Flow Cytometric Analysis of DNA Ploidy in Liquid Based Cytology for Cervical Pre-Cancer and Cancer

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1 DOI: /APJCP RESEARCH ARTICLE Flow Cytometric Analysis of DNA Ploidy in Liquid Based Cytology for Cervical Pre-Cancer and Cancer Sridhar Mishra 1, Namrata P Awasthi 1, Nuzhat Husain 1 *, Akansha Anand 1, Yashodhara Pradee 2, Roohi Ansari 3, Sarita Saxena 4 Abstract Background: DNA loidy analysis of cervical intraeithelial neolasia (CIN) and invasive cervical cancer samles by flow cytometry (FCM) has been established as an aid to rognostic assessment. Liquid based cytology (LBC) increases diagnostic secificity by using ancillary techniques that rovide information beyond morhology. The resent study was undertaken to assess DNA loidy in LBC samles as an adjunct for early detection of cervical re-cancer and cancer. Methods: DNA loidy assessment was erformed on LBC samles of 50 cases and 31 controls. Cell ellets were obtained by centrifugation and stained with Telford reagent. At least 20,000 R1 gate (G0-G1) events were acquired on a BD FACSCalibur by using a 575±10 nm filter. Results: Mean diloid G1 s were lowered significantly (<0.01) while diloid S s were significantly elevated (<0.01) in both high grade squamous intraeithelial lesions (HSILs) and squamous cell carcinomas (SCCs) as comared to controls. Receiver oerating curve (ROC) analysis of the diloid G1 was found to have significant diagnostic otential (AUC=0.682, Z=2.00, =0.046) for distinction between control and low grade squamous intraeithelial lesion (LSIL) at a cut off of 91.6 with a sensitivity and secificity of 50.0 and 87.1%, resectively. Conclusions: ROC analysis of diloid G1 and diloid S s allows discrimination between LSIL and HSIL with sensitivities and secificities of 65 and 100% and 70 and100%, resectively, and between LSIL and SCC cases with s of 71.4 and 100% and 64.3 and 100%, resectively. Keywords: Liquid Based Cytology- Flowcytometry- Low grade Squamous Intraeithelial Lesion- High grade Squamous Asian Pac J Cancer Prev, 18 (6), Introduction Cervical cancer is the second most common cancer in women worldwide, reresenting global incidence of 5, 26,600 new cases each year and 2, 65,700 death, in that India contributes 67,500 i.e., one-fourth of the world burden (Torre et al., 2015). The malignancy targets an active female oulation between yrs (Ferlay et al., 2012; WHO, 2012).Mortality in develoing countries is unaccetably high and screening rogram are not in lace (Soler et al., 2000). Liquid Based Cytology (LBC) rovides the use of ancillary techniques in addition to a good morhology and detection of cytological abnormalities (Monsonego et al., 2001). Cervical eithelial cells fixed in LBC and stained with fluorescent dye can be acquired by flowcytometer for DNA loidy measurement. Analysis of cells within a cell cycle on flowcytometer roduces a histogram which reresents DNA loidy. DNA loidy assessment by flow cytometric technique could serve as a rognostic factor that allows the estimation of the relative rogressive risk into more advanced lesions (Melsheimer et al., 2004). Cells are distributed among three major hases of cell cycle: G0/G1 hase which comrises 85% cells, S hase and G2/M hase which make 15% of the cells in a normal tissue and in majority of low grade or gradually roliferating lesions. DNA aneuloidy in S hase may indicate develoment of intermediate to late stages of the malignancies (Merkel and McGuire 1990; Ross, 1996). Precancer state results in genetic instability due to unrestricted growth of cancer cells by subsequent mutations in growing ool of roliferating cells. Structural and numerical change may occur in chromosomes resulting in overall change in the DNA content of cells, an event referred to as aneuloidy. DNA loidy assessment has been established as a rognostic factor in ovarian (Vergote et al., 1993; Kaern et al., 1994) and endometrial cancer (Erba et al., 1989; Evans and Podratz,1996), however in cervical cancer there are conflicting results (Jakobsen, 1984; Willen et al., 1993; Podratz et al., 1993). Molecular basis of aneuloidy remain undefined and divisive (Marx, 2002) with an assumtion that mitotic roteins lay role in chromosomal instability 1 Deartment of Pathology, Dr. Ram Manohar Lohia Institute of Medical Sciences, 2 Deartment of Obestrics and Gynaecology, Queen Merry Hosital, King George Medical University, 3 Deartment of Bioengineering, Integral University, 4 Deartment of Obestrics and Gynaecology, Ram Manohar Lohia Combined Hosital, Lucknow, UP, India. *For Corresondence: drnuzhathusain@ hotmail.com Asian Pacific Journal of Cancer Prevention, Vol

2 Sridhar Mishra et al (Lengauer et al., 1998; Rajagoalan et al., 2003). Another assumtion rooses that aneuloidy is itself the cause of genetic instability and cancer (Duesberg et al., 1988; Li et al., 2000).Aneuloid DNA rofiles are useful indicators for the biologic aggressiveness of cervical cancer lesions than those of diloid or olyloid rofiles (Fu et al., 1989). In this study, we have used light scatter characteristics of cells to evaluate the relationshi between DNA loidy and S hase fraction and correlated with clinicoathological arameters in cervical re cancer and cancer. Materials and Methods Patient samles and rocedures The study samle comrised of 50 cases and 31 controls from the Deartment of Obstetrics and Gynaecology, Queen Mary s Hosital, King George s Medical University and Dr.Ram Manohar Lohia Combined Hosital, Lucknow, India. Samles were collected during cervical screening of women in hosital based setting. All articiants signed an informed consent and ethical aroval was obtained from Institutional Ethics Committee before recruiting atients. All cases that fulfilled the inclusion criteria that is, females with ositive findings on coloscoy and/or visual examination of cervix were recruited for the study. Particiants were excluded from the study if they were not willing to articiate or cases with malignancy under follow u/ rior theray. Total of 90 cases were screened of which 81 were included in the study excluding 9 cases with low eithelial cell count. Cytology Samles were collected by gynaecologist in Thin re (TP) vials (Hologic, Inc. USA) containing 20 ml PreservCyt (Thin Pre, Hologic, Boxborough, MA. Cytobrush (RoversR Cervex-BrushR) was simly inserted into the endocervical canal dee enough to allow the shorter bristle to fully contact the ectocervix (keeing the last row of bristles visible). The cytobrush was slowly rotated clockwise to 180 degrees for five times avoiding excessive rotation which would distort the cell. The brush was rinsed into the PreservCyt vial by ushing the broom in the bottom of the vial 10 times, forcing the bristle aart. Smears were reared on Thinre 2000 rocessor (CYTYC Cororation) and cytologic screening was done by conventional Pa staining. All slides were evaluated by a exerienced athologist and diagnosis was made using the Bethesda System (2014). Cases with normal cytology were used as a control for DNA loidy. Samles were ket at 4 C and FCM was erformed within 1-3 weeks of collection. Flow cytometric analysis Selection of Cases: Cases in serial showing LSIL, HSIL and SCC as er Bethesda criteria with adequate cells count (100,000 cells) were included for loidy study. Samle rearation: 5-10 ml of fixed cells (fixed in PreservCyt solution) were taken in a 15 ml conical centrifuge tube, cell ellet was obtained by centrifugation at 2,000 rm for 5 min. followed by washing of cell ellet 1596 Asian Pacific Journal of Cancer Prevention, Vol 18 with hoshate buffer saline (PBS, H-7.4), three times or more deending on debris content in samles. Eithelial cell counting was erformed on Neubauer chamber. Minimum eithelial cell count required for staining and acquisition was 1x105 cells. Staining and acquisition Cells were stained with Telford reagent (Telford et al., 1991){(EDTA (Fischer Scientific, USA), RNAse A 1mg/ml, (Biochem India), Proidium Iodide 1mg/ml, (Sigma-Aldrich, Miss, USA), Triton X-100, (Sigma-Aldrich, Miss, USA) in PBS)} and incubated in dark at 4 C for 1 hour. Stained cells were filtered by 70µ cell strainer (B.D. Biosciences, USA) and cells were acquired within 1 hour on a flowcytometer (FACSCalibur: Becton, Dickinson and Co.; San Jose, CA) equied with a 488-nm blue laser. Acquisition and analysis were erformed on Cell Quest Pro software (B.D. Biosciences, USA). At least 20,000 events were acquired in R1 gate drawn around the eithelial cells based on light scatter roerties of cells. As shown in Figure 1 (A-B), and Figure 2 (A-B), two dot lot of FSC vs. SSC and FL2-W vs. FL2-A and one histogram of FL-2A vs. Count (Figure 1-C, 2-C) were utilised for acquisition. Pulse rocessing was used to exclude cell doublets from the analysis. All measurement was erformed at low flow rate. The cell cycle rofiling of all samles were done in unchanged background using same instrument and by the same observer. Cell oulations were roerly gated and the number of cells was counted in order to evaluate the eligibility of the secimen for the urose of recise DNA content analysis. Verification of instrument erformance in term of coefficient of variation (CV) and linearity of the fluorescence ulse detectors was checked by DNA QC Particle kit (B.D. Biosciences, Singaore). Cell cycle analysis and loidy Data obtained was analyzed using the ModFit LT software (DNA Modelling System) version 3.2 (Verity Software House, Inc Tosham, ME). Each histogram was assessed by the estimation of the CV for G0/G1 oulation. Histogram was considered unsuitable for interretation if its variation coefficient CV (breadth of the fluorescence signal for normal cell oulation in G0/ G1 hase at the middle of its height) exceeded 9% (Cufer et al., 1997). A samle was considered DNA diloid if, on the histogram, there was a single eak in the G0/G1 hase with DNA content of 2N (Figure 1.b). DNA aneuloidy also known as non diloidy was defined if there was at least one searate second G0/G1 oulation to the right of the first G0/G1 eak (Figure 2.b). The loidy was characterized by the DNA index (DI) and the tye of DNA content histogram (Diloid: DI= 1, Aneuloid: DI<or >1). For each samles, the ercentage distribution were recorded for diloid G1, G2, and diloid S hase, total aneuloid, including aneuloid G1, aneuloid G2, aneuloid S hase, ercentage of C.V of diloid oulation and aneuloid oulation (if resent ), DNA index (DI), total aneuloid S hase, total S hase, B.A.D (ercentage of background aggregates and debris), debris, aggregates,

3 Table 1. Diloid Cell Cycle Parameter Values (Mean ± SE) of Four Grous DOI: /APJCP Parameters Control (n=31) LSIL (n=10) HSIL (n=20) SCC (n=14) H Di G ± ± ± ± <0.001 Di G ± ± ± ± Di S 4.63 ± ± ± ± <0.001 modeled events, all cycle events, cycle events er channel and Reduced chi-square (RCS-measure of how well the model fitted the data) were estimated. Statistical analysis Data were summarized as Mean ± SE (standard error of the mean). Grous were comared by Mann-Whitney U test. Grous were also comared by Kruskal-Wallis (H) analysis of variance (ANOVA) followed by Z test. Diagnostic (sensitivity and secificity) of the cell cycle arameters was done by using the cut off s obtained from receiver oerating characteristics (ROC) curve analysis. A two-tailed <0.05 was considered statistically significant. Results DNA loidy assessment was erformed on 50 cases including 10 of Low grade Squamous Intraeithelial Lesion(LSIL), 20 of High grade Squamous Intraeithelial Lesion (HSIL) and 20 of Squamous Cell Carcinoma Figure 1. Shows the SAcquisition of Cervical Eithelial Cells on Flowcytometer, Stained with Telford Reagent. A-C shows acquisition of stained cells on FSC vs. SSC, FL2-A vs. FL2-W and FL2-A vs. Count on Cell Quest Pro software(b.d Biosciences, Singaore). Table 2. Comarison of Each Diloid Cell Cycle Parameter Value between Grous by Z Test Comarisons Di G1 Di G2 Di S Z Z Z Control vs. LSIL Control vs. HSIL Control vs. SCC LSIL vs. HSIL LSIL vs. SCC HSIL vs. SCC (SCC) and 31 controls with cervical cytology within normal limits. Analysis was ossible in all cases and controls excet for 6 cases of SCC where acquisition and analysis was not ossible due very low cellularity. The mean (±SD) age of cases was 49.3(±10.58), ranged from 25 to 70 yrs. DNA aneuloidy was observed in 0/10 cases of LSIL, 10/20 cases (50%) of HSIL and 5/14 cases (34.5 %) of SCC. None of controls shows aneuloidy. Diloid LSIL, HSIL and SCC cases had mean CV of 7.18, 7.25 and 7.18 resectively. Value of cell cycle arameters of Control and Cases: The cell cycle arameters (diloid G1, diloid G2 and diloid S hase) s of control, LSIL, HSIL and SCC are summarized in Table 1. Table 1 shows that the mean diloid G1 decreases with degree of severity while S Phase Fraction increase with increases in severity. However, diloid G2 did not show any trend with the severity. Comaring each diloid cell cycle arameter s among four grous, Kruskal-Wallis ANOVA showed significantly (<0.001) difference in s of both diloid G1 and diloid S hase (Table 1). Furthermore, Z test showed that the mean diloid G1 was significantly lower (<0.01) in both HSIL and SCC grous as comared to control grou while diloid S were significantly (<0.01) higher in both HSIL and SCC grous as comared to control grou (Table 2). However, mean diloid G2 did not differ significantly (>0.05) among the grous (Table 1 and 2). Assessment of Diloid cell cycle arameter between grous Comaring the mean of each cell cycle arameter between two grous, Mann-Whitney U test also showed significantly different and lower (16.8%) diloid G1 in cases as comared to controls (<0.001) while significantly different and higher (77.5%) diloid S in cases as comared to controls (<0.001). However, mean diloid G2 not show statistical difference between the two grous, though it was 53.5% higher in cases as comared to control (=0.423). Comarison of aneuloid cell cycle arameter between grous Aneuloidy was found in 50% of HSIL and 34.5% Table 3. Aneuloid Cell Cycle Parameter Values (Mean ± SE) of Two Grous Parameters HSIL (n=10) SCC (n=5) U Value Value An G ± ± An G ± ± An S ± ± DI 1.22 ± ± Total S hase ± ± Asian Pacific Journal of Cancer Prevention, Vol

4 Sridhar Mishra et al Table 4. Diagnostic of Diloid Cell Cycle Parameter Values to Discriminate Cases from Control Diagnostic Parameter Cutoff (%) Sensitivity (95% CI) Secificity (95% CI) PPV NPV AUC Z Control vs. LSIL Di G ( ) ( ) Di G2 > ( ) ( ) Di S > ( ) ( ) Control vs. HSIL Di G ( ) ( ) <0.001 Di G ( ) ( ) Di S > ( ) ( ) <0.001 Control vs. SCC Di G ( ) ( ) <0.001 Control vs. Cases (LSIL +HSIL + SCC) Di G2 > ( ) ( ) Di S > ( ) ( ) <0.001 Di G ( ) ( ) <0.001 Di G ( ) ( ) Di S > ( ) ( ) <0.001 of SCC cases only. The aneuloid cell cycle arameter (An G1, An G2, An S, DI and Total S hase) s are summarized in Table 3. The mean of An G1, An G2 and DI was higher while An S and Total S hase was lower in SCC as comared to HSIL. Comaring mean of each aneuloid cell cycle arameter between two grous, Mann-Whitney U test showed significantly different and higher DI in SCC as comared to HSIL (=0.027). However, mean of An G1, An G2, An S and Total S hase was not different (>0.05). Diagnostic of cell cycle arameters- Receiver Oerating Characteristics (ROC) curve analysis ROC curve analysis was erformed to evaluate Figure 1.b. Shows the Analysis of Acquired FCS File on ModFit LT 3.2 (Verity Software House). Based on ModFit analysis case was found to be of Diloid with single G0/G1 eak. Histogram statistics shown on to right. Figure 2. Shows an Aneuloid Case of HSIL on Cytomorhology Acquired on Flowcytometer, Stained with Telford Reagent. A-C shows acquisition of stained cells on FSC vs. SSC, FL2-A vs. FL2-W and FL2-A vs. Count on Cell Quest Pro software (B.D Biosciences, Singaore). Table 5. Diagnostic of Diloid Cell Cycle Parameter Values to Discriminate Cases Among Cases Diagnostic Parameter Cutoff (%) Sensitivity (95% CI) Secificity (95% CI) +PV -PV AUC Z LSIL vs. HSIL Di G ( ) ( ) Di G ( ) ( ) Di S > ( ) ( ) LSIL vs. SCC Di G ( ) ( ) Di G ( ) 50 ( ) Di S > ( ) ( ) HSIL vs. SCC Di G1 > ( ) 55 ( ) Di G2 > ( ) 90 ( ) Di S ( ) 55 ( ) Asian Pacific Journal of Cancer Prevention, Vol 18

5 Figure 2.b. Shows the Analysis of Acquired FCS File on ModFit LT 3.2 (Verity Software House). Based on ModFit analysis case was found to be Aneuloid on aearance of second G0/G1 oulation to the right of first G0/G1 eak with DNA index of diagnostic sensitivity and secificity of diloid cell cycle arameters s to discriminate the cases of LSIL, HSIL and SCC from control is deicted in Table 4. The cut off of diloid G1 at 91.6 discriminate control vs. LSIL significantly with 50.00% sensitivity (95% CI: ) and secificity (95% CI: ). Diloid G1 at a cut off of showed significant (< 0.001) diagnostic discrimination between control vs. HSIL (65.00% sensitivity and100%secificity) and control vs. SCC at a cutoff of (96.77% sensitivity and 78.57% secificity). Diloid G1 at a cut off of significantly (< 0.001) discriminate control vs. cases (LSIL + HSIL + SCC) with sensitivity and secificity of and 100% resectively (AUC= 0.775). Likewise, diloid S showed significant (<0.001) diagnostic between control vs. HSIL at a cutoff of >8.93 (65.00% sensitivity and100% secificity) and control vs. SCC at cutoff oint of >8.93 (96.77% sensitivity and 78.57% secificity). Diloid S significantly discriminate control vs. cases (LSIL + HSIL + SCC) with sensitivity and secificity of and 100% resectively (AUC= 0.775). The diagnostic sensitivity and secificity of diloid cell cycle arameter between cases (LSIL, HSIL and SCC) are summarized in Table 5. Diloid G1 and Diloid S s significantly (<0.05 or <0.01) discriminates between LSIL vs. HSIL and LSIL vs. SCC. However, none of the Diloid cell cycle arameter could significantly (>0.05) discriminates between HSIL and SCC cases. Discussion The current study attemted to assess the analysis of DNA loidy by flowcytometry on LBC samle as an ancillary test to screen women for reneolastic lesions. A susect DNA rofile with high S hase seems to be the clue for develoing malignant disease of the uterine cervix. Automation of DNA measurement with flowcytometry through a single cell susension could be useful to identify atients at risk for develoing DOI: /APJCP reneolastic (LSIL, HSIL) or neolastic lesions (SCC). More recise discrimination and rediction of malignancy is ossible with quantitative analysis of threshold for cell cycle arameters i.e. G0/G1 (Diloid), Diloid G2, Coefficient of Variation (CV), total S hase and total aneuloidy, aneuloid S hase, DNA index (DI), and RCS as also estimated in all other studies (Chhavi et al., 2010). Cytometric techniques can serve as a marker to detect neolasia and can rovide adjunct information to athological evaluation for the diagnosis of cervical cancer. DNA aneuloidy along with S Phase Fraction (SPF) analysis can be used to identify the dyslasia and give a redictive for the malignancy transformation. In our study diloid G1 and diloid S hase s were significantly higher in HSIL and SCC comared to controls. These results could not significantly differentiate low grade lesions from high grade lesions (LSIL vs. HSIL, LSIL vs. SCC and HSIL vs. SCC). ROC curve analysis of the above cell cycle arameters can more accurately differentiate and redict the malignancy beyond morhology. Though, in our study of diloid G1 and S hase were most sensitive arameters, with cut off oint at 91.6, and (di G1) and >8.93 (di S) significantly differentiate low grade and high grade lesions from controls. Although; diloid S s was not able to differentiate LSIL cases from controls at Cut off oint >7.81. Further; ROC analysis also significantly differentiate between LSIL and HSIL by estimation of diloid G1 and diloid S ; this could otentially hel us in monitoring the rogression of recancerous lesions to cancerous lesions. We have reached the 100% secificity in discrimination of LSIL cases from HSIL when analysed for di S, suggesting significance of S hase analysis in LBC of cervical remalignant lesion. Cervical lesions with higher SPF are more likely to ersist or rogress than normal diloid or with low SPF. Our result could suort the hyothesis that higher diloid S and lower diloid G1 may be associated with rogression of cervical carcinoma. Study of Chavi et al. in LBC of cervical cancer cases, found the sensitivity and secificity of diloid G0/G1 to be 96.77% and 100% in discrimination of cases from controls. In contrast to this finding, study by Singh (2000) shows that Aneuloidy was resent in 39/79 of mild, 28/36 of moderate, 11/12 of severe dyslasia, 8/57 of ASCUS and in 6/69 controls (Singh et al., 2000). Patient with low S hase fraction often shows better survival comared to the atients with high SPF (Jayat and Ratinaud, 1993). Other studies also suorted diloidy as suggestive feature for better survival when comared with aneuloid tumor (Lai et al., 1993). Many investigators have also examined the rognostic of cytohotometric DNA content analysis in cervical carcinoma (Fu et al., 1982; Strang et al., 1987a; Strang et al., 1987b; Jelen et al., 1994; Kristensen et al., 1995). However, significance of DNA analysis as a reliable diagnostic and rognostic biomarker in LBC of cervical carcinoma still remains controversial. In invasive cervical carcinoma, DNA loidy by image cytometry is a rognostic factor to estimate the risk of rogression (Antonet al., 1997; Melsheimer et al., 2001; Hornet al., 2002; Melsheimer et al., 2004; Demirel et al., 2013). Asian Pacific Journal of Cancer Prevention, Vol

6 Sridhar Mishra et al DNA loidy analysis of ovarian tumor has been roved as rognostic indicator of disease rogression (Demirel et al., 2013). In a study of Endometrial cancer of stage I atients (n=139) by flowcytometry reorted that 86% of cases were diloid while 14% were aneuloid. Studies reorted aneuloidy in CIN 1 and in normal squamous eithelium by image cytometry on larger samle size (Haroskeet al., 2001). In our study aneuloidy was found in 50% of HSIL and 35.7 % of SCC cases. This loss of aneuloidy in SCC cases may be due to low count of cervical eithelial cells in our samles due to bleeding at time of samle collection or tumor necrosis. Study carried out by Lage et al. have roven aneuloidy as a redictor of survival and was significantly associated with stage and grade of ovarian cancer. Patient grous with higher SPF seem to die earlier (Lage et al., 1992) Image cytometry have roved the diagnostic and rognostic of DNA loidy; however this technique is relatively slow, may be insensitive for near-diloid aneuloid cell with high coefficient of variation for G0/ G1 eaks. Analysis of image cytometry requires a skilled morhologist. Flow cytometry has advantage over image cytometry and rovides high-seed, fast results with high number of cells counted. High number of cell counted on flowcytometry enhances sensitivity for near-diloid aneuloid eaks. However, during DNA loidy analysis by flowcytometry simultaneous morhologic comarison of cells is not ossible. The disadvantage of flow cytometry is the requirement for single articles (cells or nuclei) which can lengthen the rocedure in order to obtain single cell susension for solid tissues or fluid with lot of aggregates/debris. 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