Mitsuaki Okodo

Vacuolated Parabasal Cells in Papanicolaou Smears Are Cellular Changes Caused by Human Papillomavirus 16 Infection

ABSTRACTIn cervical cancer screening, cytology is used as a triage test to refer high‐risk human papillomavirus (HR‐HPV)‐positive women for colposcopy, but its accuracy is inadequate. The present study aimed to demonstrate that the presence of atypical cells with large vacuoles in the cytoplasm of parabasal cells, referred to as vacuolated parabasal cells (VPCs), which are observed in the Pap smears of HPV‐positive women, is associated with specific HPV genotypes. Among 2175 patients, 310 with a single HR‐HPV infection and cytological diagnosis of high‐grade squamous intraepithelial lesions (HSIL) or atypical squamous cells not excluding HSIL (ASC‐H) were included, of which 86 were infected with HPV16. Biopsy results revealed that 69 (80.2%) patients had cervical intraepithelial neoplasia Grade 2 or higher (CIN2+). VPCs were found in 47 (54.7%) of HPV16‐infected cases, indicating a significant increase of VPCs in HPV16‐infected cases (p < 0.01). Episomal HPV16 load was quantified in 142 VPCs and 156 HSIL (ASC‐H) cells using liquid‐based cytology samples from five patients, with a median of 987 copies in VPCs, significantly higher than those in HSIL (ASC‐H) cells (176 copies; p < 0.001). VPCs in Pap specimens were identified not only as cells altered by HPV16 infection but also as CIN2+‐derived cells and HPV16‐producing cells.

Predictive Value of Various Atypical Cells for the Detection of Human Papillomavirus in Cervical Smears

It is thought that numerous genotypes of human papillomavirus (HPV) are associated with various atypical cells, such as multinucleated cells, koilocytes, binucleated cells, parakeratotic cells, and giant cells, in the cervix. We previously showed the specificity of HPV genotypes for koilocytes and multinucleated cells. Therefore, in this study, we analyzed the association among HPV genotypes and binucleated cells, parakeratotic cells, and giant cells in Papanicolaou (Pap) smears. We detected HPV genotypes and atypical cells in 651 cases of liquid-based cytology with an abnormal Pap smear. The HPV genotypes associated with atypical cells were evaluated using stepwise logistic regression with backward elimination and a likelihood ratio test for model construction. Polymerase chain reaction was used to determine the HPV genotypes in whole liquid-based cytology samples and microdissected cell samples from Pap smear slides. Binucleated cells were significantly associated with HPV genotype 42. Moreover, parakeratotic cells were significantly associated with certain HPV genotypes, such as HPV40. However, it was difficult to detect specific HPV genotypes by the manual microdissection-polymerase chain reaction method despite the presence of binucleated cells and parakeratotic cells. Thus, the presence of binucleated cells, parakeratotic cells, and giant cells in Pap smears may not be predictive of cervical lesions above low-grade squamous intraepithelial lesions or infection with highly carcinogenic HPV genotypes.

Koilocytic changes are not elicited by human papillomavirus genotypes with higher oncogenic potential

AbstractKoilocytes are considered a common cytopathological effect in patients with human papillomavirus (HPV) infection. Thus, we aimed to elucidate whether koilocytes are common to all HPV infections. Liquid‐based cytology samples from 651 patients with abnormal Papanicolaou (Pap) test results were used to analyze the presence of koilocytes and HPV genotype. HPV genotype was determined in complete liquid cytology samples and microdissected cell samples from Pap smear slides using the uniplex E6/E7 polymerase chain reaction method, which can detect 39 mucosal HPV genotypes. Koilocytes were found in 29.3% (191) of all patients. Logistical regression analysis of diverse HPV genotypes revealed that infections with low‐risk HPV types (HPV‐6b, HPV‐40, HPV‐42, HPV‐61, HPV‐74, HPV‐89, and HPV‐90), probably high‐risk HPV types (HPV‐53 and HPV‐66), and high‐risk types (HPV‐39 and HPV‐56) were significantly associated with the presence of koilocytes. However, HPV‐16, HPV‐18, and HPV‐52, which have higher oncogenic potential, were not found to be associated with koilocytes. These results were confirmed by HPV genotyping using microdissected koilocytes in 27 patients.Most common high‐risk types belonging to α‐9 and α‐7 genotypes appear to rarely induce koilocytic changes. Therefore, koilocytes may provide additional useful information for predicting the risk of progression to high‐grade lesions.

Human papillomavirus infection status of single cells isolated from cervical cytology specimens by simple manual microdissection

AbstractHuman papillomavirus (HPV) testing with cytology triage for cervical cancer screening has proven to be useful. It is considered that a significant percentage of HPV‐positive women followed by reflex cytology have had multiple‐type HPV infections rather than single‐type infections. However, the effects of multiple‐type infections on changes in the cytomorphology of exfoliated cervical cells have not been investigated. The aim of this study was to validate simple manual microdissection (MMD) maneuver and investigate the HPV infection status of single cells isolated from Papanicolaou (Pap) smears prepared from women with multiple‐type infections. Using cytology samples from 90 patients with abnormal Pap smear results, we evaluated the efficiency of the MMD procedure and determined the HPV infection status of single squamous intraepithelial lesion (SIL) cells microdissected from patients with multiple‐type infection. When validating the MMD procedure, the HPV‐positive rate was 81.5% using 119 MMD samples from the Pap smear in 61 cases with single‐type infection. This MMD procedure was able to efficiently collect single cells. Of 119 MMD samples from 29 cases with multiple‐type infection, the HPV‐positive rate was 42.9%, and most (96.1%) MMD samples exhibited only one genotype. Our MMD maneuver successfully identified HPV genotypes using single cells isolated from cytology specimens. A majority of single SIL cells prepared from multiple‐type infection cases turned out to contain only one genotype. In the future, the MMD method could be applied while studying the relationship between the morphological changes exhibited by SIL cells on Pap smear and the infected HPV genotype.

Causation of cornflake artifacts: Possible association of poor dehydration with drying before mounting in Papanicolaou stain

AbstractCornflake artifacts are artifacts that commonly occur while the mounted medium starts to evaporate before coverslipping. This study aimed to determine factors contributing to the occurrence of these artifacts in Papanicolaou (Pap) smears. Residual specimens were used after cytology to microscopically evaluate various effects on cornflake artifacts. Four SurePath™ liquid cytology (LBC) cell specimens, diagnosed as negative for intraepithelial lesions or malignancy (NILM), were used. Each LBC smear was subjected to Pap staining under four different conditions (A, without air‐drying; B, air‐drying after dehydration; C, air‐drying after xylene immersion; and D, air‐drying after dehydration and xylene immersion) using two methods: conventional and poor dehydration. Cornflake artifacts were not observed in A and B in Pap staining. By contrast, cornflake artifacts were observed in conventional and poor dehydration methods when dried after xylene immersion. When comparing the four conditions, smears B and D, which were both air‐dried after dehydration, had fewer cornflake artifacts than smear C, which was air‐dried only after xylene. Therefore, the remaining water in the cells due to poor dehydration during xylene immersion is found to result in the development of cornflake artifacts. The present study revealed that cornflake artifacts in Pap smears are caused by poor dehydration in addition to drying before mounting.

Effects of Menstrual Cycle on Various Morphologies of High-Grade Squamous Intraepithelial Lesions in SurePath™ Liquid-Based Cervical Cytology

<b><i>Introduction:</i></b> The morphology of high-grade squamous intraepithelial lesion (HSIL) on Papanicolaou (Pap) smears widely varied, including syncytial aggregates, sheets, and scattered single cells, and no particular cellular pattern is consistently observed. Therefore, this study aimed to determine whether the menstrual cycle affects the cellular pattern of HSILs, an effort to avoid false negatives due to the oversight of scattered small single HSIL cells in the cytological triage of human papillomavirus-positive women. <b><i>Methods:</i></b> A total of 147 HSIL samples of liquid-based cytology (LBC) in patients with cervical intraepithelial neoplasia grade 2 or 3 were obtained, and then, the relationship between cellular patterns, such as single-cell-like and syncytial aggregates, and menstrual cycles classified into six phases was analyzed. If a syncytial aggregate was present, the number of cells constituting the aggregate was visually counted under the microscope. <b><i>Results:</i></b> HSILs in scattered single cells and small sheets of <6 on LBC samples accounted for 43% (23/54) during the late proliferative phase of the menstrual cycle. A moderately strong statistically significant association was observed between cellular patterns and menstrual cycles (χ<sup>2</sup> [3] = 9.423, <i>p</i> < 0.05) (Cramer’s V = 0.253). The value of adjusted residuals showed a statistically significant increased proportion of single-cell-like patterns during the late proliferative phase (<i>p</i> < 0.01). <b><i>Conclusions:</i></b> The present study demonstrated that HSIL cells in Pap smears in the late proliferation phase have a high frequency of single-cell-like patterns. In human papillomavirus-positive Pap smears with a clean background and predominantly superficial cells, careful microscopic observation by targeting single HSIL cells can potentially reduce false negatives.

Cytological features of human papillomavirus‐infected immature squamous metaplastic cells from cervical intraepithelial neoplasia grade 2

AbstractIn reflex cytology, the presence of prominent nucleoli in immature metaplastic squamous cells (IM) may be underdiagnosed due to variations in interpretation. The aim of this study is to identify human papillomaviruses (HPVs) that infect IM clusters in cervical intraepithelial neoplasia 2 (CIN2) on Papanicolaou (Pap) smears to determine the cytological features of lesion‐derived cells. Thirty‐two patients with a simultaneous diagnosis of CIN2 on biopsy and high‐grade squamous intraepithelial lesions (HSIL) on cytology as well as with IM clusters on HSIL smears were included. CIN2 tissues and HSIL and IM clusters on Pap smears were isolated by manual microdissection, and HPV types were identified by PCR‐based genotyping. The nuclear area within the IM clusters was also measured. The median nuclear area of HPV‐negative IM clusters was 48 μm2, with a coefficient of variation (CV) of 0.20; those of HPV‐positive clusters were 66 μm2 and 0.34, respectively. The cut‐off values of the nuclear area and CV for HPV positivity were 62 μm2 and 0.25, respectively. IM clusters composed of cells with a nuclear area of more than twice that of neutrophils or cells with a wide variation in nuclear sizes are likely to be neoplastic cells caused by HPV.

Preferential Tissue Sites of Different Cancer-Risk Groups of Human Papillomaviruses

The oncogenic potential of human papillomavirus (HPV) may be used to determine the tissue tropism of each HPV type. Cervical cancer develops in the squamo-columar junction of the cervices, and most lesions are induced by high-risk (HR) HPV types. This suggests that HR types preferentially infect the cervix, whereas the preferential infection site for low-risk (LR) types is not well defined. The determination of HPV tropism when using cytology samples can be uncertain since it is difficult to avoid contamination of cell samples between the cervix and the vagina. Herein, cell samples were carefully collected by independently scraping the cervix and vagina, after which the HPV types were determined. HPV tissue tropism was determined by considering what HPV types were positive at only one of the sites (the cervix or the vagina) as the viruses that preferentially infected that site. This method revealed that all LR types were only identified in vaginal samples, whereas 87% of HR types were identified in cervical sites. Thus, LR types may preferentially infect the vagina, whereas HR types infect the cervix. These findings suggest that preferential tissue tropism of certain HPV types is a probable factor for malignant progression.