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

Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 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.] and II University "La Sapienza" [T. P., A. V.], Rome, Italy; and Regina Elena National Cancer Institute, Rome, Italy 00161 [G. T., P. V., C. B.]

	ABSTRACT

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Purpose: The tumor suppressor gene, FHIT, has been cloned and mapped at chromosome region 3p14.2, one of the regions most frequently deleted in cervical carcinoma. In this report, we show that the expression of the Fhit protein in relation to human papillomavirus (HPV) subtype, the type of the intraepithelial lesion, HIV-induced immunodeficiency, and the DNA content (ploidy) correlates with the biological behavior of the lesions.

Experimental Design: To investigate involvement of the FHIT gene in squamous intraepithelial lesions of low and high grade (LGSILs and HGSILs, respectively) of the uterine cervix, we examined the Fhit protein expression by immunocytochemistry in 131 cervical smears of 96 HIV-seropositive patients (42 with LGSILs and 10 with HGSILs) and 35 HIV-seronegative (5 with LGSILs) persons.

Results: Fhit protein was detected in normal cells, whereas dysplastic cells (independently of HPV infection and HPV subtypes) showed reduced expression of Fhit (P = 0.00001). Lesions from 52 HIV-seropositive patients, 42 LGSILs and 10 HGSILs, showed diploid DNA content in 63.5%, aneuploid in 32.7%, and polyploid in 3.8%, but 90% of the HGSILs showed an aneuploid DNA content, and all were infected by HPV 16/18 subtypes. 23.8% of LGSIL cases were associated with HPV 16.

Conclusions: These data clearly suggest that loss of Fhit expression occurs in the early stages of cervical carcinogenesis. Pap test represents one of the most convenient and rapid procedures available in identification of cellular changes; hence, Fhit staining might be used as an useful tool in larger population screening to detect early alteration in cellular behaviors.

	INTRODUCTION

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Cervical carcinoma is one of the most deadly neoplasms in women, particularly in developing countries (1) . The relationship between HPV³ infection and precancerous cervical lesions, such as LGSILs and HGSILs, has been demonstrated clearly (2, 3, 4) . Cytomorphological identification of cellular changes is currently the most convenient, rapid, economical, and sensitive procedure available for detection of HPV infection in the genital tract.

HPV, a DNA virus, affects both the nucleus and the cytoplasm of the infected cells with specific changes. HPV DNA may occur within epithelial cell nuclei as either unintegrated or integrated. Nuclear changes tend to be more pronounced in cases with HPV DNA integration into the epithelial cell nuclear DNA. Such lesions typically appear as dysplastic and atypical. Chromosome and ploidy alterations, hallmarks of SILs or dysplastic change, may occur in these cases. The classic manifestation of HPV infection is the presence of the koilocyte (5) .

HIV infection is an important risk factor for HPV infection and associated lesions in the anogenital tracts of women (6, 7, 8, 9) . Mechanisms by which HIV intensifies the pathogenic effect of HPV in cervical cancer cells are not yet well defined, but depressed immunological control of HPV replication or interaction between HPV and HIV may be contributing factors. It has been demonstrated that HIV has an effect on cervical immune function, reducing the number of Langerhans cells and their function (10) . These observations suggest a complex influence of HIV on induced immunosuppression, increased susceptibility to HPV infection, and facilitated evolution of the associated 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 insufficient for tumor development (12) . Other events, such as tumor suppressor gene inactivation, are probably crucial in cervical carcinoma pathogenesis. Deletions of the short arm of chromosome 3 (3p) have been detected by loss of heterozygosity studies in cervical cancer (13, 14, 15) . Regions spanning 3p13–14.3, 3p13–21.3, 3p14.2, and 3p21 have been shown to be deleted, suggesting the presence of tumor suppressor genes responsible for cervical carcinogenesis in these regions. In addition, cytogenetics studies have suggested a correlation between common chromosomal fragile sites and the integration sites of HPV (16) .

The tumor suppressor gene, FHIT, has been cloned and mapped to chromosome region 3p14.2, one of the regions most frequently deleted in cervical carcinoma. The FHIT gene encompasses a region of over 1 Mb, which includes the common FRA3B fragile region, and encodes a protein of M_r 16,800, with diadenosine triphosphate hydrolase activity (17) . Common constitutive fragile sites are chromosomal regions that show gaps and breaks after treatment with chemicals that inhibit DNA replication, usually aphidicolin. These fragile sites, which occur in all individuals, have been shown to be highly recombinogenic and to participate in deletions and translocations. Accordingly, a papillomavirus integration site, plasmid integration sites, and cancer-specific translocations have been mapped within the FRA3B fragile site in the FHIT gene (18) .

In this report, we studied the expression of the Fhit protein by immunocytochemistry of pap smears in relation to HPV subtype, the type of the SIL, HIV-induced immunodeficiency, and the DNA content (ploidy) to correlate the biological behavior of the lesions.

	PATIENTS AND METHODS

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Human Subjects and Sample Collection.
Cervical smears of 131 patients were analyzed; 96 HIV seropositive with an average age of 32.8 years (SD, 5.2; range, 24–50 years) and 35 HIV seronegative with an average age of 31.8 years (SD, 6.6; range, 21–55 years). HIV-seropositive patients were followed clinically, and HIV-seronegative patients were a category with a risk of infection. None of the patients had had previous cervical lesions. All were recruited in the Hospital "Umberto I" of the University of Rome "La Sapienza" and gave their informed consent.

The patients were interviewed for details of their gynecological history and possible HIV and HPV risk factors. All patients were submitted to gynecological visit with colposcopy, and cervical samples were obtained by exfoliation with an Ayre spatula. For each patient, three smears were collected; one was fixed in alcohol at 95% for the Papanicolau stains analysis according to the Bethesda System (19) , and two smears were fixed in acetone at 4°C for immunocytochemical analysis and DNA ploidy (with Feulgen method). The residual cellular material on the spatula was used for molecular determination of HPV by PCR. Only HIV-seropositive and -seronegative cases with cytological diagnosis of LGSIL and HGSIL (histologically confirmed) were successively evaluated by molecular analysis for HPV-PCR.

Immunocytochemistry.
All cytological smears with LGSILs and HGSILs were stained with rabbit polyclonal anti-Fhit antibody that had been raised against a glutathione S-transferase-Fhit fusion protein, without a preceding antigen retrieval 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 was used as chromogen, and slides were counterstained with hematoxylin. As negative controls, we used cell pellets from one cervical and two lung cancer cell lines that had been shown previously to be Fhit negative. As positive controls, we used similarly processed cell pellets of a Fhit-positive lung cancer cell line and a cervical cancer cell line. The degree of immunostaining was evaluated by counting diaminobenzidine-stained cervical cells in a minimum of 500 cells in different microscopic fields at a magnification of x400 and was expressed as a percentage. All cell smears were examined independently by two investigators, and complete agreement was found for determinations of Fhit protein positivity or negativity.

DNA Ploidy.
Ploidy analysis was performed only on 52 HIV-seropositive cases with LGSILs and HGSILs using the Becton Dickinson CAS 200 apparatus Ploidy Analysis (Program 2.3) on a sample stained previously by a modified Feulgen method using a commercial kit, Quantitative DNA staining kit 102300-01 (Becton Dickinson, Elmhurst, IL). The apparatus was first calibrated on mouse hepatocytes provided by the same manufacturer and then on at least 30 normotypical epithelial elements removed from a healthy area of the lateral fornix and distally smeared on the same slide. Analysis was performed on at least 100 morphologically atypical elements. When both diploid (or polyploid) and aneuploid clones were present, the lesion was regarded as aneuploid.

Molecular Detection of HPV by PCR.
All cytological cervical samples with LGSILs and HGSILs (52 HIV seropositive and 5 HIV seronegative) were routinely fixed and studied. Intraepithelial lesions were classified as viral, low-grade, and high-grade lesions according to the Bethesda System.

To avoid contamination, oligonucleotide synthesis, sample preparation, and PCR reactions were performed in distinct laboratories. Cytological samples were collected in sterile 1.5 polypropylene tubes and resuspended in 100 µl of digestion buffer with proteinase K, incubated overnight at 37°C, and boiled for 5 min.

Aliquots (10 µl) of each were used for PCR amplification. Each cytological sample was analyzed by PCR for HPV open reading frame sequences using the following primers: HPV-6/11, 5'-gAC CAg TTg TgC AAg TTTAATC-3' and 3'-CTT CCA TgC ATg TTg TCC AgC Ag-5'; HPV-16, 5'-ACC gAA ACC ggT Tag TATAAAAgC-3' and 3'-gAT CAT TTg TCT CTg gTT gCA AAT-5'; HPV-18, 5'-CAC ACC ACA ATA CTA Tgg CgCgCT-3' and 3'-CTg CTg gAT TCA ACg gTT TCT ggC-5'; and HPV-31/33/51, 5'-TgT CAA AAA CCgTTgTgTCC 3' and 3'-gAg CTg TCg TCg CTT AAT TgC TC-5'.

Every amplification experiment included one negative and one positive control for each viral type. A portion of exon 15 of the human APC gene was routinely amplified as a positive control using the following primers: APC, 5'-gTCCTTCACAgAAtgAAAgATg-3' and 3'-CTg CTT gAA gAA gAC ATA TgTTCg-5'.

The size of the amplified fragments were 399, 576, 360, 275, and 520 bp, respectively. Amplification reactions were carried out in 100 µl of reaction buffer containing 50 mM KCl, 2 mM MgCl2, 10 mM Tris (pH 8.3), 200 µM each deoxynucleotide triphosphate, 2.5 units of Taq DNA polymerase (Perkin-Elmer-Cetus, Norwalk, CT), 100 pmol of each primer, and 10 µl of proteinase K-digested sample. Samples were denatured at 95°C for 5 min, followed by 40 cycles of amplification (denaturation at 94°C for 1.5 min, annealing at 55°C for 2 min, except for HPV-6/11 and APC, where annealing was at 40°C and 57°C, respectively, and extension was at 72°C for 2 min; the final extension was prolonged to 7 min). Amplified products (15 µl) were electrophoresed through 1.6% agarose gels. The gels were analyzed by UV after staining with ethidium bromide.

Statistical Analysis.
The Mann-Whitney U test was performed to determine the statistical significance of differences in Fhit expression, and values of < 0.05 were interpreted as being statistically significant.

	RESULTS

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One hundred thirty-one patients were studied, 96 HIV seropositive and 35 seronegative. Among the 96 HIV-seropositive patients, 12 pap smears were normal, 32 had benign cellular changes, 42 showed LGSILs, and 10 HGSILs. Of the 35 seronegative patients, 12 cases showed reactive alterations by inflammatory type and 5 cases were LGSILs, but HGSILs were not found (Table 1) .

Fhit Protein Expression.
Immunocytochemical detection of Fhit protein in all pap smears was always observed in normal cells; on the contrary, dysplastic cells, independently of HPV infection and HPV subtypes, showed reduced expression of the protein (Fig. 1) . In HGSIL lesions, we observed a strong reduction or lack of Fhit protein expression.

View larger version (145K): [in this window] [in a new window] [Download PPT slide]   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 (x400).

When we examined for viral integration the 52 HIV-seropositive cases with dysplastic cells, we found that 25 of the 52 samples were associated with viral subtypes 31/33/51. Twenty-two of the 25 (91%) were LGSILs and diploid, and 3 were HGSILs, aneuploid, and also HPV-16/18 positive. Ten of 42 LGSILs (23.8%) and 8 of 10 HGSILs (80%) were HPV-16/18 positive. Unexpectedly, viral subtype 6/11 was found in the 1 aneuploid LGSIL. Strong reduction or lack of Fhit protein expression in HIV-seropositive cases was observed in 4 aneuploid LGSIL samples infected by HPV-16 and in 8 diploid LGSIL samples infected by HPV-31/33/51. The expression of Fhit protein in dysplastic cells was correlated with the cytological diagnosis (LGSIL or HGSIL), DNA ploidy analysis, and HPV subtypes. We observed complete absence of expression of Fhit protein in HGSIL dysplastic cells and HPV 18 lesions and a reduction to one-third in aneuploid/polyploid lesions. As expected, samples infected with 31/33/51 showed an increase of Fhit expression, compared with the ones infected by HPV-18 (Table 3) .

	DISCUSSION

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Low-grade lesions rarely develop into invasive carcinomas, whereas high-grade lesions proceed more frequently toward tumor progression (12) . Giovagnoli et al. (22) provided evidence of an increase in both the qualitative and quantitative evaluation of morphological atypias in cells affected by HPV infection and SILs in HIV-seropositive patients compared with the seronegative ones. The nuclei were smaller and darker; koilocytotic and dyskeratotic changes were more frequent and more clearly evident (Ref. 22 and this work). The results show a correlation among cytological diagnosis, ploidy, and immunosuppression.

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 HIV-positive patients, who are more susceptible to the development of dysplastic and successively neoplastic lesions.

It was shown previously that alterations at the FHIT locus occur in both HPV-positive and -negative cervical carcinomas (12, 13, 14) . In the present study, we have confirmed that loss of Fhit expression occurs also in the early stages of the cervical carcinogenic process.

Aberrant FHIT transcripts and loss of protein expression in cervical cancer cell lines, but not in normal cells, suggest that these alterations play an important role in cervicovaginal carcinogenesis. Loss of heterozygosity in regions of chromosome 3p were observed frequently in lung, head and neck, bladder, kidney, and breast cancer (23, 24, 25, 26, 27, 28, 29, 30) . Deletions of regions of 3p are probably early events in preneoplastic lesions of lung, proliferative benign lesions of the breast, and oral leukoplakia (31, 32, 33, 34, 35) .

Functional studies showed that replacement of Fhit in some cancer cells that lacked expression of the endogenous gene suppresses tumorigenicity (36) , demonstrating that Fhit can act as a tumor suppressor, an observation that has been confirmed in studies of Fhit knockout mice (37) . The signal pathway through which Fhit exerts its suppressive effect is not known in detail, although overexpression of exogenous Fhit by infection or transfection causes apoptosis of the Fhit-overexpressing cancer cells (38, 39, 40) .

To better understand the interaction in vivo, between two factors strongly involved in cervical carcinogenesis, FHIT gene inactivation and HPV infection, it would be particularly useful to study the corresponding animal models. Relevant recombinant mutant mouse strains have been established, Fhit knockout mice and K14-HPV 16 transgenic mice (41) ; intercrosses between these mouse models may yield new insights into the mechanisms of interaction between these two etiological factors of cervical cancer.

	ACKNOWLEDGMENTS

 
We thank Dr. Kay Huebner for helpful discussion and critical reading of the manuscript and Dr. Francesco Trapasso for general and technical assistance.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by Grant MURST-COFIN 98 from Ministero dell’Università 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 Center, Thomas Jefferson University, Room 1008, 233 South 10th Street, Philadelphia, PA 19107. Phone: (215) 503-4658; Fax: (215) 923-4498; E-mail: rmancini{at}lac.jci.tju.edu

³ The abbreviations used are: HPV, human papillomavirus; HGSIL, high-grade squamous intraepithelial lesion; LGSIL, low-grade squamous intraepithelial lesion; FHIT, fragile histidine triad; APC, adenomatous polyposis coli.

Received 12/14/00; revised 2/ 8/01; accepted 2/13/01.

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