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 Table of Contents  
SYSTEMATIC REVIEW
Year : 2021  |  Volume : 6  |  Issue : 1  |  Page : 6-17

Diagnostic techniques for human papillomavirus detection for early diagnosis of endocervical adenocarcinoma


Central Military Laboratory and Blood Bank, Prince Sultan Military Medical City, Riyadh, Kingdom of Saudi Arabia

Date of Submission17-Oct-2020
Date of Decision28-Dec-2020
Date of Acceptance14-Mar-2021
Date of Web Publication31-Jul-2021

Correspondence Address:
Abdullah Bandar Almutiri
Senior Medical Laboratory Scientific Officer (SMLSO)/Senior Specialist Histopathology, Cytopathology CT (IAC) and Medical Microbiology. Central Military Laboratory and Blood Bank, Prince Sultan Military Medical City, Riyadh 11314
Kingdom of Saudi Arabia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/KKUJHS.KKUJHS_19_20

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  Abstract 


Background: The past few decades have witnessed a shift in the rate of incidence of cervical squamous cell carcinoma (SCC) and endocervical adenocarcinoma because there has been an increase in the prevalence of the endocervical adenocarcinoma and a reduction in the SCC in countries with effective screening programs in place. Although our knowledge about the underlying reasons for this shift remains deficient, it seems that the prevalence of endocervical adenocarcinoma is on the rise partly because of greater exposure to human papillomavirus (HPV), screening delays, and more importantly, technical limitations faced in diagnosing endocervical carcinoma. Objectives: This study aims to systematically review certain diagnostic methods that detect HPV which will facilitate the early diagnosis of endocervical adenocarcinoma because adjuvant therapy has proven to be inefficient for later stages of the disease. Methodology: The relevant articles were researched online using databases such as Scopus, PubMed, ScienceDirect, ProQuest, SpringerLink, and Web of Science. The databases were electronically searched. The keywords used in the search included “human papillomavirus (HPV),” “diagnosis of endocervical adenocarcinoma,” “polymerase chain reaction (PCR),” “in situ hybridization (ISH),” and “immunohistochemistry (IHC).” The search was refined to include only those articles that were nonreviewed studies and recently published, covering the period from 2010 to the present. Results: Eight articles from different parts of the world were selected for the study and were categorized into three groups depending on the diagnostic technique used in them, which included PCR, ISH, and IHC. PCR has proven to be an efficient noninvasive method that detects HPV DNA in the early stages. ISH demonstrates high specificity and sensitivity for the detection, of which genotypes of HPVs are present in endocervical adenocarcinoma lesions. Immunohistochemical staining is a simple tool for detecting HPV, but it has limited efficiency. Conclusion: A number of diagnostic tools are available to detect HPV infection for the early diagnosis of endocervical adenocarcinoma. HPV detection tests can be used in combination with the Pap test to ensure that any abnormality is not missed during diagnosis. Nonetheless, further studies should be performed to develop novel techniques or improve existing ones, so that endocervical adenocarcinomas can be detected on time and with high accuracy, and to overcome the problems faced in the cytologic diagnosis of cervical adenocarcinomas lesions.

Keywords: Diagnosis of endocervical adenocarcinoma, human papillomavirus, immunohistochemistry, in situ hybridization, polymerase chain reaction


How to cite this article:
Almutiri AB. Diagnostic techniques for human papillomavirus detection for early diagnosis of endocervical adenocarcinoma. King Khalid Univ J Health Sci 2021;6:6-17

How to cite this URL:
Almutiri AB. Diagnostic techniques for human papillomavirus detection for early diagnosis of endocervical adenocarcinoma. King Khalid Univ J Health Sci [serial online] 2021 [cited 2021 Nov 28];6:6-17. Available from: https://www.kkujhs.org/text.asp?2021/6/1/6/322877




  Introduction Top


Squamous cell carcinoma (SCC), which starts developing in the epithelium that covers the cervix, is known to be the most commonly found form of cervical cancer, accounting for approximately 80%–90% of all cervical cancer cases. In comparison, endocervical adenocarcinoma, which begins in the glandular tissue, is not as prevalent as SCC, accounting for the remaining 10%–20% of all cervical cancer cases.[1]

Endocervical adenocarcinomas is a type of cancer that arises from the adenomatous cells that are present in the lining of the endocervical canal. These cells are responsible for secreting mucus in this route.[2] Endocervical adenocarcinoma is one of the most aggressive and lethal types of cancer. In addition, its incidence has shown a dramatic increase over recent years and its detection is more difficult compared to that of SCC. This is because its initial growth occurs in cells present in the endocervical canal, which is the route within the cervix in the endocervix, whereas the latter develops in the ectocervix.[3],[4] Therefore, these two different types of cancer of the cervix have different initiation sites for development.

In addition, endocervical adenocarcinoma shows no obvious symptoms, especially in the early phases of development.[2] However, it can be diagnosed through the examination of a Pap test specimen carried out by a pathologist.[2] Such specimens are collected by brushing or scraping the cervical lining to extract cells for microscopic examination.[5] If cells with abnormal morphology are detected in this examination, indicating cervical adenocarcinoma, a colposcopy is usually carried out by a physician. The physician will extract tissue specimens if the individual shows some symptoms indicating cervical cancer or an abnormal finding is made in the Pap test.[2] The extraction of a small portion of tissue is referred to as cervical or endocervical biopsy; alternatively, the extraction of a larger portion of cervical tissue, referred to as cervical conization, is usually conducted during colposcopy.[6]

The extracted specimen is then forwarded to a gynecologic oncologist who has expertise in identifying the microscopic characteristics of malignant tumors for the diagnosis of cervical adenocarcinoma through tissue analysis under a microscope.

If a tumor is detected, the primary tumor in the cervix and adjacent areas can be treated by surgery, which may involve procedures such as loop electrosurgical excision procedure (LEEP), laser surgery, cryosurgery, or conization.[7],[8] Some patients may require a hysterectomy to remove the cervix, uterus, or part of the vagina. In some cases, all three treatment approaches including cancers chemotherapy, radiation therapy, and surgery can be used in isolation or in combination for treating endocervical adenocarcinoma.[6],[9]

In the past few decades, the above routine diagnosis has witnessed a shift in the rate of incidence of cervical SCC and endocervical adenocarcinoma.[6] This is because there is an increase in the prevalence of the latter and a reduction in the former in countries with effective screening programs.[5] Furthermore, the Pap test has proved to be quite effective in reducing the number of patients suffering from cervical SCC. However, this test has not led to a significant reduction in the number of endocervical adenocarcinoma patients.[10] This is partly because cells affected by endocervical adenocarcinoma are not extracted during specimen collection for the Pap test as these cells occupy deeper sites in the cervix under the cervical lining, making their collection quite difficult. Nevertheless, the Pap test is sometimes unable to detect endocervical adenocarcinomas as they are small in size and are located deep in the endocervical canal. This makes these lesions difficult or even impossible to diagnose.[4],[11] Therefore, there is a need for other techniques that provide and facilitate the early diagnosis of endocervical adenocarcinoma as adjuvant therapy has proven to be inefficient for later stages of the disease. However, this can be done via the detection of human papillomavirus (HPV).[12] In addition, the treatments for these advanced stages of endocervical adenocarcinoma can be troublesome and may involve the removal of cervical tissues by conization or LEEP. These surgical procedures, depending on the extent of tissue removal, can result in the weakening of the cervix or have an adverse effect on fertility. A small increase in the number of premature deliveries has also been associated with these surgeries.[13]

Previous studies have indicated that the Pap test is not as efficient in identifying adenocarcinomas and abnormalities in glandular cells as it is in identifying abnormalities in squamous cells. To date, the evidence is insufficient to determine whether HPV tests can be utilized for cervical cancer screening. Other studies were conducted to investigate whether routine HPV tests can be used as the primary screening modality with the Pap test as a follow-up approach.[14]

In comparison with relying on a Pap test alone, carrying out an HPV test in combination with a Pap test can reduce the number of false negative results.[15] Hence, a female with a normal Pap test result and a negative HPV test result is at low risk of developing cervical cancer in the near future. Several studies have reported that if HPV and Pap tests are used in combination and the screening interval is enhanced up to 5 years, abnormalities will continue to be detected and treated early. In addition, this strategy proves to be economical as well.[16]

HPV infection is considered to be the most significant risk factor associated with cervical cancer. According to a widely accepted notion, females with cervical cancer have been infected with HPV in the past.[17],[18] The HPV infection can be detected by checking sampled cells for the presence of the DNA/RNA of HPV. In this method, the detection of the HPV infection can allow for the early diagnosis of the disease before the appearance of abnormalities in cellular morphology.[19] In relation to endocervical adenocarcinoma, sexually transmitted HPV was found to be a common causative agent.[17],[19],[20] According to many studies, endocervical adenocarcinoma is always preceded by the HPV infection. HPV-16, HPV-18, HPV-31, HPV-33, and HPV-45 are some examples of high-risk HPVs, and among these, HPV-16 and-18 are the most important as they account for two-thirds of all cervical cancer cases.[21],[22]

HPV infection may lead to alterations in cervical cells, and these cells with abnormal morphology can be detected through a Pap test or biopsy. Viral DNA/RNA can also be detected in cells through different techniques such as polymerase chain reaction (PCR). However, these approaches serve only to detect the HPV infection as it does not have observable symptoms.[20]

An efficient strategy for the detection of the HPV infection is checking for the presence of viral DNA/RNA in the cell sample.[23] There are tests that allow for the earlier detection of the HPV infection through the appearance of abnormalities such as Pap tests. Viral DNA is typically detected inside the cells; however, it does not allow for the determination of the type of high-risk HPV causing the infection. However, there is a test that specifically detects DNA related to HPV-16 and -18, which are the two highest-risk HPVs most commonly found in cervical cancers.[17],[24] Another test detects DNA from a number of different high-risk HPVs and also shows whether the DNA is of HPV-16 or -18.[17],[24] Similarly, another test can detect high-risk HPV RNAs that are mostly associated with cervical cancer.

It seems that these DNA/RNA detection tests can be used to check different types of cells for the HPV infection; yet, there is no screening test available for the detection of the HPV infection in oropharyngeal, anal, penile, vulvar, or vaginal tissues, necessitating additional studies in this area.[20]

As a result, many diagnostic methods have been developed to facilitate the early diagnosis of endocervical adenocarcinoma as adjuvant therapy has proven to be inefficient for later stages of the disease. However, HPV detection tests can be used in combination with the Pap test to ensure that any abnormality is not missed during diagnosis in addition to increasing the accuracy of the diagnosis.

The aim of is this study is to review a number of diagnostic methods for HPV detection that can facilitate and even improve the early diagnosis and identification of endocervical adenocarcinoma, mainly because for the later stages of the disease, adjuvant therapy has been identified to be ineffective.

Significance

Statistical data from reliable sources indicate that the prevalence of cancer in different regions of the world is alarming. In women, cervical cancer is recognized as one of the most common cancers. In fact, in 2018, approximately 570,000 women round the world were diagnosed with cervical cancer and approximately 311,000 women were unable to survive due to it. It is quite important to note that Swaziland is considered the area with highest patients of the disease and it is followed by Malawi. The age-standardized rates in both of them are 75.3% and 72.9%, respectively. This rate is 66.4% in Zambia and it is 62.3% in Zimbabwe.[25] Statistics related to the rate of incidence of cervical cancer in 2009 (available on Cancer Australia) showed it to be the third most prevalent gynecological cancer with 771 newly diagnosed Australian patients and constituting 1.5% of all new female cancer cases. One out of every 162 females below age 85 was at a higher risk of developing cervical cancer.[26]

In Australia, cervical cancer proved to be the most common cause of deaths from gynecological cancers in 2010 with 232 mortalities. In other words, 1.3% of women dying of cancer are victims of cervical cancer. Fortunately, quite stable survival rates have been recorded for cervical cancer in Australia during the period between 1982 and 1987 and 2006 and 2010, with 5-year relative survival rates increasing from 68% to 72.1%.[26]

In the United States, cervical cancer used to be one of the leading causes of death in female cancer patients. According to an estimate made by the American Cancer Society, approximately 12,360 individuals in the United States would be diagnosed with cervical cancer in 2014 and approximately 4020 would die from the cervical cancer.[27]

There is an obvious increase in the rate of incidence of cervical cancer. In this light, it is essential for researchers to evaluate different modalities for the diagnosis and treatment of cervical cancer. Therefore, this study may be of crucial importance in determining the value of different modalities associated with cervical cancer.


  Methodology Top


Various diagnostic methods either proposed or evaluated for the detection of different HPV genotypes by reports published in recent years have been taken into consideration in this study. Different researchers have studied the diagnostic efficiency of different assays that are based on the detection of different diagnostic markers. This study also includes an evaluation of earlier studies that had employed novel methods and biomarkers for differentiating between various genotypes of HPVs and had analyzed the efficiency of these methods for diagnosing endocervical adenocarcinoma. It is worth mentioning that this study has not taken into account all of the diagnostic techniques employed for the diagnosis of endocervical adenocarcinoma. Some techniques evaluated in this study include real-time PCR, in situ hybridization (ISH), and immunohistochemistry (IHC).

In addition to it, articles available online were searched to select suitable studies for this research. The library provided access to databases such as Web of Science, ScienceDirect, SpringerLink, PubMed, Scopus, and ProQuest. In addition to these databases, the Google Scholar database was also used. Searches were made using the keywords “human papillomavirus (HPV),” “diagnosis of endocervical adenocarcinoma,” “polymerase chain reaction (PCR),” “in situ hybridization (ISH),” and “immunohistochemistry (IHC).” The search was refined to include only those articles which that published from 2010 to the present. This was done to ensure that the latest articles that had not been reviewed earlier could be included. The full texts of articles published in the English language were accessed, and a document delivery service was utilized in case of articles whose full text was not accessible. The citation indexes of the articles were also compared while selecting articles for this study. To widen the search, references of articles were used to select relevant articles and editorials. The EndNote X7 program was utilized for referencing. In addition, the number of citations and the impact factor for the publishing journal were determined for each of the studied papers using the Web of Science database. However, only the journals that have the highest impact factor were chosen. Furthermore, the citation index was used to decide which article was the best by comparing them to each other.


  Results Top


The latest reports involving the assessment of different diagnostic techniques for the detection of HPV in samples collected from patients having endocervical adenocarcinoma were included in this study. These reports had not been reviewed earlier. The diagnostic assays that have been analysed in these studies included PCR, ISH, and IHC.

Eight research articles, all of which were published between 2010 and 2014, were included in this study. It is worth mentioning that the selected studies were quite different from one another in terms of aims and objectives, selected subjects, and significance. The results of each of these studies were, therefore, summarized separately owing to these differences.

Polymerase chain reaction

Five of the studies used PCR in their investigation of HPV control effects on endocervical adenocarcinoma reduction [Table 1].
Table 1: Human papillomavirus DNA was amplified by polymerase chain reaction

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The first study is by Park et al.[28] and it investigated the distribution and prevalence of HPV types among endocervical adenocarcinoma cases in Korean women. According to their study, HPV-DNA was detected by the use of a short PCR fragment (SPF)-10 PCR, the L1 PCR target region. Subsequently, HPV genotypes were identified through a reverse hybridization technique. This study found that out of the 196 cases submitted, 89.3% were confirmed as endocervical adenocarcinoma. The average age of the subject was 47.1 years with a standard deviation of 11.9 years. HPV DNA cases were found to account for 90.3% of these cases. HPV-18 was found to be the most prevalent, accounting for 54.2% of all cases, followed by HPV-16 (44.1%) and HPV-45 (3.4%). In addition, infections that had any positive results for high-risk HPV types included 97.7% of all the HPV-DNA cases with positive endocervical adenocarcinoma. This study concluded that HPV DNA is prevalent in endocervical adenocarcinoma among Korean women.

The second study by Nofech-Mozes et al.[29] investigated the absence and presence of high-risk HPV DNA in cervical carcinoma rare types using a PCR test. DNA was extracted from deparaffinized sections by using Roche AMPLICOR HPV Amplification Detection and control kits (Roche Molecular Systems, NJ). This kit could detect all 13 HPVDNA genotypes to be specific for polymorphic regions of L1 flanked by these primers. The AMPLICOR HPV Test's positive cut-off point was set as A450 = 0.2. There were 13 cases, of which 11 (84.6%) were positive for high-risk HPV. The positive high-risk HPV types detected were HPV-16, -18, and -45. This study suggests that a clear association exists between the rare subtypes and high-risk HPV. Despite the fact that their clinical setting and morphology are unique, most associated tumors assume the same cancer pathway characteristics of HPV.

Siriaunkgul et al.[30] provided a third study that determined the HPV distribution in endocervical adenocarcinoma cases in Thailand. Paraffinized and formalin-fixed tissues were obtained from 150 patients. The sequences of HPV were detected by a nested PCR test by using the primers MY09/MY11. Genotyping was performed using a liner array assay that was designed to identify 37 HPV genotypes including 14 high-risk types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). The results of the study indicated that the HPV virus was detected in 97% of the adenocarcinomas, which included tumors whose HPV types could not be determined, 132 single infections, and 11 multiple infections. HPV-18 was found to be the most common, accounting for 66% of all cases, followed by HPV-16 (30%) and HPV-45 (3%). HPV-16 and -18 accounted for 88% of the tumors. It was concluded that the detection of HPV in cervical adenocarcinoma is high, predominated by HPV-18 and followed by HPV-16, and that vaccination against these would prevent as much as 90% of the associated health complications.

Tornesello et al.,[21] the fourth study, examined both the viral genotypes and the HPV-16 intratypic variants in SCC and cervical adenocarcinoma among Italian women. This is because a few studies have documented the genotype distribution and prevalence of HPV-16 variants in adenocarcinoma tumors. Their study reviewed 132 SCC and 39 invasive adenocarcinomas cases that were classified based on the World Health Organization classification. The sequences of HPV were detected by a nested PCR test using primer pairs GP5+/GP6+ and MY09/MY11 system broad-spectrum consensus as well as the analysis of nucleotide sequences. The study amplified HPV-16 positive cases with E6 specificity oligonucleotides. The amplimers were eventually subjected to variant identification through direct nucleotide sequences. The study results indicated that HPV infection accounted for 72% of adenocarcinoma cases as well as 85% of the SCC cases. This study classified 140 positive cancer cases, including nine HPV genotype cases (HPV-16, 18, 31, 33, 35, 39, 45, 58, and 82). This study established that HPV-16 was the most common cause of cancer, accounting for as much as 73% and 64% of SCC and adenocarcinoma cases, respectively. The E6 nucleotide sequence analysis identified HPV-16 in 33% and 20% of adenocarcinoma and SCC cases, signifying the association between glandular origin and cancer. This study concluded that HPV-16 is particularly prevalent in SCC and invasive adenocarcinoma cases.

Finally, Baalbergen et al.[31] reviewed and characterized a group of adenocarcinomas that had been diagnosed early with primary cervical localization based on HPV typing, IHC, and clinical evaluation. Their study also sought to assess the prognostic significance as well as the prevalence of HPV genotypes associated with HPV adenocarcinomas. In total, 171 adenocarcinomas cases that had been diagnosed in Rotterdam, the Netherlands, from 1989 to 2008 were considered. A primer combination PCO3 and PCO5 was used to generate a 209 bp product. High-risk HPV was detected by a home-brewed PCR-based assay. According to the established protocols, PCR with enzyme-immuno assay readout uses a cocktail probe for 14 HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). However, high-risk HPVs were tested, and out of the 171 cases, HPV evaluation was possible for 113. Of these, 101 (89%) were found to be positive. HPV-18 was found to account for 54% of the cases; HPV-16, for 37%; and HPV-45, for 7%; and HPV-39 and HPV-53 accounted for the remainder.

This study established that the 5-year survival rate of HPV-18 victims did not differ significantly from that of HPV-16 victims. HPV-45 was found to have the worst survival rates.

In situ hybridization

Three studies on ISH were investigated, and [Table 2] summarizes their results.
Table 2: Human papillomavirus type distribution among human papillomavirus positive subjects using in situ hybridization

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The first study by Park et al.[28] estimated the distribution and prevalence of HPV types in Korean women. The HPV DNA was amplified using PCR. Subsequently, HPV genotypes were identified through reverse hybridization Line Probe Assay (SPF10 HPV LiPA version 1, Labo Biomedical Products, Rijswijk, The Netherlands, based on licensed Innogenetics technology) that enables the detection of 14 oncogenic HPV types (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, and -68) and 11 nononcogenic HPV types (HPV-6, -11, -34, -40, -42, -43, -44, -53, -54, -70, and -74). They found that out of the 196 cases submitted, 89.3% were confirmed as endocervical adenocarcinoma. The mean age was 47.1 ± 11.9 years. HPV DNA cases were found to account for 90.3% of all cases. HPV-18 was found to be the most prevalent, accounting for 54.2% of the cases, followed by HPV-16 (44.1%) and HPV-45 (3.4%). This study concluded that HPV DNA is prevalent in endocervical adenocarcinoma among Korean women, with HPV-18 being the most common type.

Sheng et al.[32] used ISH to ascertain high-risk HPV types in 27 cases of endocervical adenocarcinoma. The results were then compared with those obtained using PCR for the same specimens. Of the cases examined, 63% showed HPVDNA through the use of the INFORM HPVIII Family 16 probe (Ventana Medical Systems, Tucson, AZ, USA), which can detect 13 high-risk HPV subtypes. Following the manufacturer's guidelines, the ISH assay was performed using the BenchMark automated slide staining system (Ventana Medical Systems). On the other hand, 67% were detected by PCR. The agreement between ISH and PCT showed that 85.1% cases were positive for HPVDNA. HPV-16 was found in five cases; HPV-18, in two cases; and HPV-45, in one case. In total, 22 cases tested positive for HPV in both tests, accounting for 85.1% of all cases. Nevertheless, four cases were found to be negative through ISH and PCR. However, the study concluded that combining HPV-ISH and HPV-PCR genotyping techniques showed high sensitivity of HPV detection in formalin-fixed, paraffin-embedded (FFPE) tissues from cervical adenocarcinoma in previous and present studies. In addition, HPV-ISH is useful for recognizing the distribution of HPV in endocervical adenocarcinoma tissues.

The last study by Jones et al.[33] investigated whether adenocarcinoma present in tissues obtained by biopsy or curettage is of endocervical or endometrial origin. The study compared the expression of HPV and the IHC profile of 76 tumors. HPV ISH was carried out in 39 endometrial adenocarcinoma and 37 endocervical adenocarcinoma cases. For ISH, biotin-labeled HPV probe solutions (Dako Corp., Carpinteria, CA), which are a cocktail of HPV-6, -11, -16, -18, -31, -33, -35, -45, -51, and -52, were applied.

However, the staining patterns were subjected to Bayesian network model analysis. This study revealed that endocervical adenocarcinoma was detected similarly using IHC and ISH. Most cases were found to be positive for HPV (65%) and p16 (86%). Most endometrial adenocarcinomas were found to be negative for HPV (100%) and carcinoembryonic antigen (CEA) (97%), and positive for estrogen receptor and vimentin (90%).

Immunohistochemistry

Three studies were investigated for IHC staining. Their results were discussed and summarized in [Table 3] based on their characteristics and findings.
Table 3: Characteristics and findings of immunohistochemistry-based studies

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The first study, Park et al.,[28] estimated the distribution and prevalence of HPV types in Korean women. They used hematoxylin, eason, and immunohistochemical staining for the diagnosis and subtype of endocervical adenocarcinoma cases. The HPV DNA was amplified using PCR. Subsequently, HPV genotypes were identified through the reverse hybridization technique. Of the 196 cases submitted, 89.3% were confirmed as endocervical adenocarcinoma. However, the study used two biomarkers: p16 and progesterone receptor (PR). The results indicate that 19.6% of cases were positive for PR and 92% for p16.

The second study by Jones et al.[33] investigated whether adenocarcinoma was present in tissues obtained from the cervix or the endometrium. They compared the expression of HPV for ISH and IHC in 76 tumors. Immunostaining for p16, mammaglobin (MGB), monoclonal CEA, vimentin (Vim), estrogen receptor (ER), PR, and PAX-8 as well as HPV ISH was carried out in 39 endometrial adenocarcinoma and 37 endocervical adenocarcinoma cases. The staining patterns were subjected to Bayesian network model analysis, and tissues with known positive staining for each antibody were used as positive controls, and tissues like kidney served as internal negative controls. The results indicated that there was a similar immunohistochemical profile in numerous endocervical adenocarcinoma cases. In addition, they showed high positivity for HPV (65%) and p16 (86%). Endometrial adenocarcinoma showed high negativity for HPV (100%) and CEA (97%) and high positivity for ER and vimentin (90%).

The last study by Balan et al.[34] examined the immunohistochemical expression of p53, cyclinD1, COX-2, EGFR, and p16 in malignant and benign lesions of the components of cervical glandular tissues, which should result in the establishment of the relationship that these markers have with the HPV L1 capsid protein. All tissue fragments were subjected to immunohistochemical and histopathological examinations, which are standard laboratory procedures. The results showed that p16 can be applied for forecasting the progression risk of endocervical adenocarcinomas. Furthermore, cyclin D1 is reliable in terms of the invasive capacity, and finally, EGFR increases depending on the progression of the severity of the lesions. In addition, this study revealed that a combination of L1 capsid proteins and p16 exacerbated the risks of endocervical adenocarcinoma and its preceding lesions. The coassessment of p16 and p53 is suitable for revealing the association between p16 and HPV L1 capsid protein.


  Discussion Top


The link between HPV infection and endocervical adenocarcinoma has attracted considerable research attention.[1],[3],[4],[6],[8],[11] Theoretically, it has been assumed that an effective anti-HPV vaccine can be used to prevent endocervical carcinoma. The development of such a vaccine would eventually confirm that HPV infection is associated with the development of cervical cancer. It has been established that the conventionally used Pap test is inefficient for the detection and classification of endocervical adenocarcinoma lesions as they are present deeper in the endocervical canal and are thus beyond the range of a Pap test examination. Therefore, the development of highly efficient diagnostic methods that allow early and accurate detection of endocervical adenocarcinoma lesions is essential. Many diagnostic assays with improved accuracy for the early diagnosis of endocervical adenocarcinoma have recently been developed. These methods have also addressed the cytological challenges encountered in the detection of cervical cancer lesions. The selected techniques were evaluated with these issues in mind.

Polymerase chain reaction

There is a lack of conventional methods for detecting and diagnosing HPV. The recent advent of the PCR method has been of great importance, and its amplification technology has made it possible to detect low-level viruses in patients.[35] This method has been applied in many studies seeking to investigate the prevalence of HPV among endocervical adenocarcinoma patients. The use of this method has revealed that high-risk HPV genotypes are the major causes of cervical cancer.[36] According to most studies, the main genotypes that cause more than 70% of cancers are HPV-16 and HPV-18. The use of PCR to detect HPV among endocervical adenocarcinoma patients indicates that there is a 97.7% infection rate.[28]

Park et al.[28] concentrated on the description of the HPV type in endocervical adenocarcinoma cases in Korean patients. Their results showed that there was a positive identification of HPV-DNA among the majority of endocervical adenocarcinoma cases.[28] Baalbergen et al.[31] found that out of 171 cases, 89% were positive. However, Park et al.[28] reported some technical limitations regarding the detection of HPV-DNA. These limitations lead to false positive and false negative results. They mentioned that HPV-DNA detection in endocervical adenocarcinoma cases needs a very sensitive technique because the viral load is very low. In addition, the presence of polymerase inhibitors in the tested tissue sample may occasionally lead to false negative results. These problems could be identified by running the experiment with a diluted DNA sample. Furthermore, the presence of contaminations from other cases and HPV amplification in the tissue may lead to false positive results.[28],[37]

Nofech-Mozes et al.[29] examined the presence of high-risk HPV genotypes of a rare form of cervical carcinoma and concluded that there is a robust association between rare types of cervical carcinoma and high-risk HPV genotypes. They investigated the HPV genotype distribution among cervical adenocarcinoma cases in Thailand. Their findings showed that 79% of the adenocarcinoma cases tested positive for HPV-DNA.[30] Tornesello et al.[21] studied Italian women with adenocarcinomas and analyzed the HPV-16 viral and intratypic types. Their results indicated that there is a high prevalence of HPV-16 in cases of both invasive adenocarcinoma and SCC. In this study, the first five paraffin sections from each block were not used for the experiment. Seventy percent alcohol was used to clean the used microtome and the knives were changed after cutting each paraffin block to eliminate cross contamination between different tested samples. In addition, between each cervical sample, one empty paraffin block was cut to reduce contamination and to be used as a negative control for PCR; this indicates the quality of the study and reduces the suspicion of contamination.

The results indicate that PCR is successful in the detection of HPV among endocervical adenocarcinoma cases. In all five studies reviewed, this method was successful in detecting HPV genotypes, with HPV-16 and HPV-18 being the most common. Thus, it is considered that there is a relationship between HPV and endocervical adenocarcinoma. Given the significance of the studies that support this point, it can be generalized that all PCR-oriented tests would yield similar results. Even more convincing is the fact that these studies covered a wide range of settings, including Korean women, Italian women, Thai women, and Canadian women, suggesting the inclusivity of HPV-susceptible populations. This finding is supported by a number of similar studies[38],[39] that have found that the status of HPV is related to endocervical adenocarcinoma and is often associated with HPV-18 andHPV-16.

Given that PCR can amplify the targeted DNA regions; it can be applied to the analysis of extremely small sample amounts such as the DNA of HPV in any tissue. Furthermore, PCR should be applied in DNA analyses. Notably, the analyzed DNA can be perceived to extend to a significant number of years.[24] The ability of PCR to provide an initial diagnosis of malignant diseases such as endocervical adenocarcinoma should lead to the formulation of PCR assays for genomic DNA samples. Such a practice would result in the detection of translocation-specific malignant cells.

PCR should be applied in the detection of viral DNA. As such, it should be noted that the selected primers for the application should be compatible with the sequences that are targeted in the DNA examination of a virus. Finally, PCR should be applied in the early detection of viruses prior to disease onset to give physicians vital lead-time in treating the disease.[40]

In situ hybridization

HPV infection increases the risk factor for the development of both precancerous and cancerous cells. Studies indicate that HPV is found in more than 90% of cervical cancer cases.[41] It is possible to detect low-grade lesions. The increased evidence of a correlation between invasive carcinoma and viral integration to HPV-DNA has led to a growing trend of testing for HPV when screening for cancer. ISH is one of the methods used in HPV-DNA detection. It is a direct signal detection assay and has the advantage of maintaining the morphologic characteristic of an HPV genotype signal.[42] While there have been concerns about its low sensitivity, improved technology has led to an improvement in the sensitivity of ISH. This method has been used by numerous researchers to investigate the relationship between HPV-DNA and endocervical adenocarcinoma.[43],[44]

Park et al.[28] focused on describing the HPV genotype prevalence in Korean women with endocervical adenocarcinoma cases. They found that most endocervical adenocarcinoma cases in Korea were positive for HPV-18, HPV-16, and HPV-45. Sheng et al.[32] focused on the comparison between the specificity and the sensitivity of the PCR and ISH methods because there was an evaluation of all techniques for the purpose of detecting HPV in cervical adenocarcinoma. They found that HPV-DNA was present in cervical adenocarcinoma in previous and current studies. In addition, a high specific detection of HPV in Formalin-fixed paraffin-embedded (FFPE) tissues of cervical and formalin-fixed tissues of cervical adenocarcinoma could be achieved by combining ISH and PCR. Furthermore, ISH is a suitable technique for identifying the HPV distribution in tissues of endocervical adenocarcinoma.[45]

Jones et al.[33] intended to determine the existence of adenocarcinoma in curettage or biopsy of endometrial or endocervical origin. They found that immunohistochemical testing with multiple markers and HPV testing is a reliable tool for evaluating adenocarcinoma of both endocervical and endometrial origins. They also found that 65% of endocervical adenocarcinoma cases were positive for HPV. Furthermore, they revealed a strong association of the HPV genotype with endocervical adenocarcinoma; however, details of the HPV genotypes were not provided.

The findings of studies related to ISH indicate that HPV-DNA is present in endocervical adenocarcinoma cases. Therefore, ISH should be used for establishing nucleic acid targets, which are specific. This should be applied to cells and fixed tissues to obtain spatiotemporal information regarding genetic loci and gene expression. It is important to note that ISH provides considerable information through the visualization of the tissue results.

Immunohistochemistry

In various studies, immunohistochemical staining of potential biomarkers was performed to identify HPV positive samples. IHC is one of the most inexpensive methods available to clinical researchers. Many studies are focusing on the detection of HPV in endocervical adenocarcinoma cases. Studies are increasingly seeking to establish this relationship, which may assist in understanding whether HPV can be used as a control method for endocervical adenocarcinoma.[40] In various studies, immunohistochemical staining of potential biomarkers was performed to identify HPV positive samples and to diagnose endocervical adenocarcinoma and its subtype in the given samples. In these studies, immunohistochemical staining was conducted to evaluate the expression level of certain potential biomarkers, mainly p16, PR, and monoclonal CEAs.[46]

Park et al.[28] used two biomarkers-p16 and PR-to diagnose and subtype endocervical adenocarcinoma samples. However, p16 was found to be positive in 92% of endocervical adenocarcinoma cases and PR in 19.6% of cases. A high diffuse expression of immunohistochemical p16 can result from the binding of the retinoblastoma gene product to the HPV E7 gene product in high-grade endocervical adenocarcinoma lesions. This conclusion reveals that p16 may be useful as a panel of relevant biomarkers for endocervical adenocarcinoma that are affected by HPV.[47]

Jones et al.[33] identified p16 (86%) and monoclonal CEA (31%), followed by PAX -8 (27%) and vimentin (17%), as the most positive biomarkers, as shown in [Table 3]. Balan et al.[34] found that the use of p16, which diffused (100%), and LI capsid protein could predict the progression risk of the precursor endocervical adenocarcinoma lesions. However, in all three studies, p16 showed high-level overexpression. This high expression occurs when retinoblastoma protein is inactivated by high-risk HPV oncoprotein E7; it has been observed in other studies as well.[48]

The researchers that opted to use this method were evidently successful, indicating its reliability. Their findings concur with those of previous methods stating that HPV does play a vital role in the development of cancerous lesions. This is based on the fact that high-risk HPV-DNA was detected in the endocervical adenocarcinoma cases.[49],[50]

IHC is an excellent method that should be used for identifying the exact location of a specific protein within a tissue that is under examination. However, the major limitation of IHC lies in the fact that it cannot indicate the correspondence of staining the protein, unlike immune-blotting methods in which staining is detected using a molecular weight grid.

Limitations in cervical cancer diagnosis and their solutions

Although the techniques used for cervical cancer screening are effective, they still have certain limitations. These tests are not immune to false positive or false negative results. A false positive result is obtained when the test indicates that the patient has cancerous cells though in reality, all cells are normal. Similarly, a false negative result is obtained when the test indicates that cells are normal but in reality, cancerous cells are present.[51],[52] The Pap test has proven to be quite helpful in reducing the number of patients suffering from cervical SCC. However, this test has not significantly facilitated a reduction in the number of endocervical adenocarcinoma patients. This is partly because cells affected by cervical adenocarcinomas are not extracted during Pap test specimen collection as these cells are present in deeper sites in the cervix under the cervical lining, making their collection quite difficult.[4],[11] Another drawback of cervical cancer screening tests relates to the nature of HPV infection. These tests may identify abnormalities in the morphology of cervical tests, but they would never result in cervical cancer. As mentioned earlier, an HPV infection is mostly brief and causes transient alterations in the cells of the cervix.

If a patient exhibits some symptoms of cervical cancer or an abnormal Pap test finding is obtained, a colposcopy is performed to obtain a tissue sample to determine if the tumor has spread inside and outside the uterus. Nevertheless, in such patients where the cancer has undergone sufficient progression, a Pap test is not an efficient technique for the early diagnosis of endocervical adenocarcinoma. Therefore, the results demonstrate that HPV infection is considered to be the most significant risk factor that has been associated with cervical cancer.[17]

An HPV infection can be detected by checking sampled cells for the presence of HPV DNA or RNA. In this way, the detection of HPV can allow for the early diagnosis of the disease before the appearance of abnormalities in the cellular morphology.[19] Overall, conducting these tests proved to be successful in identifying high-risk HPV infections. Currently, the most recommended and useful procedure is PCR because it is highly sensitive to the detection of the virus.

The reports concluded that a Pap test is not as efficient in identifying adenocarcinomas and abnormalities in glandular cells as it is in identifying abnormalities in squamous cells. The findings also determined that it is very difficult to distinguish whether HPV tests can be utilized for cervical cancer screenings. Today, researchers are conducting experiments to determine whether a routine HPV test can be used as the primary screening modality with a Pap test as a follow-up approach.[14]

In comparison with relying solely on a Pap test, performing an HPV test in combination with a Pap test can result in a reduction of false negative results that can occur when using the current Pap test method alone.[15] Therefore, a female with a normal Pap test result and a negative HPV test result is at low risk for developing cervical cancer in the near future. Several researchers have reported that if HPV and Pap tests are used in combination and the screening interval is increased up to 5 years, abnormalities can continue to be detected earlier in order to be treated. Plus, this strategy proves to be economical as well.[16]

Using PCR or ISH for the early detection of HPV gives physicians significant lead-time for treating cervical cancer prior to the onset of the disease. Article findings taken into consideration for evaluating PCR and ISH have revealed that HPVDNA is found in endocervical adenocarcinoma cases. Therefore, PCR or ISH must be used to detect HPV; reports indicate a high level of congruence in the findings of PCR and IHC in detecting HPV in tissue samples collected from adenocarcinoma patients. The advantage of the PCR technique is that it shows very good sensitivity for detecting HPV DNA and requires a very small amount of sample. However, the performance of the PCR technique can be affected by contamination. On the other hand, the ISH technique cannot be easily contaminated and can detect specific HPV DNA genotypes; however, it shows less sensitivity than PCR.[53] The IHC technique is cheaper than the other techniques (PCR and ISH). However, the IHC technique is not very specific and needs a core biopsy sample to be performed, whereas in PCR and ISH, a small amount of sample is needed. [Table 4] shows a summary of the advantages and disadvantages of the evaluated techniques.
Table 4: Advantages and disadvantages of different diagnostic techniques

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Still, additional studies are highly recommended because diagnostic techniques with enhanced sensitivity not only ensure the timely diagnosis of endocervical adenocarcinoma but also address other issues related to the cytological analysis of cervical cancers lesions.


  Conclusion Top


This research was performed to review methods and techniques that detect (HPV) which will facilitate the early diagnosis of endocervical adenocarcinoma. In addition, different diagnostic techniques for HPV detection in samples obtained from endocervical adenocarcinoma patients were evaluated. A high level of congruence was seen in the findings of PCR and ISH when samples were taken from endocervical adenocarcinoma patients for HPV detection. In accordance with the systemic review, it has been identified that IHC is a simple tool that can be used for detecting HPV with limited efficiency. ISH can be used to distinguish between different HPV genotypes. However, both ISH and PCR can be used in combination with the extensively employed test, which is the current Pap test. Still, additional studies are highly recommended for sensitive diagnostic tools that not only ensure the early diagnosis of endocervical adenocarcinoma but also address other issues encountered in the cytological analysis of cervical cancers lesions.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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