Evaluation of the In Situ Hybridization Signal Patterns of Liquid-Based Cytological Human Papillomavirus Specimens for Diagnosing Squamous Intraepithelial Lesion

1 Department of Clinical Laboratory, National Hospital Organization Hamada Medical Center, Hamada 697-8511, Japan 2 Department of Obstetrics and Gynecology, National Hospital Organization Hamada Medical Center, Hamada 697-8511, Japan 3 Department of Obstetrics and Gynecology, Tokuyama Central Hospital, Shunan 745-8522, Japan 4 Department of Functional Pathology, Shimane University School of Medicine, Izumo 693-8511, Japan 5 Institute for Clinical Research, National Hospital Organization, Kure Medical Center and Chugoku Cancer Center, Kure 737-0023, Japan


Introduction
Human papillomavirus (HPV) is responsible for uterine cervical cancer [1].Screening programs for cervical cancer detection have greatly reduced the incidence of cervical cancer and cancer-related mortality.However, the cytological Papanicolaou test has relatively low sensitivity as well as a high false-negative rate and high interobserver variability.Recent studies have reported that exfoliated cells sorted for liquid-based cytology (LBC) are useful for immunohistochemical (p16) and molecular analysis methods such as in situ hybridization (ISH) or polymerase chain reaction (PCR) for detecting viral DNA in these samples [2,3].Evaluating exfoliated cells is more convenient than evaluating biopsy specimens for screening patients at high risk of cervical cancer.
Although cervical intraepithelial neoplasia (CIN) 1 lesions are regress spontaneously [4], it is known that high risk HPV integration occurs in a subset of LSILs [5], which could be an early event in carcinogenesis.Therefore, it is important to detect a marker protein or viral genomic state (including the episomal [E] and/or integrated [I] form) before the lesion progress.Under these considerations, we evaluated cytological ISH (c-ISH) with LBC specimens by using a Ventana's autostainer for detect the signal patterns and the results were evaluated along with the corresponding biopsy specimens.

Materials and Methods
The study included 33 cases who had negative for intraepithelial lesion (NILM), low-grade intraepithelial neoplasia  The patients with LSIL were followed for 3 months to 2 years and did not undergo treatment such as laser vaporation.
They underwent the biopsy procedure on the same day as or the day after the cytology specimens were obtained, and histological examination was performed as gold standard.
The results of the first cytological and histological tests were shown in Table 1.
Cytological examination was performed using split samples.Briefly, the exfoliated cervical samples were directly smeared using a bloomed brush; this was followed by Papanicolaou staining.Next, the brush was immediately suspended in ThinPrep PreservCyt Solution (Hologic Corporation, Mass, USA) for LBC specimens.
We performed ISH using c-ISH and histological ISH (h-ISH) specimens.1G-sedimented smears on slide glass using Setting Chambers (MBL, Nagoya, Japan) were prepared from ThinPrep PreservCyt Solution for c-ISH.For h-ISH, 4μm-thick sections were prepared from paraffin blocks.ISH was performed on an autostainer (BenchMarK LT; Ventana Medical systems, Tucson, Ariz, USA) according to the manufacturer's instructions, with an ISH iVIEW Blue Plus Research Kit and INFORM HPV III Family 16 Probes (B) (Ventana) for h-ISH, and with Probe (C) for c-ISH.The probe cocktail had an affinity with high-risk HPV genotypes such as 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66.The nuclear signals were punctate or diffuse, corresponding to I and E signals, respectively.To determine whether the samples identified as HPV positive by INFORM ISH were true positives, we stained a positive control section in the same run.In addition, we carefully eliminated the artifact staining according to instruction manual [6].
The comparison between the two groups was analyzed using Chi-square test.P value of <0.05 were considered statistically significant.

Results of Cytology, Histology, In Situ Hybridization (ISH) and Polymerase Chain Reaction (PCR).
For 33 patients, we performed cytological and histological examinations in combination with ISH and PCR as listed in Table 1.
In h-ISH, an E-signal (diffuse signal) and an I-signal (punctuated signal) were detected in each cell layer as previously reported [9].Dysplastic cells showing the I-signal were detected in the intermediate and superficial layers as well as in the basal layer in CIN1 cases (Figure 1).In c-ISH, the positive cells were easily detectable (Figures 2(a) to 2(f)).
Among the PCR positive cases of NILM, LSIL, ASC-US, and HSIL, a positive signal of c-ISH was detected in 0% (0/2), 84.6% (11/13), 50.0%(1/2), and 90.9% (10/11) cases, respectively.However, the morphology of the positive cells (E-signal, Figures 2(a) to 2(c); I-signal, Figures 2(d) to 2(f)), was not exactly the same as the morphology observed in the Papanicolaou smear.We classified the cells on the basis of the nuclear signal and cell size into categories such as large-sized cells (L-cell; Figures 2(a) and 2(d)), medium-sized cells (Mcells; Figures 2(b) and 2(e)), and small-sized cells (S-cells; Figures 2(c) and 2(f)).It is likely that these sizes correspond to the superficial, intermediate, and basal cells observed in the epithelial layer in biopsy specimens.

Evaluation of c-ISH Signals.
To evaluate the signal in c-ISH, we counted the E-and I-positive cells in each cell population (Table 2).The positive cells on each glass slide were counted.The pattern with E signals greater than I signals is expressed as E > I, the pattern with an equal number of E signals and I signals is expressed as E = I, and the pattern with more I signals than E signals as I > E. In the total cell (L-cell + M-cell + S-cell) fraction, the positive cell pattern of E I was observed in 10 out of 12 cases in LSIL and ASC-US.On the other hand, the pattern I > E was observed in 9 out of 11 cases in HSIL.There was a significant difference between the frequency of the E I pattern in LSIL and that of the I > E pattern in HSIL (P < 0.01 in the Chi test).This indicates the usefulness of c-ISH in diagnosing the SIL according to the pattern of signals.
The numbers of E or I cells in each cell population (Table 2) are shown in scatter plots (Figures 3 and 4).As shown in Figure 3(a), cases of the I > E pattern of LSIL ( * 1 and * 2) and E > I pattern of HSIL ( * 3 and * 4) were found to be minor cases of each lesion.Comments on these cases are provided in the Discussion section.Positive largesized cells were present in all 11 (100%) patients with LSIL and 7 of 11 (63%) patients with HSIL.On the other hand, M + S cells were observed in 6 of 11 patients (54%) with LSIL and all 11 (100%) patients with HSIL (Figure 4).The major cellular component of LSIL was revealed to be largesized cells in c-ISH.The results obtained for the large-sized cells (Figure 3(b)) with the I > E pattern cases ( * 1 and * 2) were appear to be unusual in comparison with those for the 5 E I cases ( * 6, * 7, * 8, * 11, and * 12), where regression was observed in all cases (NILM, PCR negative, and ISH negative).In * 1 (case 4), the I > E pattern of HPV   was detected (Figure 5).Although regression to NILM was observed, HPV was consistently detected (PCR positive (type 58) and ISH positive) during a period of at least 6 months; * 2 (case 15) was CIN2 with a negative ISH; * 5 (case 13) progressed to HSIL (moderate dysplasia) after 22 months.Therefore, LSIL cases showing the I > E pattern in a largesized cell fraction appear to indicate that careful diagnosis and follow-up may be necessary.

Discussion
It is important to morphological, viral, oncological, and immunological markers for the early stage of uterine cervical dysplasia.The viral-infected condition, such as the episomal or integrated form, is believed to be important because the HPV integration is known to represent a key step towards the progression of the disease [10].Therefore, we investigated HPV-infected cells to search for a diagnostic and/or prognostic marker by using the c-ISH in liquid-based specimens.PCR was also performed for the comparison.Although HPV type 52 was not the most commonly detected genotype in this study, which does not agree with the results obtained in a previous report [8], this difference is probably due to the selected LSIL patients who were followed up without any treatment.

ISRN Pathology
We used Ventana autostainer for the ISH examination using the Inform HPV III probe.Guo et al. [11] reported an ISH method, which uses the Inform HPV III probe.This method has significantly improved sensitivity compared to HPV II, and this sensitivity is comparable to PCR for detecting HPV DNA in tissue sections.Although higher specificity was reported [12,13], Alameda et al. [14] pointed out that the Ventana Inform has lower sensitivity.However, the version used is not described in the report.In this study, we detected c-ISH positive signals, which were similar to the results provided by PCR in both LSIL and HSIL.We also detected more positive signals of c-ISH than of h-ISH in PCR-positive LSIL cases (91.6% (11/12) versus 58.3%(7/12)) and PCR-positive HSIL cases (90% (10/11) versus 81.8% (9/11)).Thus, c-ISH is revealed to be more sensitive than h-ISH, especially in low-grade lesions.As discussed by Guo et al. [11], the heterogenous distribution of HPV in low-grade CIN might cause signal absence in tissue sections, resulting in false-negative results.On the other hand, in case 10 (CIN3), weak integrated signals were detected in h-ISH but not in c-ISH.Because the punctate signal pattern is known to be more frequent in CIN3 and because it becomes more difficult to recognize or interpret the ISH signals, it is possible that we did not recognize the weak signals in c-ISH.
In evaluating the ISH signals, diffuse, punctate + diffuse, and punctate patterns were described previously [2,14].As the coexistent pattern (punctate + diffuse) was found to be abundant in both h-ISH and c-ISH, we classified the c-ISH signal patterns into E I and I > E categories according to the number of E+ or I+ cells in each case.Therefore, we found that the E I pattern corresponds to LSIL, and the I > E pattern corresponds to HSIL.The differences were found to be statistically significant (P < 0.01).The signal patterns of c-ISH seem to be very useful for diagnosing the SIL.In the combination of the Papanicolaou test with the c-ISH signal patterns, HSIL with the E I pattern increased the specificity, PPV and NPV, and the I > E pattern increased the sensitivity and PPV for detection of CIN2 or more advanced lesions.And also LSIL with E I pattern increased the sensitivity and PPV, I > E pattern increased the specificity and NPV to detect the CIN1.So, the combination of Papanicolaou test with c-ISH signal patters appears to be useful in the diagnosis of CIN lesion.However, the specificity and NPV of HSIL with I > E to detect CIN2/3 was decreased to 50% (1/2).LSIL with E I pattern decreased the specificity and NPV (66.7%) to detect CIN1.Therefore, the results of signal patterns of c-ISH are useful if they are separately thought about the sensitivity and specificity in the cases of LSIL or HSIL compared with the diagnosis of CIN.
The E > I pattern cases of HSIL ( * 3 and * 4) shown in Figure 3 are discussed.In * 3 (case 25), many E+ Scells were detected.It is difficult to demonstrate a diffuse integrated pattern in ISH using Ventana system [12].Therefore, there is a possibility that c-ISH-positive S-cells undergo diffuse integrated staining.In the case of * 4 (case 26), both CIN1 and CIN3 (severe dysplasia) lesions were observed in the same histological specimen.Because the cytological specimens were composed of superficial and intermediate layered cells according to the histological analysis, the E+ cells of CIN1 appear to be easily detected in c-ISH.
We focused on the LSIL patients with an I > E pattern indicating L-sized positive cells, because an atypical persistent detection of HPV in NILM or progression to HSIL were observed during the followup of these patient.Although persistent detection of HPV is reported to be common among CIN patients at followup even in cases where cytology and histology results are obtained [2], the integration of high-risk HPV is generally a key event in cervical carcinogenesis [10].De Marchi Triglia et al. [9] reported that the presence of a punctate signal in the superficial layer in histological analysis is associated with cases without progression.Although we did not detect the Ipositive S-sized cells in c-ISH in both the case 4 and 13, I-positive basal cells were detected in h-ISH.We do not know the correlation between I-positive basal and superficial cells as well as the destiny of I-positive superficial cells now.To reveal the significance of I-positive L-cell (c-ISH) and superficial cells (h-ISH), we will investigate such cases in the future studies involving a greater number of patients.

Conclusion
We investigated c-ISH (Ventana method) for liquid-based cytology specimens and detected c-ISH-positive cells that showed E-or I-signals.From these cases, the E I pattern was found to be in LSIL, and the I > E pattern in HSIL, and the differences were revealed to be statistically significant (P < 0.01).In follow-up examination for the LSIL cases, we found the 2 patients who had large-sized cells with the I > E pattern.They showed HPV persistence and/or progression during follow-up.Therefore, presence of the I > E pattern of c-ISH large-sized cells in LSIL may be a promising marker for follow-up studies.

Table 1 :
A list of the case studied.

Table 2 :
Positive cell number of each signal (E, I) of c-ISH.