Cervical Dysplasia, Ploidy, and Human Papillomavirus Status Correlate with Loss of Fhit Expression1

1306 Vol. 7, 1306–1312, May 2001 Clinical Cancer Research

Andrea Vecchione, Nicola Zanesi, Conclusions: These data clearly suggest that loss of Fhit Giorgio Trombetta, Debora French, Paolo Visca, expression occurs in the early stages of cervical carcinogenesis. Pap test represents one of the most convenient and Tiziana Pisani, Claudio Botti, Aldo Vecchione, 2 rapid procedures available in identification of cellular Carlo M. Croce, and Rita Mancini changes; hence, Fhit staining might be used as an useful tool Department of Microbiology and Immunology, Kimmel Cancer in larger population screening to detect early alteration in Center, Thomas Jefferson University, Philadelphia, Pennsylvania cellular behaviors. 19107 [A. V., N. Z., C. M. C., R. M.]; Department of Experimental Medicine and Pathology, University “La Sapienza,” Rome, Italy [A. V.]; Centro Ricerche “Ospedale S. Pietro” [D. F., A. V., R. M.] INTRODUCTION and II University “La Sapienza” [T. P., A. V.], Rome, Italy; and Cervical carcinoma is one of the most deadly neoplasms in Regina Elena National Cancer Institute, Rome, Italy 00161 [G. T., P. V., C. B.] women, particularly in developing countries (1). The relation- ship between HPV3 infection and precancerous cervical lesions, such as LGSILs and HGSILs, has been demonstrated clearly ABSTRACT (2–4). Cytomorphological identification of cellular changes is Purpose: The tumor suppressor gene, FHIT, has been currently the most convenient, rapid, economical, and sensitive cloned and mapped at chromosome region 3p14.2, one of the procedure available for detection of HPV infection in the genital regions most frequently deleted in cervical carcinoma. In tract. this report, we show that the expression of the Fhit protein HPV, a DNA virus, affects both the nucleus and the cyto- in relation to human papillomavirus (HPV) subtype, the plasm of the infected cells with specific changes. HPV DNA type of the intraepithelial lesion, HIV-induced immunodefi- may occur within epithelial cell nuclei as either unintegrated or ciency, and the DNA content (ploidy) correlates with the integrated. Nuclear changes tend to be more pronounced in biological behavior of the lesions. cases with HPV DNA integration into the epithelial cell nuclear Experimental Design: To investigate involvement of the DNA. Such lesions typically appear as dysplastic and atypical. FHIT gene in squamous intraepithelial lesions of low and Chromosome and ploidy alterations, hallmarks of SILs or dys- high grade (LGSILs and HGSILs, respectively) of the uter- plastic change, may occur in these cases. The classic manifes- ine cervix, we examined the Fhit protein expression by tation of HPV infection is the presence of the koilocyte (5). immunocytochemistry in 131 cervical smears of 96 HIV- HIV infection is an important risk factor for HPV infection seropositive patients (42 with LGSILs and 10 with HGSILs) and associated lesions in the anogenital tracts of women (6–9). and 35 HIV-seronegative (5 with LGSILs) persons. Mechanisms by which HIV intensifies the pathogenic effect of Results: Fhit protein was detected in normal cells, HPV in cervical cancer cells are not yet well defined, but whereas dysplastic cells (independently of HPV infection depressed immunological control of HPV replication or interaction between HPV and HIV may be contributing factors. It has ؍ and HPV subtypes) showed reduced expression of Fhit (P 0.00001). Lesions from 52 HIV-seropositive patients, 42 been demonstrated that HIV has an effect on cervical immune LGSILs and 10 HGSILs, showed diploid DNA content in function, reducing the number of Langerhans cells and their 63.5%, aneuploid in 32.7%, and polyploid in 3.8%, but 90% function (10). These observations suggest a complex influence of the HGSILs showed an aneuploid DNA content, and all of HIV on induced immunosuppression, increased susceptibility were infected by HPV 16/18 subtypes. 23.8% of LGSIL cases to HPV infection, and facilitated evolution of the associated were associated with HPV 16. anogenital lesions. Furthermore, persistent infection of high oncogenic risk HPV types 16/18 associated with HGSIL development has been observed in HIV-positive women (10, 11). Although HPV infection has an important role in cervicovaginal carcinogenesis, some authors suggest that it is insuffi- Received 12/14/00; revised 2/8/01; accepted 2/13/01. cient for tumor development (12). Other events, such as tumor The costs of publication of this article were defrayed in part by the suppressor gene inactivation, are probably crucial in cervical payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to carcinoma pathogenesis. Deletions of the short arm of chromo- indicate this fact. some 3 (3p) have been detected by loss of heterozygosity studies 1 Supported by Grant MURST-COFIN 98 from Ministero dell’ Universita`e Ricerca Scientifica e Tecnologica and partially by USPHS Grants CA 77738, CA 51083, and CA 56336 from the National Cancer Institute. 2 To whom requests for reprints should be addressed, at Kimmel Cancer 3 The abbreviations used are: HPV, human papillomavirus; HGSIL, Center, Thomas Jefferson University, Room 1008, 233 South 10th high-grade squamous intraepithelial lesion; LGSIL, low-grade squa- Street, Philadelphia, PA 19107. Phone: (215) 503-4658; Fax: (215) 923- mous intraepithelial lesion; FHIT, fragile histidine triad; APC, adenom- 4498; E-mail: [email protected]. atous polyposis coli.

Table 1 Cytological diagnoses HPV by PCR. Only HIV-seropositive and -seronegative cases Diagnosis HIVϩa HIVϪb Total with cytological diagnosis of LGSIL and HGSIL (histologically confirmed) were successively evaluated by molecular analysis Within normal limits 12 (40%) 18 (60%) 30 Benign cellular changes 32 (72.7%) 12 (27.3%) 44 for HPV-PCR. LGSIL 42 (89.4%) 5 (10.6%) 47 Immunocytochemistry. All cytological smears with HGSIL 10 (100%) 0 10 LGSILs and HGSILs were stained with rabbit polyclonal anti- Total 96 35 131 Fhit antibody that had been raised against a glutathione S- a HIV seropositive. transferase-Fhit fusion protein, without a preceding antigen re- b HIV seronegative. trieval step. The antibody was used at a 1:2500 dilution for 2 h at room temperature. After this incubation, smears were treated with biotinylated antirabbit antibody and streptavidin-biotin- peroxidase (Immunotech Delta Kit protocol). Diaminobenzidine in cervical cancer (13–15). Regions spanning 3p13–14.3, 3p13– was used as chromogen, and slides were counterstained with 21.3, 3p14.2, and 3p21 have been shown to be deleted, suggest- hematoxylin. As negative controls, we used cell pellets from one ing the presence of tumor suppressor genes responsible for cervical and two lung cancer cell lines that had been shown cervical carcinogenesis in these regions. In addition, cytogenet- previously to be Fhit negative. As positive controls, we used ics studies have suggested a correlation between common chro- similarly processed cell pellets of a Fhit-positive lung cancer mosomal fragile sites and the integration sites of HPV (16). cell line and a cervical cancer cell line. The degree of immu- The tumor suppressor gene, FHIT, has been cloned and nostaining was evaluated by counting diaminobenzidine-stained mapped to chromosome region 3p14.2, one of the regions most cervical cells in a minimum of 500 cells in different microscopic frequently deleted in cervical carcinoma. The FHIT gene en- fields at a magnification of ϫ400 and was expressed as a compasses a region of over 1 Mb, which includes the common percentage. All cell smears were examined independently by FRA3B fragile region, and encodes a protein of Mr 16,800, with two investigators, and complete agreement was found for deter- diadenosine triphosphate hydrolase activity (17). Common con- minations of Fhit protein positivity or negativity. stitutive fragile sites are chromosomal regions that show gaps DNA Ploidy. Ploidy analysis was performed only on 52 and breaks after treatment with chemicals that inhibit DNA HIV-seropositive cases with LGSILs and HGSILs using the replication, usually aphidicolin. These fragile sites, which occur Becton Dickinson CAS 200 apparatus Ploidy Analysis (Program in all individuals, have been shown to be highly recombinogenic 2.3) on a sample stained previously by a modified Feulgen and to participate in deletions and translocations. Accordingly, a method using a commercial kit, Quantitative DNA staining kit papillomavirus integration site, plasmid integration sites, and 102300-01 (Becton Dickinson, Elmhurst, IL). The apparatus cancer-specific translocations have been mapped within the was first calibrated on mouse hepatocytes provided by the same FRA3B fragile site in the FHIT gene (18). manufacturer and then on at least 30 normotypical epithelial In this report, we studied the expression of the Fhit protein elements removed from a healthy area of the lateral fornix and by immunocytochemistry of pap smears in relation to HPV distally smeared on the same slide. Analysis was performed on subtype, the type of the SIL, HIV-induced immunodeficiency, at least 100 morphologically atypical elements. When both and the DNA content (ploidy) to correlate the biological behav- diploid (or polyploid) and aneuploid clones were present, the ior of the lesions. lesion was regarded as aneuploid. Molecular Detection of HPV by PCR. All cytological PATIENTS AND METHODS cervical samples with LGSILs and HGSILs (52 HIV seroposi- Human Subjects and Sample Collection. Cervical tive and 5 HIV seronegative) were routinely fixed and studied. smears of 131 patients were analyzed; 96 HIV seropositive with Intraepithelial lesions were classified as viral, low-grade, and an average age of 32.8 years (SD, 5.2; range, 24–50 years) and high-grade lesions according to the Bethesda System. 35 HIV seronegative with an average age of 31.8 years (SD, 6.6; To avoid contamination, oligonucleotide synthesis, sample range, 21–55 years). HIV-seropositive patients were followed preparation, and PCR reactions were performed in distinct lab- clinically, and HIV-seronegative patients were a category with a oratories. Cytological samples were collected in sterile 1.5 risk of infection. None of the patients had had previous cervical polypropylene tubes and resuspended in 100 ␮l of digestion lesions. All were recruited in the Hospital “Umberto I” of the buffer with proteinase K, incubated overnight at 37°C, and University of Rome “La Sapienza” and gave their informed boiled for 5 min. consent. Aliquots (10 ␮l) of each were used for PCR amplification. The patients were interviewed for details of their gyneco- Each cytological sample was analyzed by PCR for HPV open logical history and possible HIV and HPV risk factors. All reading frame sequences using the following primers: HPV-6/ patients were submitted to gynecological visit with colposcopy, 11, 5Ј-gAC CAg TTg TgC AAg TTTAATC-3Ј and 3Ј-CTT and cervical samples were obtained by exfoliation with an Ayre CCA TgC ATg TTg TCC AgC Ag-5Ј; HPV-16, 5Ј-ACC gAA spatula. For each patient, three smears were collected; one was ACC ggT Tag TATAAAAgC-3Ј and 3Ј-gAT CAT TTg TCT fixed in alcohol at 95% for the Papanicolau stains analysis CTg gTT gCA AAT-5Ј; HPV-18, 5Ј-CAC ACC ACA ATA according to the Bethesda System (19), and two smears were CTA Tgg CgCgCT-3Ј and 3Ј-CTg CTg gAT TCA ACg gTT fixed in acetone at 4°C for immunocytochemical analysis and TCT ggC-5Ј; and HPV-31/33/51, 5Ј-TgT CAA AAA CCgTT- DNA ploidy (with Feulgen method). The residual cellular ma- gTgTCC 3Ј and 3Ј-gAg CTg TCg TCg CTT AAT TgC TC-5Ј. terial on the spatula was used for molecular determination of Every amplification experiment included one negative and

Table 2 Different end points studied in 52 HIV-seropositive cases Cytomorphological CD4 Fhit expression diagnosis Ploidy cells/␮l in dysplastic HPV-6/11 HPV-16 HPV-18 HPV-31/33/51 LGSIL Aa 170 20 Neg Neg Neg Neg LGSIL A 240 40 Neg Pos Neg Neg LGSIL A 210 40 Neg Pos Neg Neg LGSIL A 290 60 Pos Pos Neg Pos LGSIL A 223 10 Neg Pos Neg Neg LGSIL A 198 0 Neg Neg Neg Neg LGSIL A 280 15 Neg Pos Neg Neg LGSIL A 369 25 Neg Pos Neg Neg LGSIL D 495 50 Neg Pos Neg Pos LGSIL D 481 60 Neg Neg Neg Pos LGSIL D 330 55 Neg Neg Neg Pos LGSIL D 280 60 Neg Neg Neg Pos LGSIL D 240 55 Neg Neg Neg Pos LGSIL D 290 50 Neg Neg Neg Pos LGSIL D 570 50 Neg Neg Neg Pos LGSIL D 629 15 Neg Neg Neg Pos LGSIL D 750 0 Neg Neg Neg Pos LGSIL D 501 50 Neg Neg Neg Neg LGSIL D 298 60 Neg Neg Neg Pos LGSIL D 269 40 Neg Neg Neg Pos LGSIL D 280 30 Neg Neg Neg Pos LGSIL D 290 30 Neg Neg Neg Pos LGSIL D 228 25 Neg Pos Neg Neg LGSIL D 472 40 Neg Neg Neg Neg LGSIL D 79 0 Neg Neg Neg Pos LGSIL D 400 40 Neg Neg Neg Pos LGSIL D 950 20 Neg Pos Neg Neg LGSIL D 590 25 Neg Neg Neg Neg LGSIL D 780 45 Neg Neg Neg Pos LGSIL D 459 40 Neg Neg Neg Neg LGSIL D 205 15 Neg Neg Neg Neg LGSIL D 563 30 Neg Neg Neg Pos LGSIL D 789 45 Neg Neg Neg Pos LGSIL D 467 40 Neg Neg Neg Pos LGSIL D 678 50 Neg Neg Neg Pos LGSIL D 698 35 Neg Neg Neg Neg LGSIL D 296 40 Neg Neg Neg Neg LGSIL D 354 25 Neg Neg Neg Pos LGSIL D 364 30 Neg Neg Neg Pos LGSIL D 654 35 Neg Neg Neg Neg LGSIL D 578 45 Neg Neg Neg Neg LGSIL P 429 13 Neg Pos Neg Neg HGSIL A 125 5 Neg Pos Pos Pos HGSIL A 85 10 Neg Pos Pos Pos HGSIL A 210 0 Neg Neg Pos Neg HGSIL A 185 0 Neg Pos Pos Neg HGSIL A 145 0 Neg Neg Pos Neg HGSIL A 215 0 Neg Neg Neg Neg HGSIL A 169 0 Neg Neg Pos Neg HGSIL A 235 0 Neg Neg Neg Neg HGSIL A 214 0 Neg Pos Pos Neg HGSIL P 146 0 Neg Neg Pos Neg a A, aneuploid; D, diploid; P, polyploid; Neg, negative; Pos, positive.

one positive control for each viral type. A portion of exon 15 of deoxynucleotide triphosphate, 2.5 units of Taq DNA poly- the human APC gene was routinely amplified as a positive merase (Perkin-Elmer-Cetus, Norwalk, CT), 100 pmol of control using the following primers: APC, 5Ј-gTCCTTCACA- each primer, and 10 ␮l of proteinase K-digested sample. gAAtgAAAgATg-3Ј and 3Ј-CTg CTT gAA gAA gAC ATA Samples were denatured at 95°C for 5 min, followed by 40 TgTTCg-5Ј. cycles of amplification (denaturation at 94°C for 1.5 min, The size of the amplified fragments were 399, 576, 360, annealing at 55°C for 2 min, except for HPV-6/11 and APC, 275, and 520 bp, respectively. Amplification reactions were where annealing was at 40°C and 57°C, respectively, and carried out in 100 ␮l of reaction buffer containing 50 mM extension was at 72°C for 2 min; the final extension was KCl, 2 mM MgCl2, 10 mM Tris (pH 8.3), 200 ␮M each prolonged to 7 min). Amplified products (15 ␮l) were elec-

Fig. 1 A LGSIL from a HIV-seropositive patient. Dark staining, normal superficial squamous cells, strongly positive for Fhit staining. The other cells are LGSIL negative for Fhit (ϫ400).

trophoresed through 1.6% agarose gels. The gels were ana- only 15 of 52 HIV-positive patients (15.6%) showed CD4ϩ lyzed by UV after staining with ethidium bromide. values Ͻ250 cells/␮l. A CD4ϩ value Ͻ250 cells/␮l is com- Statistical Analysis. The Mann-Whitney U test was per- monly considered clinical AIDS. In this group, 14 showed SIL formed to determine the statistical significance of differences in lesions. We observed aneuploid lesions in 17 patients, and 14 of Fhit expression, and values of ␣Ͻ0.05 were interpreted as them, all with LGSILs and HGSILs, showed CD4ϩ levels Ͻ250 being statistically significant. cell/␮l. Fhit Protein Expression. Immunocytochemical detec- RESULTS tion of Fhit protein in all pap smears was always observed in One hundred thirty-one patients were studied, 96 HIV normal cells; on the contrary, dysplastic cells, independently of seropositive and 35 seronegative. Among the 96 HIV-seropos- HPV infection and HPV subtypes, showed reduced expression itive patients, 12 pap smears were normal, 32 had benign cel- of the protein (Fig. 1). In HGSIL lesions, we observed a strong lular changes, 42 showed LGSILs, and 10 HGSILs. Of the 35 reduction or lack of Fhit protein expression. seronegative patients, 12 cases showed reactive alterations by We identified HPV subtypes by PCR reaction and observed inflammatory type and 5 cases were LGSILs, but HGSILs were that 4 LGSIL samples in HIV-seronegative patients were asso- not found (Table 1). ciated with viral subtypes 31/33/51 and 1 with HPV-16/18. Fhit Fhit expression was evaluated in all cases. HPV subtypes protein expression was decreased in all 5 cases (48%). were detected in dysplastic cells of 52 HIV-seropositive patients When we examined for viral integration the 52 HIV-sero- and 5 HIV-seronegative patients. Ploidy analysis and CD4ϩ positive cases with dysplastic cells, we found that 25 of the 52 lymphocytes were analyzed in 52 HIV-seropositive patients samples were associated with viral subtypes 31/33/51. Twenty- characterized by LGSILs and HGSILs (Table 2). Of these le- two of the 25 (91%) were LGSILs and diploid, and 3 were sions, 33 (63.5%) exhibited a diploid DNA content, 17 (32.7%) HGSILs, aneuploid, and also HPV-16/18 positive. Ten of 42 aneuploid, and 2 (3.8%) polyploid. Nine of 10 HGSILs of LGSILs (23.8%) and 8 of 10 HGSILs (80%) were HPV-16/18 HIV-positive cases showed an aneuploid DNA content, and all positive. Unexpectedly, viral subtype 6/11 was found in the 1 were infected by HPV-16/18 subtypes. Ten of 42 LGSILs of aneuploid LGSIL. Strong reduction or lack of Fhit protein HIV-positive cases (23.8%) were associated with HPV-16. Fur- expression in HIV-seropositive cases was observed in 4 aneu- thermore, 33 samples in this group (78.6%) had a diploid DNA, ploid LGSIL samples infected by HPV-16 and in 8 diploid and 8 (19%) had aneuploid DNA content; 6 of 8 aneuploid cases LGSIL samples infected by HPV-31/33/51. The expression of were associated with HPV-16. Fhit protein in dysplastic cells was correlated with the cytolog- We studied the level of CD4ϩ-positive lymphocytes, and ical diagnosis (LGSIL or HGSIL), DNA ploidy analysis, and

Table 3 Fhit expression in 52 HIVϩcasesa Table 4 Fhit expression in LGSILsa of HIVϩ cases No. of cases Dysplastic cells, Fhit positive No. of cases Dysplastic cells, Fhit positive Cytological diagnosis Ploidy LGSIL 42 34.6 Ϯ 17 Diploid 33 37.3 Ϯ 15.8 HGSIL 10 1.5 Ϯ 3.4 Aneuploid/polyploid 9 24.8 Ϯ 18.7 ␣Ͻ0.000001 ␣ϭ0.0048 Ploidy HPV-16 Diploid 33 37.3 Ϯ 15.8 ϩ 10 36.1 Ϯ 17 Aneuploid/polyploid 19 12.5 Ϯ 3.4 Ϫ 32 29.8 Ϯ 16.9 ␣Ͻ0.001 ␣ϭ0.223 HPV-18 HPV-31/33/51 ϩ 8 1.9 Ϯ 3.7 ϩ 23 28 Ϯ 13.8 Ϫ 44 33 Ϯ 18.1 Ϫ 19 40 Ϯ 17.8 ␣Ͻ0.0001 ␣ϭ0.010 HPV-31/33/51 a Values are expressed as the mean percentage of positive cells. ϩ 25 37.4 Ϯ 19.3 Ϫ 27 19.7 Ϯ 17.4 ␣Ͻ0.001 a Values are expressed as the mean percentage of positive cells. Interestingly, in the HIV-positive patients we found frequent association with the high oncogenic-risk HPV subtypes 16/18. For this reason, we consider viral typing important in HPV subtypes. We observed complete absence of expression of HIV-positive patients, who are more susceptible to the devel- Fhit protein in HGSIL dysplastic cells and HPV 18 lesions and opment of dysplastic and successively neoplastic lesions. a reduction to one-third in aneuploid/polyploid lesions. As ex- It was shown previously that alterations at the FHIT locus pected, samples infected with 31/33/51 showed an increase of occur in both HPV-positive and -negative cervical carcinomas Fhit expression, compared with the ones infected by HPV-18 (12–14). In the present study, we have confirmed that loss of (Table 3). Fhit expression occurs also in the early stages of the cervical We measured the level of Fhit expression only in LGSILs carcinogenic process. (Table 4) because the average number of dysplastic cells was Aberrant FHIT transcripts and loss of protein expression in close to zero in HGSILs (Table 3) because of a large number of cervical cancer cell lines, but not in normal cells, suggest that koilocytes. In Table 4, we show that the number of Fhit-positive these alterations play an important role in cervicovaginal carci- dysplastic cells in aneuploid/polyploid LGSILs is lower than in nogenesis. Loss of heterozygosity in regions of chromosome 3p diploid cells (␣ϭ0.0048). Even in LGSIL dysplastic cells were observed frequently in lung, head and neck, bladder, infected by HPV-31/33/51, we found fewer Fhit-positive cells kidney, and breast cancer (23–30). Deletions of regions of 3p than in noninfected ones (␣ϭ0.010). are probably early events in preneoplastic lesions of lung, proliferative benign lesions of the breast, and oral leukoplakia DISCUSSION (31–35). The high-risk HPV subtypes 16, 18, 31, 33, and 51 are Functional studies showed that replacement of Fhit in some involved in cervical carcinoma etiopathogenesis (10, 11), al- cancer cells that lacked expression of the endogenous gene though these viruses are by themselves insufficient for complete suppresses tumorigenicity (36), demonstrating that Fhit can act transformation of the cervicovaginal epithelium (1, 8, 12). In as a tumor suppressor, an observation that has been confirmed in precancerous lesions, the ploidy may dramatically change; the studies of Fhit knockout mice (37). The signal pathway through presence of aneuploid clones has been shown in many tumors which Fhit exerts its suppressive effect is not known in detail, (20). Another important factor in viral infection is the relation- although overexpression of exogenous Fhit by infection or ship between ploidy and different HPV subtypes. Goppinger et transfection causes apoptosis of the Fhit-overexpressing cancer al. (21) showed that 84% of HPV-16/18 lesions were aneuploid. cells (38–40). It has been shown that HPV-16/18 aneuploid lesions lead to a To better understand the interaction in vivo, between two poor prognosis, whereas HPV-16/18 diploid lesions lead to a factors strongly involved in cervical carcinogenesis, FHIT gene good prognosis. Ninety % of cervical cancers are SILs. inactivation and HPV infection, it would be particularly useful Low-grade lesions rarely develop into invasive carcinomas, to study the corresponding animal models. Relevant recombi- whereas high-grade lesions proceed more frequently toward nant mutant mouse strains have been established, Fhit knockout tumor progression (12). Giovagnoli et al. (22) provided evi- mice and K14-HPV 16 transgenic mice (41); intercrosses be- dence of an increase in both the qualitative and quantitative tween these mouse models may yield new insights into the evaluation of morphological atypias in cells affected by HPV mechanisms of interaction between these two etiological factors infection and SILs in HIV-seropositive patients compared with of cervical cancer. the seronegative ones. The nuclei were smaller and darker; koilocytotic and dyskeratotic changes were more frequent and ACKNOWLEDGMENTS more clearly evident (Ref. 22 and this work). The results show We thank Dr. Kay Huebner for helpful discussion and critical a correlation among cytological diagnosis, ploidy, and immu- reading of the manuscript and Dr. Francesco Trapasso for general and nosuppression. technical assistance.

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