Journal of Cytology
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ORIGINAL ARTICLE  
Year : 2011  |  Volume : 28  |  Issue : 4  |  Page : 159-164
Defining the validity of classical and non-classical cellular changes indicative of low-grade squamous intraepithelial lesion encompassing human papillomavirus infection in relation to human papillomavirus deoxyribonucleic acid testing


1 Department of Cytology, Institute of Cytology and Preventive Oncology (DHR), Noida, UP, India
2 Department of Molecular Oncology, Institute of Cytology and Preventive Oncology (DHR), Noida, UP, India

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Date of Web Publication20-Oct-2011
 

   Abstract 

Background: Human papillomavirus (HPV) infection as of now has been beyond doubt to be the causative agent for cervical carcinoma. Its morphological identification in Pap smear is important.
Aim: To define the validity of classical and non-classical cellular changes indicative of low-grade squamous intraepithelial lesion (SIL) encompassing HPV infection in relation to positivity for 'high risk' HPV16 as well as for 'low risk' HPV6/11.
Materials and Methods : A total of 3000 Papanicolaou smears were screened, of which 150 were reported as low grade-SIL encompassing HPV infection (LSIL-HPV). Subsequently cervical scrapes from these 150 subjects, along with equal number of normal women as controls, were collected and processed for HPV deoxy-ribonucleic acid testing by polymerase chain reaction (PCR).
Results: On the basis of cytomorphological characteristics in Pap smears, HPV infection were categorized into the following two groups: Classical (koilocytic) changes (CC) encountered in 30 women and non-classical changes (NCC) encountered in 120 women. It was observed that 21 (70%) CC and 46 (38.3%) NCC of HPV infection were positive for HR-HPV16; however only 12 cases (10%) of NCC and two cases (6.6%) of CC were positive for LR-HPV 6/11. Majority (41.7%) of HPV positive cases were reported in the age group of 25 to 30 years and HPV positivity decreased with the increasing age.
Conclusion: Classical cellular changes are not the only diagnostic features for HPV infection in Pap smear, non-classical diagnostic features also support the diagnosis of HPV infection and may be positive for HR-HPV16.

Keywords: HPV deoxyribonucleic acid; koilocytes; LSIL-HPV; Pap smear; PCR

How to cite this article:
Kashyap V, Hedau S, Bhambhani S. Defining the validity of classical and non-classical cellular changes indicative of low-grade squamous intraepithelial lesion encompassing human papillomavirus infection in relation to human papillomavirus deoxyribonucleic acid testing. J Cytol 2011;28:159-64

How to cite this URL:
Kashyap V, Hedau S, Bhambhani S. Defining the validity of classical and non-classical cellular changes indicative of low-grade squamous intraepithelial lesion encompassing human papillomavirus infection in relation to human papillomavirus deoxyribonucleic acid testing. J Cytol [serial online] 2011 [cited 2020 Nov 29];28:159-64. Available from: https://www.jcytol.org/text.asp?2011/28/4/159/86340



   Introduction Top


The Papanicolaou smear is a routine screening method for detecting cervical cancer and squamous intraepithelial lesions (SILs) including cytologic changes induced by human papillomavirus (HPV) infection. [1] HPV is considered as the principal sexually transmitted causal agent in the development of cancer of the uterine cervix and in women incidence of HPV infection of the cervix varies with the age. [2] HPV infection can be diagnosed by specific morphological changes in the host cells, by the virus infection, through cytology, histopathology, colposcopy, immunocytochemistry and by molecular methods. Non-oncogenic HPVs as well as oncogenic HPVs have the capacity to induce cytologic changes, but only integrated HPVs are able to produce the oncogenic transformation of the cells. [3] In Papanicolaou (Pap) smears diagnosis of HPV infection is based on classical changes (CC) (koilocyte) as well as on non-classical cellular changes (NCC). Diagnostically, koilocyte is an excellent indicator of HPV infection where intermediate and superficial cells show paranuclear halo with eccentric single or binucleated hyperchromatic nuclei with mild irregularity of outline. These are characteristic of the last stage of viral replication and infective genome released as koilocytes are shed, so koilocyte is regarded as the most pathognomic feature with high degree of specificity. However, it is not always present in HPV infected cells, especially those from dysplastic lesion. [4] Non-classical features, which are reflected in cells like rounding of cell margin, mild nuclear hyperchromasia and eccentric nuclei, might be indicator for HPV infection. [5] With reliable techniques of polymerase chain reaction (PCR) and Hybrid Culture II, HPV deoxyribonucleic acid (DNA) can be readily amplified in cervical scrapes to confirm the HPV positivity. [6] The aim of this study was to assess the validity of cytomorphological CC and NCC of HPV infection in  Pap smear More Detailss with their positivity for high-risk HPV type16 and low risk HPV 6/11.


   Materials and Methods Top


Patient population

The patient population comprised of 3000 Pap smears collected from women, between the age of 21 to 65 years attending gynecologic out patient departments of Lok Nayak hospital, New Delhi. Cervical smears were obtained by scraping the cells from ectocervix with the help of an Ayre's spatula and immediately smeared the cellular material on a glass slide and fixed in ethanol. The Pap smears were stained according to method of Papanicolaou and classified as per The Bethesda System 2001. [7] Cases diagnosed as LSIL-HPV infection were specifically selected for this study and on the basis of cellular characteristics of HPV infection the cytological changes were categorized into the following two groups-CC (koilocytes) and NCC. CC were defined as presence of typical koilocytes, which were squamous cells with cytoplasmic condensation at the periphery giving a hollow central portion, and eccentrically placed dark nucleus. In NCC koilocytes were not seen however small rounded cells appeared in chain with unevenly distributed and heterogenous cytoplasm, nuclei were small pyknotic and slightly eccentric placed. Cervical scrapes of 150 such LSIL-HPV cases along with same number of scrapes from normal healthy women (controls), whose Pap smears were reported as normal and there was no evidence of CC or NCC of HPV infection, were collected in chilled phosphate buffer saline and stored in -20°C and further processed for HPV DNA analysis. Informed consent was obtained from all the patients and clinico-epidemiological details were taken from their clinical records. The age of 150 selected patients ranged from 25 to 45 years with mean age 34.3 years (SD ± 6.3). The patients were investigated by colposcopy and guided biopsies were done wherever required.

DNA extraction and detection of HPV DNA sequences by polymerase chain reaction

High molecular weight genomic DNA from freshly collected cervical scrapes were isolated using standard Proteinase K digestion, phenol chloroform extraction and ethanol precipitation method routinely being employed in our laboratory. [8],[9] The quality and concentration of DNA was measured either on an ethidium bromide-stained 1% agarose gel using Hind III-digested lambda marker [Figure 1]a or by standard spectrophotometric methods.
Figure 1: (a) Estimation of quantity and quality of phenol-chloroform extracted genomic DNA from cervical scrapes as visualised on an ethidium bromide-stained 1% agarose gel. Lane M: Hind III-digested ë-DNA molecular weight marker. Lane 1-10: Genomic DNA samples from cervical scrapes. (b) PCR amplification of HPV L-1 consensus showing the amplimer of 450 bp along with b-globin showing 268bp. Lane M: Hae III-digested öx174 DNA molecular weight marker. Lane 1: Positive control (plasmid DNA), Lane 2: HPV negative cell line (C33aDNA), Lane 3-9: Cervical scrape DNA samples. Lane 3-5,7 and 9 showing positive by HPV L-1 consensus PCR. (c) PCR amplification of HPV 16 showing the amplimer of 217 bp along with β-globin showing 268bp. Lane M: Hae III-digested öx174 DNA molecular weight marker. Lane 1: Positive control (plasmid DNA), Lane 2: HPV negative cell line (C33aDNA), Lane 3-8: Cervical scrape DNA samples. Lane 3,4,6 and 8 showing positive by HPV 16 PCR

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For the detection of HPV DNA, PCR methodology was employed using most common L1 consensus primers MY 09/11 primers derived from HPV genome. HPV 16 plasmid DNA or HeLa DNA served as positive controls, whereas HPV negative cell lines, C33a DNA or human placental DNA served as negative control. Amplification of b-globin gene served as internal controls to examine quality, integrity and successful amplification of cervical scrapes DNA.

Conventional polymerase chain reaction using L1 consensus MY 09/11 primers

Approximately, 100 to 200 hg genomic DNA was utilized for PCR according to the routinely followed protocol of our laboratory [10] on a DNA Engine Tetrad (MJ Research, USA). Detection of HPV was carried out using consensus primers (MY09/MY11) located within the conserved L 1 region of HPV genome (forward primer, 5'-GCM CAG GGW CAT AAY AAT GG-3', reverse primer 5'-CGT CCM ARR GGA WAC TGA TC-3' where M = A+C, W = A+T, Y = C+T, R = A+G). HPV 16, HPV 6 and HPV 11 typing was done by type-specific primers HPV 16 (F) 5'-AAG GCC AAC RAA TAG TCA C-3'; (R) 5'-CTG CTT TTA TAC TAA CCG G-3'; HPV 6 (F) 5'-TAG TGG GCC TAT GGC TCG TC-3'; (R) 5'-TCC ATT AGC CTC CAC GGG TG-3'; HPV 11 (F) 5'-GGA ATA CAT GCG CCA TGT GG-3'; (R) CGA GCA GAC GTC CGT CCT CG-3'. b-globin gene sequences (forward primer, 5'-GAA GAG CCA AGG ACA GGT AC-3', reverse primer, 5'-CAA CTT CAT CCA CGT TAC ACC - 3') were used as internal controls.

Briefly, the method involved a 25 ml reaction mixture containing 100 to 200 hg DNA, 10 mM Tris-Cl (pH 8.4), 50 mM KCl, 1.5 mM MgCl 2 , 12.5 mM of each dNTP (dATP, dCTP, dGTP and dTTP), 5pmoles of each oligonucleotide primer and 0.5U AmpliTaq Gold DNA polymerase (Perkin-Elmer Biosystems, Foster City, CA,USA). The temperature profile used for amplification constituted an initial denaturation at 95°C for 9 minutes followed by 39 cycles with denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds and extension at 72°C for 30seconds which was extended for 4 to 7 minutes in the final cycle. The oligonucleotide primers were either synthesised in an automated Applied Biosystems DNA Synthesizer (Model 381A, Applied Biosystems Inc., Foster City, CA, USA), and HPLC purified or commercially got synthesised from Microsynth GmbH, Balgach, Switzerland.

Statistical analysis

Chi square test was employed to find out the association between cytological changes of LSIL-HPV and HPV DNA test. All the diagnostic tests (sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated by applying the Epi-Info version 6.0 software.


   Results Top


A total of 3000 Pap smears from same number of women were screened and classified as per The Bethesda System 2001. [7] Amongst them 2780 (92.6%) reported as negative for intraepithelial lesion or malignancy, 20 (0.6%) atypical squamous cells, 175 (5.8%) low-grade SIL encompassing HPV infection in 150 (5%) and mild dysplasia in 25 (0.8%), 10 (0.3%) high grade SIL and 15 (0.5%) as malignant cells.

The cytomorphological characteristics of 150 cases of LSIL-HPV infection were broadly divided in two groups. Group I had CC of koilocytosis in 30 (20%) cases where Pap smears showed presence of koilocytes, which were epithelial squamous cells where cell margins get blurred; cytoplasm condensed at the periphery and was delineated into ecto and endoplasm. Central portion of the infected cells become hollow giving a wire loop appearance. The nucleus was hyperchromatic, enlarged and eccentrically placed with paranuclear halo on one side. Nuclei varied from single to binucleated [Figure 2]a- d.
Figure 2 (a-d): These image show cells with varying degree of cytoplasmic vacuolation and peripheral cytoplasmic thickening. Cytoplasm clearing is apparent. Nuclei are slightly large, cells are often binucleated and show hyperchromasia (koilocyte), (Pap stain, × 400)

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Out of 30 cases, 27 (90%) cases were HPV L 1 positive DNA where β-globin showed amplification of 268 bp product [Figure 1]b in all cases. The correlation between CC with HPV 16 positivity was found statistically significant (P<0.01). The sensitivity between LSIL-HPV and HPV DNA test was 25.7% (95%CI. 17.9-35.3), specificity of Pap test was 93.3% (95%CI-80.7-98.3), positive predictive value 90.0% (95%CI. 72.3-97.4) and negative predictive value was 35.0% (95%CI. 26.7-44.3) [Table 1]. Amongst 30 cases 21(70%) were positive for HPV 16, two (6.6%) were positive for HPV 6/11 and 4 (13.3%) were HPV L 1 positive but negative for HPV 6/11/16 which could be positive for any other HPV type. In PCR method HPV 16 plasmid or HPV 16 positive cervical tumor DNA and human placental DNA or HPV negative C33a DNA were used as positive and negative control respectively [Figure 1]c.
Table 1: Relation between cytology group and HPV DNA test


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Group-II had NCC of HPV infection in 120 (80%) cases, where cells showed early stage of infection. There were small rounded cells that appeared in chains and groups. Their cytoplasm was unevenly stained and texture too was heterogeneous, sometimes gave impression of a dried smear. Nuclei were small, pyknotic and slightly eccentrically placed [Figure 3]a-d. Out of 120 cases, 78 (65%) were HPV L1 positive DNA where b-globin showed amplification of 268 bp product in all cases. A total of 46 (38.3%) cases were positive for HPV 16, 12 (10%) were positive for HPV 6/11 and 20 (16.6%) were HPV L1 positive but negative for HPV 6/11/16 and could be positive for other HPV type. The correlation between CC with HPV 16 positivity was found statistically significant (P<0.01).The cytological changes predictive of infection by HPV showed a sensitivity of 31.3% (95%CI, 20.9-44.0), specificity of 89.2% (95%CI,79.9-94.6), positive predictive value of 70.0% (95%CI, 50.4-84.6) and negative predictive value of 61.7% (95%CI, 52.3-70.3) [Table 2] when compared with PCR for HR-HPV 16 DNA. Remaining 45 cases were not adequate for PCR due to insufficient amount of DNA template consisting three cases of CC and 42 cases of NCC. Amongst controls only 15 (10%) were positive for HR-HPV 16 and four (2%) for LR-HPVs 6/11.
Table 2: Cytological features of LSIL-HPV with HPV DNA types


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Figure 3 (a-d): These images show small rounded cells in chains and groups. Their cytoplasm was unevenly stained, Nuclei were small, pyknotic and slightly eccentrically placed (incomplete koilocytes), (Pap stain, × 400)

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Correlation of HR-HPV 16 positivity with age

Of the 150 cases of HPV infection detected in Pap smears, 105 were HPV DNA positive and amongst them 67 were positive for HR-HPV 16. Amongst HPV 16 positive cases 29 (43.2%) women were between the age of 25 and 30 years, 17 (25.3%) were 31 to 35 years, 12 (17.9%) were 36 to 40 years and 10 (14.9%) were between 41 and 45 years. Majority (41.7%) of HPV positive cases were reported in the age group of 25 to 30 years and HR-HPV 16 positivity decreased as the age increased. No statistical significance (P=0.72) was observed between the age group and HPV DNA test [Table 3].
Table 3: The correlation of HPV DNA positivity with age


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   Discussion Top


Papillomaviruses are small DNA viruses, approximately 55 nm in diameter. They are epitheliotropic and infect epithelial cells of skin, oropharyngeal and anogenital mucosa. In cervix, HPV infection involves epithelial cells and viruses replicate fully only in mitotically active cells. [11] Most women have HPV infection at some point of their lives, could often have repeated infection and majority of these infections are cleared spontaneously. [12] HPV infections of the cervix fall in two groups: HPV 6, 11, 42, 43 and 44 are associated with condylomata acuminate and low grade CIN which usually does not progress is termed as non-oncogenic HPVs. Low grade CIN that progresses, high grade CIN and invasive carcinomas are associated mainly with HPV type 16,18,31,33,35,39,45,51,52,56,58 and so called oncogenic HPVs. The two HPV genotypes HPV 16 and HPV 18 have been identified as the high-risk genotypes for the detection of cervical precancerous lesions missed by cytological examination with Pap smear, however, HPV 16 is the commonest type amongst women with and without cervical dysplasia and HPV 18 is more oncopotent. HPV infection can be latent or productive. In latent infection no clinical or morphologic sign are present indicating that the squamous epithelium has been infected. The only manner in which infection can be detected is by identification of HPV DNA using molecular techniques. In the productive or replicative type of infection, viral replication occurs independent of the replication of host cellular DNA. The productive type of infection produces infective virions. Assembly of the virion occurs in superficial cells often resulting in koilocytic morphology as the cells cytoplasm comes to be filled with viral particles. [13] However, NCC showed incomplete koilocytes and the cells near the surface may have nuclei that are somewhat smaller and pyknotic with thickening of cytoplasm.

In the present study cytologic changes of HPV infection were described either as CC or NCC. 27 of 30 (90%) CC and 78 of 120 (65%) NCC were HPV positive which is quite comparable with the findings of Bollmann et al. [5] who reported 46.3% association of non-classic changes with HPV positivity. Majority of CC, having koilocytes and binucleation, were found significantly associated with presence of HR-HPV 16 in 21 of 30 (70%) cases and can be strong predictor for HPV DNA positivity as also earlier reported by Rotelo-Martin et al. [14] In this study 70% CC were positive for HR-HPV 16 in comparision with 38% NCC (incomplete koilocytes), which also supports sensitivity of koilocytes for HR-HPV DNA positivity. The 38% HR-HPV 16 positivity in NCC support the findings of Bollmann et al. [5] who reported 46.3% HPV positivity of non-classical signs, however, higher percentage (78%) of High risk HPV DNA in low grade SILs was also detected by Nuovo. [15] Since HPV infection is transient infection and many times NCC positive for LR-HPV 6/11 disappears very often in repeat smears, eccentric and pyknotic nuclei in Pap smears might reveal the presence of Low risk HPV 6/11.

Age is significantly associated with an increased risk of HPV infection and young sexually active women, in particular, show considerable fluctuation in their HPV status and HPV type overtime. [16],[17] This study revealed that 43.2% women between the ages of 25 to 30 years were positive for HR-HPV 16. The HPV positivity decreased with the increasing age and reported as low as 14.9% for HR-HPV 16 in the women aging between 41 and 45 yrs. Women <35 years of age are likely to acquire genital infections with oncogenic HPV; with HPV in the majority of cases they will disappear spontaneously, while oncogenic HPV in women >35 years of age tend to persist and become clinical with risk of developing cancer. [3] It is concluded from this study that Classical cellular changes are not the only diagnostic features for HPV infection in Pap smear, the non-classical diagnostic features also support the diagnosis of HPV infection and may be positive for LR-HPV 6/11 or HR-HPV16. HPV DNA testing by PCR permits the sensitive genetic analysis of small amount of cells and HPV DNA test alone is significantly more sensitive than and as specific as the Pap test. In economically less developed countries, the costs of DNA probes, other reagents and materials needed for HPV testing exceed the economic resource so the classic Pap test remain the optimal method for cervical cancer screening. The presence of CC and NCC in a cervical smear can be first indication to perform an HPV DNA test for high risk HPV 16 infection in women, if not subjected for HPV screening till the age of 45 years or during sexually active phase of life.


   Acknowledgements Top


Authors are thankful to untiring efforts of a dedicated team of ICPO's Medical Social Workers without which this study would have been impossible.

 
   References Top

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3.Feichter G, Meisels A. Task force consensus report on HPV- related changes of the lower female genital tract Acta Cytol 2002;46:630-2.  Back to cited text no. 3
    
4.Koss LG, Melamed MR. Koss' squamous carcinoma of the uterine cervix and its precursors in: diagnostic cytology and its histopathologic bases. 5 th ed. Vol. 1. Philadelphia: Lippincott Williams and Wilkins; 2005. p. 287.  Back to cited text no. 4
    
5.Bollmann M, Bankfalvi A, Trosic A, Speich N, Schmitt C, Bollmann R. Can we detect cervical human papillomavirus (HPV) infection by cytomorphology alone? Diagnostic value of non-classic cytological signs of HPV in minimally abnormal Pap tests. Cytopathology 2005;16:13-21.  Back to cited text no. 5
    
6.Gjoen K Sauer T, Oslen A, Orstavik I. Correlation between polymerase chain reaction and cervical cytology for detection of human papillomavirus infection in women with and without dysplasia. APMIS 1997;105:71-5.  Back to cited text no. 6
    
7.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. JAMA 2002;287:2114-9.  Back to cited text no. 7
    
8.Das BC, Sharma JK, Gopalkrishna V, Das DK, Singh V, Gissmann L,et al. A high frequency of human papillomavirus DNA sequences in cervical carcinomas of Indian women as revealed by Southern blot hybridization and polymerase chain reaction. J Med Virol 1992,36:239-45.  Back to cited text no. 8
    
9.Sambrook J, Russell DW. Isolation of high molecular weight DNA from mammalian cells using Proteinase K and Phenol. In: Molecular cloning: A laboratory manual Protocol 1. 3rd ed. Vol. 1, Cold Spring Harbour Laboratory Press; New York 2001, p 6.6-6.11.  Back to cited text no. 9
    
10.Das BC, Sharma JK, Gopalkrishna V, Luthra UK. Analysis by polymerase chain reaction of the physical state of human papillomavirus type 16 DNA in cervical preneoplastic and neoplastic lesions. J Gen Virol 1992b;73:2327-36.  Back to cited text no. 10
    
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12.Syrajanen KJ. Spontaneous evolution of intraepithelial lesions according to grade and type of the implicated human papillomavirus (HPV) Eur J Obstet Gynecol Reprod Biol 1996;65:45-53.  Back to cited text no. 12
    
13.Geisinger KR, Stanley MW, Raab SS, Silvermann JF, Aboti A. Squamous intraepithelial lesions. Modern cytopathology, New York: Churchill Livingstone; 2004. P. 111  Back to cited text no. 13
    
14.Roteli-Matins CM, Alves VA, Santos RT, Martinez EZ, Syrjanen KJ, Derchain SF. Value of morphological criteria in diagnosing cervical HPV lesions confirmed by in situ hybridization and hybrid capture assay. Pathol Res Pract 2001;197:677-82.  Back to cited text no. 14
    
15.Nuovo GJ. Detection of human papillomavirus in Papanicolau smears: correlation with pathologic findings and clinical outcome. Diagn Mol Pathol 1998;7:158-63.  Back to cited text no. 15
    
16.Rosenfeld W, Rose E, Vermund SH, Schreiber K, Burk RD. Follow-up evaluation of cervico vaginal human papillomavirus infection in adolescents. J Pediatr 1992;21:307-11.  Back to cited text no. 16
    
17.Smith JS, Melendy A, Rana RK, Pimenta JM. Age-specific prevalence of infection with human papillomavirus in females: a global review. J Adolesc Health 2008 Oct;43(4 Suppl): S5-25,  Back to cited text no. 17
    

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Correspondence Address:
Veena Kashyap
Department of Cytology, Institute of Cytology and Preventive Oncology (DHR), Plot I-7, Sector-39, Noida, UP
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9371.86340

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    Figures

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