Method of detection of carcinogenic human papillomavirus

Details Method of detection of carcinogenic human papillomavirus Method of detection of carcinogenic human papillomavirus

1997-12-30

2001-04-17

Arthur, Lisa B. (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S006120, C435S091200

Reexamination Certificate

active

06218104

ABSTRACT:

TECHNICAL FIELD OF INVENTION
This invention relates to a method for the specific detection of the DNA of papillomaviruses in clinical samples. In particular, the test aims to differentiate, in the shortest possible time, whether cells from the anogenital region contain types of papillomavirus that are associated with benign lesions. Such differentiation has important implications in patient evaluation and follow-up.
BACKGROUND
Cancer of the cervix is the most common cancer in women (~25% of all female cancer). Moreover, the incidence is increasing in younger women. Indeed, approximately 2% of routine cervical smears show abnormal cytology, indicating an epidemic. Such an epidemic is current in many western and developing countries. Sexual activity appears to be an important predisposing factor in the epidemiology of cardinogenesis and precancerous lesions. An early age of sexual intercourse and multiplicity of sexual partners are associated statistically with an increased risk of malignancy [Harris et al.,
Br. J. Cancer
42: 359-63, 1980]. The consorts are often men with penile warts (“high risk males”), and a very high proportion (>90%) of cervical carcinoma tissue contain detectable DNA sequences for known varieties of the human papillomavirus (HPV). This supports a growing body of evidence implicating certain types of HPV as the sexually transmitted factor involved in the development of squamous-cell carcinoma of the cervix [zur Hausen et al.,
Progr. Med. Virol.
30: 170-86, 1984; zur Hausen,
Prog. Med. Virol.
32: 15-21, 1985; zur Hausen,
Cancer
59: 1692-6; Campion et al.,
Lancet
1: 943-6, 1985]. The prevalence of cervical cancer and precancerous lesions is becoming increasingly more common in younger women. Without treatment it can be fatal, the death rate being ~100 per million women per year in Western countries. Fortunately, if detected at an early stage, effective treatment is available that can eliminate the fatal consequences.
The immediate management and subsequent follow-up of young women with abnormal cytological smears who still wish to bear children presents many problems. This has been compounded by uncertainty about the interpretation of smears with features of papillomavirus infection (“kollocytes”) as well as dysplasia. Moreover, the current cytological testing tool for cervical cancer screening, the Pap smear, has a false negative rate of ~20%. Significant numbers of dysplastic lesions regress spontaneously, others fail to progress, while a few progress rapidly. Thus, from an ill-defined cloud of morphological abnormalities occasional cancers develop. At present there is no clear way to predict whether cancer will result if a Pap smear happens to be abnormal. Clinical examination of many of these patients has failed to find warty lesions (condylomata accuminata) on the external genitalia or, indeed, on the cervix itself. The more difficult procedure of colposcopy, after the application of 3% acetic acid, is, in fact, required, revealing the presence of flat (“non-condylomatous”) warts (which are invisible to the naked eye). These are the expected premalignant lesions. Histophathological progression of the wart to carcinoma in situ and frank malignancy has been well described [e.g., Dyson et al.,
J. Clin. Path.
37: 126-31, 1984]. An increasingly prevalent problem is the occurrence of invasive cancer within 3 years of a negative Pap smear [Berkowitz et al.,
Gynecol. Oncol.
8: 311, 1979; Holman et al.,
Med. J. Aust.
2: 597, 1981]. Whereas the presence of papillomavirus replication may be confirmed in cervical condylomata by detection of virus particles or the group-specific antigen, neither particles nor antigen have, however, been found in squamous cell carcinoma tissue.
In contrast to the uncertainty and controversy that surrounds the interpretation of tests based on morphology, the new techniques in molecular biology can be utilised to bypass such problems and provide more objective information. By using nucleic acid hybridization techniques the viral DNA can be identified directly and at an earlier stage of infection. Indeed, using these approaches, HPV types have been found in both benign and premalignant lesions.
At present ~50 types of the papillomavirus have been distinguished in human infection. Different ones infect different epithelial areas. The particular types of HPV that commonly infect the genital tract include those assigned the numbers 6, 11, 16, 18 and several rarer types (31, 33, 35, 39, 43 and 44). HPV6 [de Villlers et al.,
J. Virol.
40: 932-5, 1981] and HPV11 [Gissmann et al.,
J. Virol.
44: 393-400, 1982; Gissman et al.,
Proc. Natl. Acad. Sci. USA
80: 560-3, 1983; Dartmann et al.,
Virology
151: 124-30, 1986] have been associated with benign condylomata accuminata, the classical lesion of the anal and genital tract [Gissmann et al.,
J. Invest. Dermatol.
83: 26s-8s, 1984]. In contrast, HPV16 [Durst et al.,
Proc. Natl. Acad. Sci. USA
80: 3812-5, 1983] and HPV18 [Boshart et al.,
EMBO J.
3: 1151-7, 1984; Cole and Danos,
J. Mol. Biol.
193: 599-608, 1987] are more often detected in dysplastic flat lesions of the vulva and cervix, and squamous carcinoma of the cervix and penis [Crum et al.,
Cancer
49: 468-71, 1982; Campion et al.,
Lancet i:
943-6, 1985].
Thus the types of HPV that infect the anogenital area can be assigned to two categories as follows:
1. “Low-risk”; HPV 6 and HPV11, with type 6 being the most common of all anogenital types.
2. “High-risk”; HPV16, HPV18, HPV31, HPV33, HPV35, HPV39 HPV43 and HPV44.
The frequency of occurence of the higher risk types is in decreasing order. Thus, within the high risk category, HPV16 is most common (45-60%), HPV18 is next most common (20-30%) and the others are rarer, the last 4 being discovered only recently and reported in 1986 (total frequency for all of these rarer types is, collectively, ~15%). Other rarer types are likely to be discovered in due course.
In support of a role for HPVs in cervical cancer the following findings are noteworthy:
(i) DNAs of known high risk HPVs have been detected in ~90% of cervical adenocarcinomas and squamous cell carcinomas [Zachow et al.,
Nature
300: 771-3, 1982; Gissmann et al., 1984, ibid].
(ii) High risk HPV DNA has been found in metastases arising from cervical tumours [Lancaster et al.,
Am. J. Obstet. Gynecol.
154: 115-9, 1986].
(iii) Instead of being present in cells in the usual episomal form, DNAs of high risk HPVs have been found integrated into human genomic DNA [Schwartz et al.,
Nature
314: 111-4, 1985; Lehn et al.,
Proc. Natl. Acad. Sci. USA
82: 5540-4, 1985; Kreider et al.,
Nature
317: 639-41, 1985; Matsukura et al.,
J. Virol.
58: 979-82, 1986; Schneider-Gadicke and Schwartz,
EMBO J.
5: 2285-92, 1986; Di Luca et al.,
J. Gen. Virol.
67: 583-9, 1986]. Such integration has been suggested to be necessary for malignant conversion of the cells, supported by findings of integration also in precarcinoma tissue [Shirasawa et al.,
J. Gen. Virol.
67: 2011-5, 1986].
(iv) The integration pattern usually interrupts or deletes specific regions of the HPV16 or 18 DNA, but consistently leaves intact the E6 and E7 openreading frames (ORFs) ([Pater and Pater,
Virology
145: 313-8, 1985], which continue to express, at least in cell lines derived from cervical carcinomas [Smotkin and Wettstein,
Proc. Natl Acad. Sci. USA
83: 4680-4, 1986; Androphy et al.,
EMBO J.
6: 989-92, 1987; Baker et al.,
J. Virol.
61: 962-71, 1987; Takebe et al.,
Biochem. Biophys. Res. Commun.
143: 837-44, 1987].
(v) A splice donor exists in the E6 ORF of HPV16 and 18 (but not HPV6 and 11) which can result in the generation of an ORF which when translated resembles epidermal growth factor [zur Hausen,
Lancet
489
-
91
,
1986
].
(vi) Integration in cervical cell lines (HeLa, CaSki, SiHa, SW756, etc) is often near proto-oncogenes [Dürst et al.,
Proc. Natl. Acad

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