**7. HPV detection methods**

an isolate SCC cause on oral cavity. Some studies argue that HPV is a mere supporting causer [1, 61] while another study indicates that tumors positive for oncogenic types of HPV may show better survival [62], mainly in oropharynx, where HPV positive tumors are associated with a specific morphology (basaloid squamous cell carcinoma, a subtype of conventional

HPV-16 has been found in 90% of head and neck cancers and in 50% of oropharynx [1, 18, 53,

Potentially malignant disorders such as leukoplakia, erytroplakia, proliferative verrucous leukoplakia and lichen planus may progress to OSCC [1] and upon biopsy, there may be

Surgical resection is the treatment of choice when the lesion is placed on oral cavity while chemoradiotherapy is used when the oropharynx is the afflicted site [22] or if it is a tumor in

Surgery may impair some functions as speech, swallowing, and chewing and abruptly change quality of life. To maintain swallowing and speech, an alternative course is ablative surgery (microvascular free tissue), but this is not regarded to be as effective as surgical resection. In advanced stage when metastasis is located on upper aerodigestive tract, treatment should be

Recurrent OSCC is challenging as the risk of complication is increased due to fibrosis and tissue

Radiotherapy may be primary, adjuvant or neoadjuvant. It is regarded as primary for unre‐ sectable tumor or for patients who cannot undergo surgery, adjuvant as a post-surgery complementary method, and neoadjuvant when performed before surgery to facilitate tumor

HPV infection may lead to cell immortalization by means of infection of the mucosa and skin basal epithelial cells, which are the only ones that keep in the cell cycle [8, 61]. It may be by itself a causative agent of malignant transformation or when associated with other unclear cofactors [61]. However, some researchers have argued that HPV is not able to cause malignant

Some factors should come into play to immortalize cells: virus type, synergetic action among physics, biological and chemical agents and genetic constitution, which are able to modify the natural course of the disease. But if the exposed person has a favorable condition and acquires

Oral HPV has been diagnosed in OSCC and it is believed that it has been involved in oral carcinogenesis by transforming the keratinocytes through a mechanism involving E6 and E7

high risk HPV it becomes easier to integrate viral DNA into human genome [61].

carcinoma) and positivity for p16 using immunohistochemistry [63].

64]. However, some authors have not found such association [64].

multimodal, combining surgery and chemoradiotherapy [46].

transformation, despite the studies which point to the contrary [12].

already areas of an actual OSCC [65].

a very advanced stage [66].

34 Trends in Infectious Diseases

hipovascularization [51].

**6. HPV oncogenic potential**

resection [54].

The identification of various types of HPV is a recent technological advance due to the growth impossibility in tissue cultures and research animals.

Diagnoses methods vary from simple to sophisticated ones, ranging from light microscopy to DNA expression, with low to high sensitivity. Light microscopy and in situ hybridization are considered low sensitivity methods because it only tests positive when there are more than 10 viral DNA copies per cell. Among the intermediate sensitivity methods we find southern blot, do blot and reverse hybridization with a positive detection result when there is from 1 to 10 DNA copies per cell. High sensitivity methods, such as PCR, needs less than 1 viral DNA copy per cell for microorganism detection [70].

#### **7.1. Light microscopy**

This method provides some data, even though it has low sensibility and it does not inform the HPV type. The most common HPV induced changes are epithelial thickening, prominent keratohyalin granules, hyperkeratosis, nuclear dysplasia, hyperchromasia, double nucleation of superficial and intermediated cell, perinuclear cytoplasmic halos, and atypical immature metaplasia [9].

#### **7.2. Electron microscopy**

HPV particles may be identified by electron microscopy (EM), but not the HPV type. EM can detect the presence of virion on koilocytic and dyskeratotic cell nuclei, but it is a limited method to investigate infection, because high risk HPV do not reproduce and as such cannot be identified through EM [5].

#### **7.3. Molecular methods**

Molecular methods can be divided into two types: non-hybridization, such as in situ amplifi‐ cation, southern and dot blot hybridization and the amplified, such as target amplification, signal amplification and probe amplification. Target amplification is best exemplified by PCR. Signal amplification may be represented by hybrid technique sample. Probe amplification which is a compound-probe is added to a probe generating signal (Ligase Chain Reaction) according to literature [5].

#### **7.4.** *In Situ* **Hybridization (ISH)**

*In situ* hybridization using biotinylated probes is a common method for detecting HPV in oral epithelium. It is practical and economical for screening for HPV in clinical pathology labora‐ tories. In situ hybridization also permits direct comparison of viral DNA location with histologic morphology [9, 71]. Although this technique is highly sensitive in cases in which individual nuclei contain a high copy number of the target DNA, as is likely to occur in most active infections, the method of ISH often fails to detect cases in which subgenomic fragments of the viral DNA have been incorporated into the host genome and the infection is nonpro‐ ductive of intact viral particles. So when there is low viral DNA amount, it leads to low sensitivity [71, 72]

behavior. Clinical examinations have to be done periodically and smoking and chronic

Oral HPV Related Diseases: A Review and an Update 37

It is crucial that the population in general be informed about HPV prevention as a control strategy and early diagnosis promotion. Raising awareness of HPV through education is essential to develop population perception about risk factors, mainly those related to sexual

In view of the increasing figures of cervix cancer, the US Food and Drugs Administration (FDA) approved in 2006 a vaccine against HPV [75]. There is a bivalent vaccine that contains L1 HPV-16/18 protein which generates a huge number of genotype specific antibody [75]. For HPV 16/18 vaccine efficiency is round 96% on cervix cancer [69]. There is also a tetravalent vaccine that works in the same way as the bivalent, but provides further immunity enclosing HPV types 6, 11, 16, 18 [75]. Both of them use virus- like particles in their composition [9].

The vaccine(s) stimulates humoral response, but it also stimulates B cell immune memory response, which persists for five years [69]. After a 5 years follow up, it has demonstrated 100%

As HPV's physiopathology is very similar on the affected sites, whether they are the skin, cervix, penile, anus or oral mucosa, there is no reason to doubt that the vaccine which works well on the cervix would also prove effective for the prevention of oral mucosa lesions [75].

As HPV-16 seems to be an important risk factor for the progress of malignant lesions (because it is found in most OSCCs), it might be possible that the vaccine would prevent or even treat

HPV vaccine seems to be less effective on women who have already been exposed to the virus, hence the public health focus on vaccinating girls before their first sexual relation to prevent warts and more disaster lesions in the future [8, 9]. Some countries have promoted vaccination for any females from 9 to 26 years who have never had sexual experience before [76]. Other countries promote vaccination for females up to 45 years old [77]. Vaccines for men aged 11 up to 26 years old in order to prevent genital warts and anal cancer was approved in 2011 by

HPV is as frequent in men as in women; however, it is often asymptomatic in males, what makes them a HPV reservoir to cervix and non- cervical lesions in females, transmitted mainly by sexual activity [69]. Current studies have been done in order to further assess the natural

A therapeutic vaccine is under study, one which could be used as adjuvant on surgery or radiotherapy, to clean up microscopic waste of the lesion, thus generating immune response.

In view of the potential risks of HPV and the potential benefits of the vaccine (some not yet fully established) some researchers favor the extended use of the vaccine to all age groups of both sexes, regardless of previous sexual practice, as a form to interrupt the transmission cycle

activity[18]. It is also important to discourage early sexual initiation [69].

alcoholism must be abolished.

of efficacy on persistent infection prevention [8].

the US Advisory Committee on Immunization Practices [78].

history of the HPV infection in men [79, 80].

them [8].

### **7.5. Southern blot and dot blot hybridization**

Southern Blot classifies and identifies new viral types. It is a labor-intensive process that requires well trained skills and depends on a new generation of equipments. This technique requires the total length of a DNA molecule, and offers additional information about viral integration and subtype [70].

Dot blot is a simplified southern blot, requiring less sophisticated facilities, but it is rarely performed because of its low sensitivity. It is often used as detection kits available on the market [70].

### **7.6. Target amplification**

The classic example of target amplification is PCR. This is the best subtype detection method due to the high sensitivity [9]. It is commonly used as diagnostic tool for HPV DNA epide‐ miological investigation, but because of the high cost this method cannot be used in a routine clinical practice. PCR has a high sensitivity and it is very effective for both malignant and pre malignant lesions identification, and material can be collected with oral swab or wash [7, 9].

#### **7.7. Hybrid Capture technology (HC)**

Using signal amplification with microplate chemiluminescent detection, this method identifies nucleic acid due to its high sensitivity [9]. HC is a very important tool to detect high risk HPV, and the method has identified HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 types [9]. This technique is able to identify 5000 viral copies per sample [73].

#### **7.8. Probe amplification**

Probe amplification methods differ from target amplification in that the amplification products contain a sequence only present in the initial probes. It is used currently as an important diagnostic application, the detection of high-risk genotypes of human papillo‐ ma viruses (HPV) [74].

#### **7.9. Education and vaccine prophylaxis**

One simple and effective prophylactic measure is patient education. It must be clear to patients that even after treatment the virus remains on the oral mucosa, so it is imperative to maintain good oral hygiene, condom use in all sexual relations and refrain from promiscuous sexual behavior. Clinical examinations have to be done periodically and smoking and chronic alcoholism must be abolished.

tories. In situ hybridization also permits direct comparison of viral DNA location with histologic morphology [9, 71]. Although this technique is highly sensitive in cases in which individual nuclei contain a high copy number of the target DNA, as is likely to occur in most active infections, the method of ISH often fails to detect cases in which subgenomic fragments of the viral DNA have been incorporated into the host genome and the infection is nonpro‐ ductive of intact viral particles. So when there is low viral DNA amount, it leads to low

Southern Blot classifies and identifies new viral types. It is a labor-intensive process that requires well trained skills and depends on a new generation of equipments. This technique requires the total length of a DNA molecule, and offers additional information about viral

Dot blot is a simplified southern blot, requiring less sophisticated facilities, but it is rarely performed because of its low sensitivity. It is often used as detection kits available on the

The classic example of target amplification is PCR. This is the best subtype detection method due to the high sensitivity [9]. It is commonly used as diagnostic tool for HPV DNA epide‐ miological investigation, but because of the high cost this method cannot be used in a routine clinical practice. PCR has a high sensitivity and it is very effective for both malignant and pre malignant lesions identification, and material can be collected with oral swab or wash [7, 9].

Using signal amplification with microplate chemiluminescent detection, this method identifies nucleic acid due to its high sensitivity [9]. HC is a very important tool to detect high risk HPV, and the method has identified HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 types [9]. This

Probe amplification methods differ from target amplification in that the amplification products contain a sequence only present in the initial probes. It is used currently as an important diagnostic application, the detection of high-risk genotypes of human papillo‐

One simple and effective prophylactic measure is patient education. It must be clear to patients that even after treatment the virus remains on the oral mucosa, so it is imperative to maintain good oral hygiene, condom use in all sexual relations and refrain from promiscuous sexual

sensitivity [71, 72]

36 Trends in Infectious Diseases

market [70].

integration and subtype [70].

**7.6. Target amplification**

**7.8. Probe amplification**

ma viruses (HPV) [74].

**7.7. Hybrid Capture technology (HC)**

**7.9. Education and vaccine prophylaxis**

technique is able to identify 5000 viral copies per sample [73].

**7.5. Southern blot and dot blot hybridization**

It is crucial that the population in general be informed about HPV prevention as a control strategy and early diagnosis promotion. Raising awareness of HPV through education is essential to develop population perception about risk factors, mainly those related to sexual activity[18]. It is also important to discourage early sexual initiation [69].

In view of the increasing figures of cervix cancer, the US Food and Drugs Administration (FDA) approved in 2006 a vaccine against HPV [75]. There is a bivalent vaccine that contains L1 HPV-16/18 protein which generates a huge number of genotype specific antibody [75]. For HPV 16/18 vaccine efficiency is round 96% on cervix cancer [69]. There is also a tetravalent vaccine that works in the same way as the bivalent, but provides further immunity enclosing HPV types 6, 11, 16, 18 [75]. Both of them use virus- like particles in their composition [9].

The vaccine(s) stimulates humoral response, but it also stimulates B cell immune memory response, which persists for five years [69]. After a 5 years follow up, it has demonstrated 100% of efficacy on persistent infection prevention [8].

As HPV's physiopathology is very similar on the affected sites, whether they are the skin, cervix, penile, anus or oral mucosa, there is no reason to doubt that the vaccine which works well on the cervix would also prove effective for the prevention of oral mucosa lesions [75].

As HPV-16 seems to be an important risk factor for the progress of malignant lesions (because it is found in most OSCCs), it might be possible that the vaccine would prevent or even treat them [8].

HPV vaccine seems to be less effective on women who have already been exposed to the virus, hence the public health focus on vaccinating girls before their first sexual relation to prevent warts and more disaster lesions in the future [8, 9]. Some countries have promoted vaccination for any females from 9 to 26 years who have never had sexual experience before [76]. Other countries promote vaccination for females up to 45 years old [77]. Vaccines for men aged 11 up to 26 years old in order to prevent genital warts and anal cancer was approved in 2011 by the US Advisory Committee on Immunization Practices [78].

HPV is as frequent in men as in women; however, it is often asymptomatic in males, what makes them a HPV reservoir to cervix and non- cervical lesions in females, transmitted mainly by sexual activity [69]. Current studies have been done in order to further assess the natural history of the HPV infection in men [79, 80].

A therapeutic vaccine is under study, one which could be used as adjuvant on surgery or radiotherapy, to clean up microscopic waste of the lesion, thus generating immune response.

In view of the potential risks of HPV and the potential benefits of the vaccine (some not yet fully established) some researchers favor the extended use of the vaccine to all age groups of both sexes, regardless of previous sexual practice, as a form to interrupt the transmission cycle and as a preventive strategy in controlling and avoiding the risks posed by HPV, including various types of cancer in different locations in the human body.

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