Unexpected high prevalence of HPV 90 infection in an underserved population: is it really a low-risk genotype?
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.
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)
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.
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, 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.
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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: email@example.com).
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.
Please note: Illustration(s) are not available due to copyright restrictions.
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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|
|Date:||Nov 1, 2013|
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