Developed in 1989, the Bethesda System (TBS) has largely replaced previous classifications of Papanicolaou (Pap) smears from the uterine cervix. The system is binary, dividing smears into two groups - low-grade, squamous, epithelial lesions (LSIL) or high-grade, squamous, epithelial lesions (HSIL). A third category, atypical squamous cells (of undetermined significance ASC-US or cannot exclude HSIL ASC-H), is used to classify minimal cellular changes that do not satisfy the criteria for the low- or high-grade categories. The TBS terminology system for Pap smears has been blamed for the increased numbers of colposcopy referrals, by creating the category of ASCUS. The need of a "I don't know what it is" category is felt by most cytopahtologists as necessary, even by those that abhor the ASCUS label, using other names. TBS has been revised, resulting in some modifications. This is the link to Bethesda 2001.

The presence of koilocytes is the trademark of a self-limiting viral (HPV induced, of low and high risk types) STD epithelial lesion, for which unfortunately no efficacious treatment is available. We think that it has nothing to do directly with intra-epithelial precursor lesions of squamous cell cervical carcinoma, except that is caused by the same family of viruses, with a fundamental difference: HSIL lesions and malignancy (rare events) are preceded by integration of some sequences of the viral DNA into the host genome, specially of the so-called high risk types, followed by other genetic changes, while the infectious lesions (extremely frequent events) are caused by episomal complete virions. Needless to say, both the precursor high-grade epithelial lesions and the cancer are not infective.

HPVs have evolved to accomplish the task of controlling host cell proliferation and differentiation to the end of producing more infectious virions. Coincident with the viral life cycle, however, is the risk that the viral genome will be disrupted and its DNA integrated into the host cell chromosomes (11).

The results of a study done by Cullen (12), in 1991, indicated that detectable integration of HPV DNA, regardless of type, occurs infrequently in cervical intraepithelial neoplasia, findings confirmed by Kalantari et al (13), in 2001. The absence of HPV 16 DNA integration in some carcinomas implied that integration is not always required for malignant progression. In contrast, the consistent integration of HPV 18 DNA in all cervical cancers examined may be related to its greater transforming efficiency in vitro and its reported clinical association with more aggressive cervical cancers (12). HPV genomes are frequently integrated into the host cell genome, some reports suggesting random distribution of chromosomal integration sites, in HPV-related cancers cells (12, 14), usually accompanied by the loss of expression of the viral E2 gene, releasing suppression of viral E6/E7 oncogenes, a key factor for oncogenic progression (15, 16). However, more recent reports suggest specific non random sites, specially at fragile sites, such as FRA13C, FRA3B/FHIT, and FRA17B (17, 18, 19).
The fact that high-risk HPV E6 can inactivate tumor suppressor p53 and E7 can inactivate pRB provides important insight into HPV oncogenesis at the molecular level. It appears to be a very attractive model for the molecular pathogenesis of HPV-related cervical carcinoma. Furthermore, the inability of low-risk HPV E6 and E7 proteins to inactivate or bind to tumor suppressors may in part explain why cervical lesions associated with HPV-6 and HPV-11 rarely progress to invasive cancer(20). The literature on Human papillomavirus oncogenesis was reviewed by zur Hausen and de Villiers in 1994 (21), and by McGlennen in 2000( 11).

The use of systems with three divisions for dysplasia or intra-epithelial lesions, creates a therapeutic dilemma because a single diagnostic category (usually the intermediate grade) may contain both self-limiting and progressive lesions. My answer to the riddle: keep the self-limiting infective viral lesion completely apart, and use a binary sytem for the precursor lesions: low potential for malignancy lesions and high potential for malignancy lesions. At follow up most of low potential lesions (so judged by morphological criteria) should regress, being infective viral lesions without koilocytes, and some of the high potential ones will progress to cancer if not ablated.

The lesions that are cytologically or histologically classified as precursor lesions of the cervical squamous cell carcinoma, or SIL, squamous intra-epithelial lesions, represent a heterogeneous clinical entity, associated with many types of Human papillomaviruses (HPVs), and have a variable biological behavior. The low grade lesions (LSIL), HPV condiloma and CIN 1, are caused by any HPV type,  and are most probably transient and self-limited in at least 50% of the times, and regress spontaneously,  specially in women younger than 30-year of age. According to Östor (1), about 50% have a neoplastic behavior, persisting or progressing to high grade lesions (HSIL). The sudy of Östor has considerable problems due to the low sensitivity and reproducibility of cytology. A significant number of such lesons is associated with HPV 31 and 33, the so called "intermediate risk" HPVs (2).

The high grade lesions (HSIL), CIN 2 and 3, and carcinoma in situ, are almost always caused by the so-called "high risk" HPVs, specially types 16 and 18. In at least 50% of the time they occur without previous documentation of a LSIL lesion. Laura A Koutsky et al., of the University of Washington, Seattle,  in 1992 (3), studied prospectively a cohort of 241 women who presented for evaluation of sexually transmitted disease and had negative cervical cytologic tests. The women were followed every four months with cytologic and colposcopic examinations of the uterine cervix and tests for HPV DNA and other sexually transmitted diseases. Cervical intraepithelial neoplasia grade 2 or 3 was confirmed by biopsy in 28 women. All 24 cases of cervical intraepithelial neoplasia grade 2 or 3 among HPV-positive women were detected within 24 months after the first positive test for HPV.

Some epidemiological studies, have looked at the so-called "interval cancers", that develop shortly after the demonstration of a cytologically negative smear. According to the group of Janerich et al. (4)  in a population-based study of all women in Connecticut who developed cervical cancer between 1985 and 1990, a total of 118 of 481 (24.5%) participants were diagnosed with cervical cancer within 3 years of their last true-negative Papanicolaou smear. Adenocarcinomas occurred in 38 cases (32.2%). These data suggest that rapidly occurring cervical cancer may be a manifestation of endocervical carcinomas that have been inadequately screened. Hildesheim et al., (10) from the NIH, demonstrated that compared with normal-onset cases, rapid-onset cases tended to be younger (P =.001) and were more likely to be white (P =.002), diagnosed with adenocarcinomas or adenosquamous carcinomas (P =.001), and diagnosed with early-stage disease (P =.001). 

Until quite recently the paradigm was the progression of low grade to high grade lesions, following a cytological and histological spectrum (5, 6). This paradigm has been refuted by several studies, including the ones by Koutsky et al. (3) , and Schiffman et al. (7): they demonstrated that low grade lesions are almost always self-limited, and transient, while the high grade lesions are de novo lesions. Cervical cancer appears as a rapidly expansive clone inside the high grade lesion developed some time before. J. H. Robertson, B. Woodend, and J. Hutchinson (8), said this as early as 1994: in their study, cervical smears were reviewed from 62 women who developed squamous carcinoma of the cervix up to 18 years later. Their findings indicate that the prevention of cervical cancer by screening depends very largely on the detection of severe dyskaryosis. In their series there was no evidence that mild dyskaryosis (the British equivalent to LSIL) was a forerunner of invasive disease. Cytology during the evolution of squamous carcinoma is not characterised by a dyskaryosis which progressively increases in severity. Instead the findings support new concepts that cervical cancer generally arises from an aggressive CIN 3 lesion widely present in the cervix, and in their series, established years before invasion occurs. They concluded that would be more useful to report cytology as showing either a low or high grade abnormality rather than distinguishing between different degrees of dyskaryosis. In line with this new approach, Nancy B. Kiviat and Laura  A. Koutsky, in an editorial of the Journal of the National Cancer Institute, in March 20, 1996, (9) ask for high priority for detecting the high grade lesions, in the cervical cancer prevention strategies, a position we strongly endorse.

What clinical role has the DNA-based identification of HPV types, remains to be determined. We predict that specially the "I don't know what it is" or ASC-US or ASC-H situations benefit from it, as recently demonstrated by the results of the ALTS trial (22): Hybrid Capture 2 testing for cancer-associated HPV DNA was a viable option in the management of women with ASCUS. It had greater sensitivity to detect CIN3 or above and specificity comparable to a single additional cytologic test indicating ASCUS or above. Manos et al (23), in 1999 achieved similar results: for women with ASCUS Pap tests, HPV DNA testing of residual specimens collected for routine cervical liquid based cytology helped identify those who had underlying HSIL. By testing the specimen collected at initial screening, the majority of high-risk cases can be identified and referred for colposcopy based on a single screening. Similar conclusions were reached in a study done in France by Bergeron (24). Because a very high percentage (over 80%) of women with a LSIL diagnosis from Pap smears are positive for HPV DNA by Hybrid Capture 2 testing, there is limited potential for this assay to direct decisions about the clinical management of women with LSIL (25). Another role of HPV DNA testing should be the follow up of lesions treated by conization, but the testing should wait for at least 6 months to be performed, to avoid false positives.

The use of HPV DNA testing as a primary screening strategy (used alone or in combination with cytology) should be tested in a large prospective trial, specially in countries with resources to do so, but there will be a problem: what to do with a woman with HPV DNA positive test and negative cytology and colposcopy? My personal opinion: repeat in one year to document persistent positivity. The persistently high risk HPV infected transformation zone implies a very significant risk for cancer, and should be ablated.

To end up, read this interesting piece of writing on ASCUS madness... .

References

1. Östor, AG. Natural history of cervical intraepithelial neoplasia: a critical review. Int J Gynecol Pathol 1993;12:186-192.

2. Lorincz, AT, Reid R, Jenson AB, Greenberg MD, Lancaster W, Kurman RJ. Human papillomavirus infection of the cervix: relative risk associations of 15 common anogenital types. Obstet Gynecol 1992;79: 328-337.

3. Koutsky, LA, Holmes KK, Critchlow CW, Stevens CE, Paavonen J, Beckmann AM, DeRouen TA, Galloway DA, Vernon D, Kiviat NB. A cohort study of the risk of cervical intraepithelial neoplasia grade 2 or 3 in relation to papillomavirus infection. N Engl J Med 1992;327: 1272-1278.

4. Schwartz PE, Hadjimichael O, Lowell DM, Merino MJ, Janerich D. Rapidly progressive cervical cancer: the Connecticut experience. Am J Obstet Gynecol. 1996;175:1105-1109.

5. Koss, LG. Concept of genesis and development of carcinoma of the cervix. Obstet Gynecol Surv 1969;24:850-860.

6. Richart, RM, Barron BA. A follow-up study of patients with cervical dysplasia. Am J Obstet Gynecol 1969, 105, 386-393.

7. Schiffman, MH, Bauer HM, Hoover RN, Glass AG, Cadell DM, Rush BB, Scott DR, Sherman ME, Kurman RJ, Wacholder S, et al. Epidemiologic evidence showing that human papillomavirus infection causes most cervical intraepithelial neoplasia. J Natl Cancer Inst 1993, 85, 958-964.

8. Robertson, JH, Woodend B, Hutchinson J. Cytological changes preceding cervical cancer. J Clin Pathol 1994, 47, 278-279.

9. Kiviat, NB, Koutsky LA. Do our current cervical cancer control strategies still make sense? J Natl Cancer Inst 1996, 88, 317-318.

10. Hildesheim A, Hadjimichael O, Schwartz PE, Wheeler CM, Barnes W, Lowell DM, Willett J, Schiffman M. Risk factors for rapid-onset cervical cancer. Am J Obstet Gynecol 1999;180:571-577.

11. McGlennen RC. Human papillomavirus oncogenesis. Clin Lab Med 2000;20:383-406.

12. Cullen AP, Reid R, Campion M, Lorincz AT. Analysis of the physical state of different human papillomavirus DNAs in intraepithelial and invasive cervical neoplasm. J Virol 1991;65:606-612.

13. Kalantari M,Blennow E, Hagmar B, Johansson B. Physical state of HPV16 and chromosomal mapping of the integrated form in cervical carcinomas. Diagn Mol Pathol 2001;10:46-54.

14. Luft F, Klaes R, Nees M, Durst M, Heilmann V, Melsheimer P, von Knebel Doeberitz M. Detection of integrated papillomavirus sequences by ligation-mediated PCR (DIPS-PCR) and molecular characterization in cervical cancer cells. Int J Cancer 2001;92:9-17.

15. Wells SI, Francis DA, Karpova AY, Dowhanick JJ, Benson JD, Howley PM. Papillomavirus E2 induces senescence in HPV-positive cells via pRB- and p21(CIP)-dependent pathways. EMBO J 2000;19:5762-5771.

16. Tonon SA, Picconi MA, Bos PD, Zinovich JB, Galuppo J, Alonio LV, Teyssie AR. Physical status of the E2 human papilloma virus 16 viral gene in cervical preneoplastic and neoplastic lesions. J Clin Virol 2001;21:129-134.

17. Thorland EC, Myers SL, Persing DH, Sarkar G, McGovern RM, Gostout BS, Smith DI. Human papillomavirus type 16 integrations in cervical tumors frequently occur in common fragile sites. Cancer Res 2000;60:5916-5921.

18. Corden SA, Sant-Cassia LJ, Easton AJ, Morris AG. The integration of HPV-18 DNA in cervical carcinoma. Mol Pathol 1999;52:275-282.

19. Hidalgo A, Monroy A, Arana RM, Taja L, Vazquez G, Salcedo M. Chromosomal imbalances in four new uterine cervix carcinoma derived cell lines. BMC Cancer. 2003;3(1):8.

20. Scheffner M, Romanczuk H, Munger K, Huibregtse JM, Mietz JA, Howley PM. Functions of human papillomavirus proteins. Curr Topics Microbiol Immunol 1994; 186:83–99.

21. zur Hausen H, de Villiers EM. Human papillomaviruses. Annu Rev Microbiol 1994;48:427-447.

22. Solomon D, Schiffman M, Tarone R; ALTS Study group. Comparison of three management strategies for patients with atypical squamous cells of undetermined significance: baseline results from a randomized trial. J Natl Cancer Inst 2001;93:293-299.

23. Manos MM, Kinney WK, Hurley LB, Sherman ME, Shieh-Ngai J, Kurman RJ, Ransley JE, Fetterman BJ, Hartinger JS, McIntosh KM, Pawlick GF, Hiatt RA.Identifying women with cervical neoplasia: using human papillomavirus DNA testing for equivocal Papanicolaou results. JAMA. 1999;281:1645-1647

24. Bergeron C, Jeannel D, Poveda J, Cassonnet P, Orth G. Human papillomavirus testing in women with mild cytologic atypia. Obstet Gynecol 2000;95:821-827.

25. No authors. Human papillomavirus testing for triage of women with cytologic evidence of low-grade squamous intraepithelial lesions: baseline data from a randomized trial. The Atypical Squamous Cells of Undetermined Significance/Low-Grade Squamous Intraepithelial Lesions Triage Study (ALTS) Group. J Natl Cancer Inst 2000;92:397-402.

The Nov 21, 2002 issue of The New England Journal of Medicine ( Vol 347 #21 pp1645-1651) brings an extremely important paper by Laura A. Koustky et al., A Controlled Trial of a Human Papillomavirus Type 16 Vaccine.
The Editorial by Christopher P. Crum - The Beginning of the End for Cervical Cancer? pp 1703-1705 concludes on a very optimistic tone: "...If the promise implicit in the study by Koutsky et al. is realized, we could, in our lifetime, see the gradual but progressive dismantling of the barriers to preventing cervical cancer. The captives of our current system - both patients and their caregivers - may set free." Amen!