Aaron Shibemba

Allen C Bateman

BJ Kocjan

C Firnhaber

Carla J Chibwesha

Dirk P Dittmer

F Miething

FX Bosch

Groesbeck P Parham

I Cannavo

J Ferlay

JM Walboomers

JS Smith

K Bowa

Katundu Katundu

L Denny

M Steinau

Mulindi Mwanahamuntu

NG Campos

P Guan

Pascal Polepole

S Huang

VV Sahasrabuddhe

Virology Journal

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Bateman et al. Virology Journal  (2015) 12:2 DOI 10.1186/s12985-014-0234-8SHORT REPORT Open AccessIdentification of human papillomaviruses fromformalin-fixed, paraffin-embedded pre-cancer andinvasive cervical cancer specimens in Zambia: across-sectional studyAllen C Bateman1,2*, Katundu Katundu1, Pascal Polepole3, Aaron Shibemba3, Mulindi Mwanahamuntu1,3,Dirk P Dittmer4, Groesbeck P Parham1,3,5 and Carla J Chibwesha1,5AbstractBackground: The most common human papillomavirus (HPV) genotypes isolated from cervical cancer in selectAfrican countries are HPV-16, HPV-18, HPV-35, and HPV-45, but the most common genotypes in Zambia areunknown. The overall objective of this study was to assess the potential impact of current HPV vaccines inpreventing cervical cancer in Zambia, by determining the combined prevalence of HPV-16 and/or HPV-18 ininvasive cervical cancer (ICC) and high-grade pre-cancer [cervical intraepithelial neoplasia 2 or 3 (CIN2/3)] cases.Findings: We compared DNA extraction techniques to determine which assay performs well in the Zambiancontext, where unbuffered formalin is used to fix specimens. We then tested specimens with the Abbott RealTimeHigh-Risk HPV test to estimate the prevalence of HPV-16/18 in formalin-fixed, paraffin-embedded ICC and CIN2/3specimens. DNA extraction using heat (without xylene) was more successful than xylene-based extraction. Over80% of specimens tested using heat extraction and the Abbott RealTime HPV test were positive for HPV. HPV-16and/or HPV-18 were identified in 65/93 (69.9%) ICC specimens positive for HPV and in 38/65 (58.5%) CIN2/3specimens positive for HPV.Conclusions: To our knowledge this is the first report to identify HPV genotypes in cervical cancers in Zambia. Acombined HPV-16/18 prevalence of 69.9% in ICC specimens suggests that current vaccines will be highly protectiveagainst cervical cancer in Zambia.Keywords: Cervical cancer, Formalin-fixed paraffin-embedded, Human papillomavirus, ZambiaFindingsBackgroundCervical cancer is the second most common cancer inwomen worldwide [1] and is the most common femalemalignancy referred to the University Teaching Hospital(UTH) in Lusaka [2]. Human papillomaviruses (HPVs)are responsible for over 90% of cervical cancers [3,4].Fifteen HPV types have been associated with cervicalcancer [5] and are collectively known as oncogenic or* Correspondence: bateman.allen@gmail.com1Centre for Infectious Disease Research in Zambia, Plot 5032 Great NorthRoad, Lusaka, Zambia2Department of Medicine, UNC School of Medicine, University of NorthCarolina at Chapel Hill, Chapel Hill, North Carolina, USAFull list of author information is available at the end of the article© 2015 Bateman et al.; licensee BioMed CentrCommons Attribution License (http://creativecreproduction in any medium, provided the orDedication waiver (http://creativecommons.orunless otherwise stated.high-risk types. The two most common are HPV-16 andHPV-18, which are associated with 60–70% of cervicalcancer worldwide [5]. HPV-16 and HPV-18 are the twogenotypes included in currently licensed HPV vaccines(Cervarix® and Gardasil®). Cost-effectiveness modelingestimates that HPV-16/18 vaccines can decrease the life-time risk of cervical cancer in women in Central andEastern Africa by 36–45% [6]. However, these vaccinescan only prevent cervical cancer in areas where HPV-16and HPV-18 are primary causes. The most commongenotypes isolated from cervical cancer in select Africancountries are HPV-16, HPV-18, HPV-35, and HPV-45[7,8], but common genotypes in Zambia are unknown.al. This is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andiginal work is properly credited. The Creative Commons Public Domaing/publicdomain/zero/1.0/) applies to the data made available in this article,Bateman et al. Virology Journal  (2015) 12:2 Page 2 of 4Studies of pre-cancer specimens in South Africa andZambia reported a wide diversity of HPV genotypes [9,10],generating concern that current HPV vaccines might nothave a large preventive effect in Zambia. However, mostpre-cancerous lesions do not develop into cancer, and todetermine if HPV-16 and HPV-18 cause invasive cervicalcancer (ICC) requires testing ICC specimens.To assess the impact that current vaccines can have inpreventing cervical cancer in Zambia, we first comparedDNA extraction techniques to determine which performedbetter in the Zambian context. We then estimated thecombined prevalence of HPV-16 and/or HPV-18 informalin-fixed, paraffin-embedded (FFPE) ICC and cervicalintraepithelial neoplasia 2 or 3 (CIN 2/3) specimens fromUTH. Our main outcomes were the percentages of speci-mens positive for HPV-16 and/or HPV-18.MethodsUTH and the adjacent Cancer Diseases Hospital in Lusakaare the primary centers for cancer diagnosis and treatmentin Zambia. FFPE cervical specimens obtained during rou-tine clinical care from 2007–2012 and stored at the UTHPathology Department were used for this study. Unbuf-fered formalin is used for routine specimens at UTH,which likely leads to more degraded DNA than if bufferedformalin were used [11]. Specimens were anonymized atthe time of sectioning, and HIV status was unavailable. Tominimize the potential for DNA contamination, gloveswere worn throughout the procedure, the microtome andsurrounding station were cleaned with DNAzap (LifeTechnologies, Carlsbad, CA, USA) or bleach before begin-ning work and between each specimen, and a new micro-tome blade and pair of gloves was used for each block.Non-HPV-containing specimens (spleen or liver) weresectioned every 15 specimens and tested; all of thesecontrols were negative for HPV, as expected. The first andlast sections of each cervical specimen block were 3 μmthick and were mounted on slides, stained with H&E, andreviewed by a pathologist at UTH to confirm the diagno-sis. The middle sections (n = 3 to 9, depending on thetissue size) were 10 μm thick and were placed into 2.0 mlScrew Cap Micro Tubes (Sarstedt, Nümbrecht, Germany)and taken to the CIDRZ Central Laboratory for DNAextraction and HPV testing.We compared two extraction techniques: heat-dependent and xylene-dependent. The heat extractiontechnique was adapted from Steinau et al. [12]. To eachtube, 180 μl of ATL lysis buffer from a DNeasy Bloodand Tissue Kit (Qiagen, Valencia, CA) was added andheated at 99°C for 30 minutes. Proteinase K (20 μl) wasadded, tubes were incubated at 56°C with shaking at1,000 rpm for 16 hours, 400 μl of Buffer AL:ethanolmixture was added, and the DNA was further purifiedwith the Qiagen kit.The xylene extraction technique was adapted fromKocjan et al. [13]. To each specimen, 1.2 ml xylene wasadded, followed by vortexing and centrifugation at max-imum speed for 5 minutes. The supernatant was removed,1.6 ml of 100% ethanol was added to the pellet, followedby vortexing and centrifugation for 5 minutes. The speci-men was washed two additional times and the pellet wasair-dried for 15 minutes and resuspended in 180 μl of buf-fer ATL from a QIAamp DNA Mini Kit (Qiagen). Protein-ase K (20 μl) was added, samples were incubated overnightat 56°C with shaking at 1,000 rpm, 200 μl of buffer AL wasadded, and DNA was further purified with the Qiagen kit.For both extraction techniques, DNA was recovered in asingle elution step with 110 μl of AE solution.Nucleic acid concentrations were determined using aNanodrop ND-1000 spectrophotometer (Thermo FisherScientific, Wilmington, DE, USA), and DNA extracts werefrozen at −80°C until further analysis.Extracted DNA was tested using the Abbott RealTimeHigh Risk HPV assay (Abbott Laboratories, Abbott Park,IL, USA), which uses real-time PCR to amplify and de-tect 14 high-risk genotypes [14]. RealTime also differen-tiates between HPV-16, HPV-18, and non-HPV16/18genotypes (other high-risk). RealTime contains primersand probes specific for the human β-globin gene, to moni-tor DNA extraction and amplification. A cycle threshold(Ct) value of <35 was considered positive for each of theRealTime channels.Data were entered into Microsoft Excel™ (Redmond, WA,USA), cleaned with SAS™ version 9.2 (SAS Institute Inc.,Cary, NC, USA), and analyzed using SAS™ and Open Epi(www.openepi.com). p < 0.05 was considered significant.ResultsTo determine the most effective DNA extraction tech-nique, 27 FFPE specimens (aged 3–5 years old) were ex-tracted using the two techniques. The mean nucleic acidconcentration (standard deviation) following heat extrac-tion was 117.4 ng/μl (85.4 ng/μl), while that followingxylene extraction was 38.2 ng/μl (35.9 ng/μl) (p < 0.0001,Wilcoxon signed-rank test).The 27 FFPE specimens were tested with the RealTimeassay. RealTime successfully identified HPV in the vastmajority of specimens: one or more HPV was identifiedin 23/27 with heat extraction and 21/27 with xyleneextraction (Table 1). In addition, heat extraction resultedin lower Ct values than xylene extraction (Table 1). There-fore, the remaining FFPE specimens were tested usingheat extraction.Including the initial 27 specimens, 189 FFPE speci-mens were tested, including 114 ICC (109 squamous cellcarcinoma and 5 adenocarcinoma) and 75 CIN 2/3. Of114 ICC specimens tested, 88% were valid results (Table 2).Of the valid ICC results, HPV was identified in 93 (93%),Table 1 RealTime results and Ct values from DNAextracted from ICC and CIN2/3 specimens using heat orxylene treatmentHeat XyleneRealTime result n nInvalida 2 2No HPV detected 2 4Single HPV infection 18 19Dual HPV infections 4 2Triple HPV infections 1 0Total 27 27Abbott Fluorophore/channeln MeanCt valuen MeanCt valuep valuebβ-globin internal control 25 27.7 24 30.6 <0.001HPV-16 14 23.8 12 25.3 0.349HPV-18 3 26.3 3 29.9 0.222other HR HPV 13 28.1 9 31.0 0.156aβ-globin internal control was either negative or had a Ct count >35.bStudent’s t-test.Results represent three separate experiments of 9 specimens each (27 FFPEspecimens total; 17 ICC and 10 CIN 2/3). Ct values above 35 were excluded.Bateman et al. Virology Journal  (2015) 12:2 Page 3 of 4including 78 single infections and 15 co-infections. Includ-ing specimens with multiple HPV genotypes identified,52% of specimens were positive for HPV-16 and 25% werepositive for HPV-18; together, 65/93 (70%; 95% CI: 60%–78%) were positive for HPV-16 and/or HPV-18 (Table 2).Three (60%) of the adenocarcinoma specimens had validresults; of these, HPV-16 and/or HPV-18 was identified in2/3 (67%).Of 75 CIN 2/3 specimens tested, 92% were validresults (Table 2) and HPV was identified in 65/69 (94%),including 48 single infections and 17 co-infections. Includ-ing specimens with multiple HPV genotypes identified,Table 2 HPV results of ICC and CIN2/3 specimens testedby heat extraction and the RealTime assayICC CIN 2/3n Prevalence(95% CI)n Prevalence(95% CI)Total tested 114 -- 75 --Total with valid resultsa 100 -- 69 --Positive for any HPVb 93 -- 65 --HPV-16 positive 48 51.6% (41.6–61.5) 36 55.4% (43.3–66.8)HPV-18 positive 23 24.7% (17.1–34.4) 5 7.7% (3.3–16.8)Other HR HPVpositive38 40.9% (31.4–51.0) 43 66.2% (54.0–76.5)HPV-16 and/or HPV-18positive65 69.9% (59.9–78.3) 38 58.5% (46.3–69.6)aInvalid results mean the β-globin internal control was not reactive or had a Ctvalue of >35.bIncluding specimens with multiple HPV types identified. The number ofspecimens that were positive for HPV was used as the denominator forprevalence estimates.38/65 (59%; 95% CI: 46%–70%) of specimens were positivefor HPV-16 and/or HPV-18 (Table 2).DiscussionTo our knowledge this is the first report to identify HPVgenotypes in ICC in Zambia. A combined HPV-16/18prevalence of 69.9% in ICC specimens suggests thatcurrent vaccines will be protective against cervical can-cer in Zambia, and that the proportion of ICC attribut-able to HPV-16/18 in Zambia is similar to that in SouthAfrica, Kenya, and the overall worldwide proportion [5].The combined HPV-16/18 prevalence in CIN 2/3 speci-mens (58.5%) was lower than that in ICC specimens, butthe difference was not significant (p = 0.14, two-tailed chi-square test). This is consistent with previous studies inZambia and South Africa that reported a wide diversity ofHPV types in pre-cancer samples [9,10]. The heat extrac-tion technique performed better than the xylene extrac-tion technique in our study, which is consistent withprevious studies that report that DNA extraction withoutxylene is more successful than with xylene [12,15]. TheAbbott RealTime assay identified HPV in over 80% ofthe specimens. The use of unbuffered formalin at UTHlikely means that the Abbott RealTime (which has a 150base pair amplicon) or other assays with relatively shortamplicons will be most sensitive for identifying HPV inthis context.Strengths of this study include the successful develop-ment of assays to identify HPV from FFPE specimens inthe Zambian context and the ability to estimate theimpact of HPV vaccines on both pre-cancer and cancer.Weaknesses include the relatively small number of spec-imens sampled from a single referral center. However,because UTH is the primary center for cancer diagnosisand treatment in Zambia, we believe that our estimateof the prevalence of HPV-16/18 in cervical cancers isthe best available for the country to date.With an estimated 69.9% of ICC specimens at UTHcontaining HPV-16 and/or HPV-18, current HPV vaccinescan have a substantial preventive impact on cervical cancerin Zambia. These data provide a rationale to advocate fordelivery of HPV vaccines throughout Zambia. In addition,the methods described here can be used to examine therole of HPV in other cancers in Zambia, such as head andneck cancers [16,17], and the combined prevalence ofHPV16/18 in ICC can be used to examine the effect ofHPV vaccines, by testing FFPE specimens in the future.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsACB, GPP, DPD, and CC conceived of and designed the study; ACB, KK, PP,AS, and MM were involved in the acquisition of data; ACB, CC, and GPPanalyzed and interpreted the results and drafted the article; all authorscontributed to revising the article and approving the final submitted version.Bateman et al. Virology Journal  (2015) 12:2 Page 4 of 4AcknowledgementsWe thank Chilufya Kasongamulilo for technical assistance with the AbbottRealTime HR HPV assay.This study was funded by an NCI supplemental award to the UNCLineberger Cancer Center (CA16086) and a seed grant from the Program inGlobal Oncology of the UNC Lineberger Cancer Center. Investigator supportwas provided through Fogarty International Center Award R25TW009340 tothe UNC Hopkins Morehouse Tulane Fogarty Global Health Fellows Program,National Cancer Institute Award 1D43CA153784, and NIH grants DE018304and CA019014. No funding sources had involvement in any aspect of thestudy, including the study design, the collection, analysis and interpretationof data, the writing of the report, or the decision to submit the article forpublication.Author details1Centre for Infectious Disease Research in Zambia, Plot 5032 Great NorthRoad, Lusaka, Zambia. 2Department of Medicine, UNC School of Medicine,University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.3University of Zambia Teaching Hospital, Lusaka, Zambia. 4Program in GlobalOncology, UNC Lineberger Comprehensive Cancer Center and School ofMedicine, UNC, Chapel Hill, North Carolina, USA. 5Department of Obstetricsand Gynecology, UNC School of Medicine, UNC, Chapel Hill, North Carolina,USA.Received: 17 October 2014 Accepted: 22 December 2014References1. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates ofworldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer.2010;127:2893–917.2. Bowa K, Wood C, Chao A, Chintu C, Mudenda V, Chikwenya M. A review ofthe epidemiology of cancers at the University Teaching Hospital, Lusaka,Zambia. Trop Doct. 2009;39:5–7.3. Bosch FX, Lorincz A, Munoz N, Meijer CJ, Shah KV. The causal relationbetween human papillomavirus and cervical cancer. J Clin Pathol.2002;55:244–65.4. Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV,et al. Human papillomavirus is a necessary cause of invasive cervical cancerworldwide. J Pathol. 1999;189:12–9.5. Smith JS, Lindsay L, Hoots B, Keys J, Franceschi S, Winer R, et al. Humanpapillomavirus type distribution in invasive cervical cancer and high-gradecervical lesions: a meta-analysis update. Int J Cancer. 2007;121:621–32.6. Campos NG, Kim JJ, Castle PE, Ortendahl JD, O’Shea M, Diaz M, et al. Healthand economic impact of HPV 16/18 vaccination and cervical cancerscreening in Eastern Africa. Int J Cancer. 2012;130:2672–84.7. Guan P, Howell-Jones R, Li N, Bruni L, de Sanjose S, Franceschi S, et al.Human papillomavirus types in 115,789 HPV-positive women: a meta-analysisfrom cervical infection to cancer. Int J Cancer. 2012;131:2349–59.8. Denny L, Adewole I, Anorlu R, Dreyer G, Moodley M, Smith T, et al. Humanpapillomavirus prevalence and type distribution in invasive cervical cancerin sub-Saharan Africa. Int J Cancer. 2014;134:1389–98.9. Sahasrabuddhe VV, Mwanahamuntu MH, Vermund SH, Huh WK, Lyon MD,Stringer JS, et al. Prevalence and distribution of HPV genotypes amongHIV-infected women in Zambia. Br J Cancer. 2007;96:1480–3.10. Firnhaber C, Zungu K, Levin S, Michelow P, Montaner LJ, McPhail P, et al.Diverse and high prevalence of human papillomavirus associated with asignificant high rate of cervical dysplasia in human immunodeficiency virus-infected women in Johannesburg, South Africa. Acta Cytol. 2009;53:10–7.11. Miething F, Hering S, Hanschke B, Dressler J. Effect of fixation to thedegradation of nuclear and mitochondrial DNA in different tissues.J Histochem Cytochem. 2006;54:371–4.12. Steinau M, Patel SS, Unger ER. Efficient DNA extraction for HPV genotypingin formalin-fixed, paraffin-embedded tissues. J Mol Diagn. 2011;13:377–81.13. Kocjan BJ, Seme K, Poljak M. Comparison of the Abbott RealTime High RiskHPV test and INNO-LiPA HPV Genotyping Extra test for the detection ofhuman papillomaviruses in formalin-fixed, paraffin-embedded cervicalcancer specimens. J Virol Methods. 2011;175:117–9.14. Huang S, Tang N, Mak WB, Erickson B, Salituro J, Li Y, et al. Principles andanalytical performance of Abbott RealTime High Risk HPV test. J Clin Virol.2009;45 Suppl 1:S13–7.15. Cannavo I, Loubatier C, Chevallier A, Giordanengo V. Improvement of DNAextraction for human papillomavirus genotyping from formalin-fixedparaffin-embedded tissues. Biores Open Access. 2012;1:333–7.16. Kocjan BJ, Maver PJ, Hosnjak L, Zidar N, Odar K, Gale N, et al. Comparativeevaluation of the Abbott RealTime High Risk HPV test and INNO-LiPA HPVGenotyping Extra test for detecting and identifying human papillomavirusesin archival tissue specimens of head and neck cancers. Acta DermatovenerolAlp Pannonica Adriat. 2012;21:73–5.17. Steinau M, Saraiya M, Goodman MT, Peters ES, Watson M, Cleveland JL,et al. Human papillomavirus prevalence in oropharyngeal cancer beforevaccine introduction, United States. Emerg Infect Dis. 2014;20:822–8.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color ﬁgure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submit

Identification of human papillomaviruses from formalin-fixed, paraffin-embedded pre-cancer and invasive cervical cancer specimens in Zambia: a cross-sectional study

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Identification of human papillomaviruses from formalin-fixed, paraffin-embedded pre-cancer and invasive cervical cancer specimens in Zambia: a cross-sectional study

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