STI) knowledge and awareness with the intention of influencing risk perceptions amongst those sexually active, may not effectively translate into an increase in prevention behaviors. To reach the public health goal of reducing STI prevalence, barriers to engaging in STI prevention need to be addressed, including preventive strategies [105].

Education should not only be considered an extra activity, but an action that redirects practices at health promotion as a whole. Suggests that preventive knowledge about the natural history of cervical and non cervical cancer and, such as use of HPV vaccination in both sex will decrease the incidence of HPV associated cancers and has the potential to be of great significance to high-risk female and male populations, the largest group to suffer from HPV-associated cancers within this greater population [105].

In addition, molecular tools are a relatively new division of laboratory medicine that detects, characterizes, and/or quantifies nucleic acids to assist in the diagnosis of human disease. Molecular assays augment classical areas of laboratory medicine by providing additional diagnostic data either in a more expeditious manner or by providing results that would not be obtainable using standard methodologies. These methods are used for detecting infectious agents have several advantages when compared to classical approaches, because these methods generally do not require growth in culture media, like HPV, which cannot be grown in conventional cell cultures [106].

In this context, researchers benefit from having a variety of molecular diagnostic tests at their fingertips; however, the clinical laboratories in the United States have a more limited selection of FDA-approved tests for HPV. Many of the HPV diagnostic kits available in regions such as Europe and Canada have not been approved for clinical use in the United States. Meanwhile, laboratories may choose to use non-approved tests as analyte specific reagents (ASRs) or home brews, although more extensive validation is required in these cases [59].

Regardless of the technical method used, careful consideration is necessary in the evaluation of diagnostic techniques for HPV screening. Most infectious disease tests strive for the highest possible analytical sensitivity, and PCR is typically the optimal method to achieve that standard. However, the more important standard for HPV screening is not analytical sensi‐ tivity but clinical sensitivity and specificity. Clinical utility of HPV screening is based on the prediction of cervical cancer, not simply the presence of the virus. Especially in young adult populations, detecting the HPV virus has little clinical use because the vast majority of these cases will self-resolve and never develop into cancer. The high sensitivity of PCR is thus a detriment in HPV screening, because PCR can detect even miniscule amounts of virus that may have no clinical significance [59].

**Acknowledgements**

**Author details**

Brazil

**References**

244–65.

(S1) S25-33

2007.

Angela Adamski da Silva Reis1

and Molecular Biology, Brazil

Aparecido Divino da Cruz3

The authors thank S. Quail for English support.

ogy and Obstetrics 2006; 94 (1) S3-S7.

nal of Pathology 1999;189(1)12-9.

ogy of human cancer. Vaccine 2006; 24 (3) S3/1-10.

This chapter was written by the researchers of the HPV Study Group in Goiânia-GO, Brazil.

, Daniela de Melo e Silva2

1 Federal University of Goiás - Biological Sciences Institute - Department of Biochemistry

2 Federal University of Goiás - Biological Sciences Institute - Department of General Biology,

[1] Villa LL. Biology of genital human papillomavirus. International Journal of Gynecol‐

[2] Bosch FX, Lorincz A, Munoz N, Meijer CJ, Shah KV. The causal relation between hu‐ man papillomavirus and cervical cancer. Journal of Clinical Pathology 2002;55(4)

[3] Steenbergen RD, de Wilde J, Wilting SM, Brink AA, Snijders PJ, Meijer CJ. HPVmediated transformation of the anogenital tract. Journal of Clinical Virology 2005;32

[4] Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. The Jour‐

[5] Muñoz N, Castellsagué X, de González AB, Gissmann L. Chapter 1: HPV in the etiol‐

[6] International Agency for Research on Cancer: Monographs on the Evaluation of Car‐ cinogenic Risks to Humans-Human papillomaviruses. Lyon: IARC Press (90) 16–36,

3 Pontifical Catholic University of Goiás.- Department of Medicine and Biology, Brazil

, Cláudio Carlos da Silva3

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47

and

There are many advantages to the PCR technology for such screening applications, including automation capabilities, turnaround time, multiplexing, sensitivity/specificity, multiple specimen types, and small-specimen volume. The major concern with the in vitro amplification technologies is the potential for contamination [107]. PCR require that laboratories determine a threshold of detection representing a clinically significant result. Likewise, the detection of non cancer-causing, low-risk strains of HPV has virtually no clinical utility. Knowing that a low-risk HPV strain is present does not have an impact on the clinical management of a patient with cutaneous or mucosal warts. In order to prevent superfluous laboratory testing, clinicians should also heed the ASCCP guidelines for the management of women with or without cytological abnormalities [59].

The clinical laboratory must evaluate many factors in the adoption of an appropriate HPV test, including consideration of the population being served. In underprivileged areas, for example, HPV screening tests with less than optimal clinical sensitivities and specificities may still far surpass current cervical cancer screening methods. As new data emerge from recently established HPV screening methods, researchers and clinicians will continue to strive toward the goal of early and accurate detection of cervical cancer [59].
