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Unexpected high prevalence of HPV 90 infection in an underserved population: is it really a low-risk genotype?

A handful of human papillomavirus (HPV) genotypes are the causative agents of most cervical intraepithelial lesions and carcinomas. (1-3) Well-defined, high-risk HPV (HR-HPV) genotypes, particularly HPVs 16 and 18, account for most cervical cancer cases worldwide. (4) HPV 90 is a rarely reported HPV genotype associated with anogenital lesions, but it has not been studied in the United States. Few data are available regarding its prevalence, oncogenic potential, and association with cervical intraepithelial lesions or cancer. Characterization of individual HPV genotypes using various genotyping techniques heightens our understanding regarding their specific contribution toward cervical intraepithelial lesions. This knowledge has been pivotal in the development of vaccination strategies and will play a key role in patient surveillance in the post-HPV vaccination era.

While providing support-in-kind pathology services to community outreach clinical partners, our team found an unexpected high rate of HPV 90 infection in a cohort of Latino women. This led us to investigate the prevalence, distribution, and disease association of HPV 90 infection in this underserved, inner-city population.

MATERIALS AND METHODS

Study Population and Cytology Classification

The study was conducted with approval of the Institutional Review Board of The Methodist Hospital Research Institute (IRB1210-0221). The study included a total of 808 women who were referred to our institution from 84 charity clinics in the greater Houston area for abnormal Papanicolaou tests from December 2009 to April 2011. The vast majority of the women were Latino, and none of them had medical insurance or qualified for the Medicare or Medicaid programs at the time of the study. Liquid-based Papanicolaou tests (SurePath, BD Diagnostics, Burlington, North Carolina) were performed during the evaluations, and the findings were interpreted according to the criteria set by the Bethesda System (TBS 2001) (5) with minimal modification to include an additional category of low-grade squamous intraepithelial lesion, cannot exclude high-grade squamous intraepithelial lesion (LSIL-H). The available corresponding biopsies of HPV 90-positive specimens were reviewed.

HPV Genotyping by DNA Microarray

HPV DNA was extracted from the residual SurePath specimens and amplified with polymerase chain reaction in the L1 region of the HPV genome, with human beta actin as the reference gene. The amplified HPV L1 gene and human beta actin gene were labeled with Cy5 and hybridized with an HPV DNA chip (GG HPV DNA Genotyping Chip Kit, GoodGene Inc, Seoul, Korea) that simultaneously detects 47 HPV genotypes. The signal was visualized using a GenePix 4000B Microarray Scanner (Molecular Devices Inc, Sunnyvale, California). The detection limit of the HPV DNA microarray chip is between 10 and 100 copies of HPV DNA per sample. DNA microarray provided high sensitivity for HPV subtypes in the specimens and identified cases infected with multiple HPV subtypes.

HPV Genotyping by DNA Sequencing

In all samples, HPV DNA microarray results were further confirmed by conventional direct DNA sequencing methods. The sequence data obtained by automated DNA sequencing were analyzed using BLAST search (http://www.ncbi.nlm.nih.gov/ BLAST/, accessed in August 2012) for HPV genotypes. DNA sequencing identified the most dominant genotype in a given specimen, and it served as a confirmatory assay.

HPV In Situ Hybridization in Cervical Biopsies

In 3 of the patients with abnormal cytology and HPV 90 infection, in situ hybridization (ISH) for HR-HPV was performed on the available current biopsies according to the protocol provided by the manufacturer (INFORM HPV III Family 16 (B), Ventana Medical Systems Inc, Tucson, Arizona). The cocktail of probes was designed to indiscriminately detect any of 12 HR-HPV genotypes, including 16, 18, 31, 33, 35, 39, 45, 51, 56, 58, and 66. HPV-ISH was also performed on available tissue blocks from other women with HPV 90 infection that were negative on cytology.

Data Analysis

The 2009 recommendations from the expert working group at the International Agency for Research on Cancer (IARC) categorized HPV into 4 groups: carcinogenic (group 1), probably carcinogenic (group 2A), possibly carcinogenic (group 2B), and not classifiable (group 3). (6) The HPV genotypes in IARC groups 1 and 2A are commonly referred to as HR-HPV and are included in the Digene Hybrid Capture 2 test. These genotypes include 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68A/68B. In this study, IARC group 2B (including genotypes 5, 8, 26, 30, 34, 53, 66, 67, 69, 70, 73, 82, 85, and 97) and group 3 (including 6, 11, 90, and others) will be referred to as intermediate-risk HPV(IR-HPV) and low-risk HPV (LR-HPV), respectively. (4)

RESULTS

HPV genotyping and sequencing data were obtained from all 808 samples. Our study population showed a 93% overall HPV infection rate (754 of 808), with 64% infected with HRHPV, 17% with IR-HPV, and 12% with LR-HPV genotypes. Coinfection with multiple HPV genotypes was observed in 39% of these women. HPV 90 was detected in 32 women (4%; Figure 1) with an average age of 39 years (range, 23-84 years), slightly older than the average of 36.5 years in the cohort. A vast majority of the women had HPV 90 as the single infecting HPV genotype (31 of 32 women; 96.8%), with only one woman being coinfected with HPV 13 (Table). Although negative concurrent cytology results were documented in 29 patients (90.6%), cytologic abnormalities were identified in 3 patients (9.4%), including 2 women with LSIL (Figure 2, A and C) and 1 woman with LSIL-H (Figure 2, B).

Concurrent biopsies in 2 of the 3 patients with abnormal cytology confirmed the LSIL with HPV cytopathic effect. The other patient did not have tissue available for correlation.

In the 32 cases with HPV 90 infection, 14 cases had biopsy or resection tissue available for an HPV-ISH test. No HRHPV was evidenced in concurrent biopsies in 2 of the 3 cases with cytologic abnormalities (Figure 2, D). Two women with current negative Papanicolaou tests had positive HPV-ISH in tissue biopsied 2 years prior. All remaining tissues examined were negative for HPV-ISH.

COMMENT

The Papillomaviridae family includes 16 different genera, and those associated with mucosal tumor development belong to the alpha genus. Their circular genome of about 7900 bp contains 7 to 8 open reading frames that can be divided into 3 regions: long control region (viral gene expression regulation), early (E, involved in viral gene expression, replication, and survival), and late (L, encodes functional proteins). (4) Members of the same genus must share a minimum of 60% nucleotide sequence identity across the L1. (7) Virtually all papillomaviruses carcinogenic to humans belong to the alpha genus (in particular, alpha 5, 6, 7, 9, and 11), a fact that might indicate a strong phylogenetic influence in their oncogenic capacity. (6) It has been proposed that this oncogenic potential disparity among members of the same group is at least in part due to an ability to degrade p53 (a tumor suppressor) by virtue of E6 expression. (7)

HPV 90 belongs to the alpha genus, species 15. It was first described in 1998 by Feoli-Fonseca et al (8) in a negative Papanicolaou test specimen from a patient infected with human immunodeficiency virus. Since then, a few reports have documented its occurrence in humans, mostly in patients with negative Papanicolaou tests, occasionally with diagnoses of atypical squamous cells of undetermined significance and LSIL. HPV 90 was also rarely detected in normal oral mucosa. (9-15)

Epidemiologic evidence of carcinogenesis is essentially nonexistent for HPV 90, (4) and determining its carcinogenic potential is very challenging from an epidemiologic standpoint. (6) As a consequence, the current IARC classification considers HPV 90 as not classifiable (group 3) because of a lack of sufficient data. When compared with the extensive studies done on HR-HPV genotypes, HPV 90 is usually excluded from most of the large-scale HPV genotyping studies, even in some with relatively expanded HPV panels. Hence, its true prevalence and contribution to cervical intraepithelial lesions have not been established. The data regarding HPV 90 in North America are essentially absent.

To the best of our knowledge, the current study is the first investigation that centers on the prevalence, distribution, and cervical disease association of HPV 90 in a North American population. By using a comprehensive HPV detection panel in conjunction with DNA sequencing technology, we identified an unexpected, high prevalence of HPV 90 infection in 4% of our study population, which was even higher than that of many well-studied genotypes (ie, HPVs 11, 32, 33, 43, 14, 44, and 68b; Figure 2) in the same cohort. Scant studies from other continents reported a wide range of HPV 90 infection rates among a variety of patient populations. In West Germany, Kovacs et al (12) reported a 0.5% infection rate of HPV 90 in a routine screening population with multiple HPV infection. Chan saenroj et al (13) detected HVP 90 infection in 24 of 1662 Thai women (1.4%) at one hospital in Bangkok. A slightly higher infection rate (5 of 298; 1.7%) was observed in Kuwaiti women with abnormal Papanicolaou tests. (15) Among Turkish women who visited a gynecology clinic, Dursun et al (14) detected HPV 90 infection in 3% of them (12 of 403), a rate close to that in our current study. It is worth noting that those studies investigated HPV 90 infection by using different detection methods in very diverse populations, which makes the comparisons extremely difficult.

Our correlation study showed cytologic abnormalities in 3 of the 32 cases with HPV 90 infection, confirmed with available biopsies in 2 of them. The scant previous studies had contradictory observations regarding cervical disease associated with HPV 90 infection. Although HPV 90 infection was linked to 3 women with atypical squamous cells of undetermined significance and 2 with LSIL in a Kuwaiti study,[15] there were no cytologic abnormalities identified in women with HPV 90 infection in studies from Turkey (14) and Thailand. (13)

An outstanding feature of HPV 90 infection observed in our cohort was that the vast majority of women (31 of 32 cases; 96.8%) had HPV 90 as a single infecting agent. This was in contrast to a high rate of multiple HPV infection in this cohort (39%), with as many as 16 genotypes in a single sample. All 3 cases with cytologic abnormalities were infected with HPV 90 as the only genotype.

The abnormal cytology with concurrent single HPV genotype infection invites the consideration of HPV 90 as a hypothetical causal agent of cervical intraepithelial lesions. A reasonable argument is that these cervical lesions might be a residual effect of previous HR-HPV infection that had since cleared, in which HPV 90 acted only as a bystander. However, the absence of other HPV genotypes and presence of unequivocal viral cytopathic effect in concurrent cytologic and histologic specimens strongly argue against the bystander notion. In an effort to further explore the possible association, we performed ISH tests for HR-HPV on available specimens from cervical biopsies or loop electrosurgical excision procedure in the women with HPV 90 infection. In the 3 women with cytologic abnormalities, there was no HR-HPV detected by HPV-ISH in the 2 women with available tissues. These results suggest HPV 90 may not be a truly noncarcinogenic genotype as conventionally believed, and may be a causative agent for cervical intraepithelial lesions.

It is unknown whether HPV 90 causes cervical lesions in the same manner as the well-studied HR-HPV genotypes. Because most of the women with single HPV 90 infection do not develop cervical dysplasia, the pathogenesis may be multifactorial and include host immunological status, viral mutation, and other coexisting infections. As mentioned earlier, HPV E6-induced degradation of p53 is thought to be an essential activity by which HR-HPV contributes to cervical cancer development. It was recently demonstrated in Western blot assay that the K16N mutation of HPV 90 E6 enables it to fully degrade p53. This mutated form also degraded p53 as effectively as HPV 16 E6 did in a single-transfected cell assay. (16) It is possible that the HPV 90 associated with the 3 cases of cervical intraepithelial lesions might not be the wild type of HPV 90, but rather a potent HPV 90 mutant that may act as powerfully as HPV 16 in tumorigenesis. Unfortunately, we could not discriminate the wild type from a mutant HPV 90 in the current study, and further investigation is certainly warranted.

With the advances in molecular techniques now more accessible to pathology laboratories, we are able to expand our search and characterization of lesser-known HPV genotypes. Although it might not be cost-effective to apply comprehensive HPV genotyping panels as a screening method in the general population, they may be valuable in monitoring the emergence of previously underrecognized HPV genotypes of clinical importance. (1,3,17) Moreover, as HPV vaccination becomes a widespread common practice, HPV genotype changes in our population will become more relevant. Understanding the potentially changing trends in HPV composition will help guide the development of appropriate preventative and therapeutic strategies in the emerging post-HPV vaccination era.

Our results should be interpreted with caution for several reasons. First, our cohort consists of a high-risk, underserved population, with 6.9% of the patients having high-grade intraepithelial or above cervical lesions. HPV 90 could be overrepresented in this particular population. In addition, HPV 90 is a relatively rare genotype, and the number of cases in this study was less than desirable to achieve a statistical conclusion. As well, this is a cross-sectional study with a relatively short period of follow-up, owing to the difficulties in long-term follow-up of the patients in the group. And lastly, there were no cervical cancer cases in our cohort for analyzing the oncogenic effect of this particular HPV genotype.

To the best of our knowledge, this is the first study that investigated the prevalence and disease association of HPV 90 with a comprehensive HPV genotyping panel, DNA sequencing, and ISH techniques in a North American population. In this study, we demonstrated the prevalence of HPV 90 infection and its association with cervical intraepithelial lesions in an underserved, inner-city Latino population. The association raises the possibility of HPV 90 as a causative agent for cervical intraepithelial lesions in some women. The prevalence and oncogenic role of HPV90 may have been underestimated in this study because of the relatively low infection prevalence and lack of follow-up. Further study is needed to determine the prevalence and distribution of HPV 90 in the general population, as well as its oncogenic effect in cervical dysplasia and cancer.

CAPTION: Figure 1. Distribution of human papillomavirus (HPV) genotypes in cytology diagnostic categories demonstrates an unexpectedly high prevalence of HPV 90 (4%). Approximately 9.4% of the women infected with HPV 90 had abnormal cytology in the same specimen. Abbreviations: ASC-H, atypical squamous cells, cannot exclude high-grade intraepithelial lesion; ASCUS, atypical squamous cells of uncertain significance; HSIL, high-grade intraepithelial lesion; LSIL, low- grade intraepithelial lesion; LSIL-H, low-grade intraepithelial lesion, cannot exclude high-grade intraepithelial lesion; NILM, negative for intraepithelial lesion.

Caption: Figure 2. Microphotographs show an unequivocal human papillomavirus (HPV) cytopathic effect on Papanicolaou test (A) and follow-up biopsy (C, hematoxylin-eosin stain) in a woman infected with HPV 90. Atypical squamous cells cannot rule out high-grade intraepithelial lesion was observed on Papanicolaou test in another case of HPV 90 infection (B). No high-risk HPV was detected on concurrent biopsies by in situ hybridization (D) (original magnifications X600 [A and B] and X200 [C and D]).

We thank Dr Woo-Chul Moon, MD, PhD; Dr Myung-Ryul Oh, PhD; and Dr Jin-Kyung Lee, PhD, for sponsoring the human papillomavirus genotyping tests, and Philip Randall, MBA, for scientific editing of the manuscript. Mr Randall's time and effort are funded through the Department of Pathology and Genomic Medicine at The Methodist Hospital.

References

(1.) Garnett GP, Waddell HC. Public health paradoxes and the epidemiological impact of an HPV vaccine. J Clin Virol. 2000;19(1-2):101-111.

(2.) Schiffman M, Glass AG, Wentzensen N, Rush BB, Castle PE, Scott DR, et al. A long-term prospective study of type-specific human papillomavirus infection and risk of cervical neoplasia among 20,000 women in the Portland Kaiser Cohort Study. Cancer Epidemiol Biomarkers Prev. 2011;20(7):1398-1409.

(3.) Wentzensen N, Schiffman M, Dunn T, Zuna RE, Gold MA, Allen RA, et al. Multiple human papillomavirus genotype infections in cervical cancer progression in the study to understand cervical cancer early endpoints and determinants. Int J Cancer. 2009;125(9):2151-2158.

(4.) International Agency for Research on Cancer. Human Papillomaviruses. Vol. 100B. Lyon, France: International Agency for Research on Cancer; 2009.

(5.) Solomon D, Davey D, Kurman R, Moriarty A, O'Connor D, Prey M, et al. The 2001 Bethesda System: terminology for reporting results of cervical cytology. IAMA. 2002;287(16):2114-2119.

(6.) Schiffman M, Clifford G, Buonaguro FM. Classification of weakly carcinogenic human papillomavirus types: addressing the limits of epidemiology at the borderline. Infect Agent Cancer. 2009;4:1-8.

(7.) Daf S, Jena L, Kumar S. Comparative phylogenetic analysis of E6 and E7 proteins of different 42 strains of HPV. JK Sci. 2010;12(1):6-10.

(8.) Feoli-Fonseca JC, Oligny LL, Filion M, Simard P, Russo PA, Yotov WV. JC9813-A putative novel human papillomavirus identified by PCR-DS. Biochem Biophys Res Commun. 1998;250(1):63-67.

(9.) Klaassen CH, Prinsen CF, de Valk HA, Horrevorts AM, Jeunink MA, Thunnissen FB. DNA microarray format for detection and subtyping of human papillomavirus. J Clin Microbiol. 2004;42(5):2152-2160.

(10.) Terai M, Burk RD. Identification and characterization of 3 novel genital human papillomaviruses by overlapping polymerase chain reaction: candHPV89, candHPV90, and candHPV91. J Infect Dis. 2002;185(12):1794-1797.

(11.) Migaldi M, Pecorari M, Forbicini G, Nanni N, Grottola A, Grandi T, et al. Low prevalence of human papillomavirus infection in the healthy oral mucosa of a Northern Italian population. J Oral Pathol Med. 2012;41(1):16-20.

(12.) Kovacs K, Varnai AD, Bollmann M, Bankfalvi A, Szendy M, Speich N, et al. Prevalence and genotype distribution of multiple human papillomavirus infection in the uterine cervix: a 7.5-year longitudinal study in a routine cytology-based screening population in West Germany. J Med Virol. 2008;80(10):1814-1823.

(13.) Chansaenroj J, Lurchachaiwong W, Termrungruanglert W, Tresukosol D, Niruthisard S, Trivijitsilp P, et al. Prevalence and genotypes of human papillomavirus among Thai women. Asian Pac J Cancer Prev. 2010;11(1):117-122.

(14.) Dursun P, Senger SS, Arslan H, Kuscu E, Ayhan A. Human papillomavirus (HPV) prevalence and types among Turkish women at a gynecology outpatient unit. BMC Infect Dis. 2009;9(191):1-6.

(15.) Al-Awadhi R, Chehadeh W, Jaragh M, Al-Shaheen A, Sharma P, Kapila K. Distribution of human papillomavirus among women with abnormal cervical cytology in Kuwait. Diagn Cytopathol. 2013;41(2):107-114.

(16.) Fu L, Van Doorslaer K, Chen Z, Ristriani T, Masson M, Trave G, et al. Degradation of p53 by human Alphapapillomavirus E6 proteins shows a stronger correlation with phylogeny than oncogenicity. PLoS One. 2010;5(9):e12816.

(17.) Schiffman M, Castle PE. The promise of global cervical-cancer prevention. N Engl J Med. 2005;353(20):2101-2104.

Gabriela Quiroga-Garza, MD; Haijun Zhou, MD, PhD; Dina R. Mody, MD; Mary R. Schwartz, MD; Yimin Ge, MD

Accepted for publication January 15, 2013.

Published as an Early Online Release February 20, 2013.

From the Department of Pathology and Genomic Medicine, The Methodist Hospital, Houston, Texas (Drs Quiroga-Garza, Zhou, Mody, Schwartz, and Ge); and the Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, New York (Drs Mody and Ge).

The authors have no relevant financial interest in the products or companies described in this article.

Presented in part at the 105th Annual Meeting of the United States & Canadian Academy of Pathology in 2012; Vancouver, Canada.

Reprints: Yimin Ge, MD, Department of Pathology and Genomic Medicine, The Methodist Hospital, 6565 Fannin St, Suite M227, Houston, TX 77030 (e-mail: yge@tmhs.org).

Clinicopathologic Features of Human Papillomavirus 90 (HPV 90)
Infection

Case No.      Age, y    Colposcopy Findings

     1          40      NP
     2          84      NP
     3          29      Acetowhite
     4          64      NP
     5          50      NP
     6          32      Thin plaque, acetowhite
     7          70      NP
     8          33      Acetowhite, abnormal vessel,
                        cervix friable
     9          31      NP
    10          35      Negative
    11          35      Ectropion, squamous metaplasia
    12          50      Negative
    13          24      Squamous metaplasia
    14          63      NP
    15          24      HPV changes at 6:00
    16          49      Acetowhite
    17          42      Mosaicism
    18          40      Negative
    19          26      Squamous metaplasia
    20          36      Acetowhite, mosaicism
    21          37      Acetowhite
    22          27      NP
    23          38      Negative
    24          28      NP
    25          25      Acetowhite
    26          26      Acetowhite
    27          42      NP
    28          33      NP
    29          23      Acetowhite
    30          39      Punctations
    31          43      Negative
    32          35      Negative

Case No.      Age, y     Papanicolaou      HPV Genotype
                             Test

     1          40           NILM               90
     2          84           NILM               90
     3          29           NILM               90
     4          64           NILM               90
     5          50           NILM               90
     6          32           NILM               90
     7          70           NILM               90
     8          33           NILM               90

     9          31           NILM               90
    10          35           NILM               90
    11          35           NILM               90
    12          50           NILM               90
    13          24           LSIL               90
    14          63           NILM               90
    15          24           NILM               90
    16          49           NILM               90
    17          42           NILM               90
    18          40           NILM               90
    19          26           NILM               90
    20          36           NILM               90
    21          37           NILM               90
    22          27           NILM               90
    23          38           NILM               90
    24          28           LSIL               90
    25          25           NILM               90
    26          26           NILM               90
    27          42          LSIL-H              90
    28          33           NILM               90
    29          23           NILM               90
    30          39           NILM               90
    31          43           NILM               90
    32          35           NILM             90, 13

Case No.      Age, y    HR-HPV ISH

     1          40      NTA
     2          84      NTA
     3          29      NTA
     4          64      NTA
     5          50      NTA
     6          32      Negative
     7          70      NTA
     8          33      Positive

     9          31      Negative
    10          35      NTA
    11          35      NTA
    12          50      NTA
    13          24      NTA
    14          63      NTA
    15          24      Negative
    16          49      Negative
    17          42      NTA
    18          40      NTA
    19          26      Negative
    20          36      NTA
    21          37      NTA
    22          27      Negative
    23          38      Negative
    24          28      Negative
    25          25      NTA
    26          26      Negative
    27          42      Negative
    28          33      Positive
    29          23      Negative
    30          39      Negative
    31          43      NTA
    32          35      NTA

Abbreviations: HR, high risk; ISH, in situ hybridization; LSIL,
low-grade intraepithelial lesion; LSIL-H, low-grade
intraepithelial lesion, cannot exclude high grade intraepithelial
lesion; NILM, negative for intraepithelial lesion; NP, not performed;
NTA, no tissue available.


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Title Annotation:human papillomavirus
Author:Quiroga-Garza, Gabriela; Zhou, Haijun; Mody, Dina R.; Schwartz, Mary R.; Ge, Yimin
Publication:Archives of Pathology & Laboratory Medicine
Article Type:Report
Geographic Code:1USA
Date:Nov 1, 2013
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