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Human Papillomaviruses and thePolymerase Chain Reaction

AT the Eighth International PapillomavirusWorkshop in Taos, New Mexico, in March, scientistsfrom all over the world gathered to discuss the linkbetween these viruses and human cancer. For

anogenital cancers, the consensus view favoured anaetiological role for the viruses. Support for this

opinion was drawn from epidemiological studies

showing that specific virus types (HPV 16 and 18) arepresent in many squamous cancers of the genital tract;it was also drawn from a large body of experimentalevidence showing that HPV 16 (unlike HPV 6) hastransforming properties and can immortalise primaryhuman keratinocytes in vitro.1 However, there wasgeneral agreement that the relation between virus andtumour was not as straightforward as it had seemedwhen the association was first reported 13 years ago.Why should there be such reservations about the

role of papillomaviruses? One reason is the conflictingresults of many of the epidemiological studies that aresaid to indicate the oncogenicity of HPV 16. In areview2 of the evidence, leading epidemiologists drewattention to the extraordinary range of prevalencesassigned to HPV 16 by twenty different laboratories in

1. Zur Hausen H, Schnieder A. The role of papillomaviruses in human anogenital cancer.In: Salzman NP, Howley PM, eds. The papovaviridae 2. The papillomaviruses,New York: Plenum, 1987: 245-63.

2. Munoz N, Bosch X, Kaldor JM. Does human papillomavirus cause cervical cancer?The state of the epidemiological evidence. Br J Cancer 1988; 57: 1-5.

various parts of the world. The recorded prevalence ofHPV 16 in cervical cancer biopsy specimens in theUK, Germany, the USA, and Japan ranged from 18to 92%, while the prevalence of HPV 16 in normalcervical epithelium ranged from 0 to 34 6%. Thesedata might indicate that the prevalence of HPV 16varies in different parts of the world. Another view,preferred by Munoz and her colleagues is that theresults reflect differences in study design (eg, selectionbias, sample size, choice of controls) and in sensitivityand specificity of the hydridisation techniques used(eg, Southern blot, dot blot, in-situ filter

hybridisation). A disturbing finding was reported bySchiffman at a workshop on papillomaviruses inFrance last year.3 He sent identical biopsy material forDNA analysis to several laboratories and obtaineddifferent reports on the prevalence of HPV 6, 11, and16 in the sample. Whatever the reason for thesefindings, the epidemiological evidence linking HPVwith cervical cancer must be interpreted with cautionif spurious associations are to be avoided.The influence of study design and DNA technology

on HPV prevalence surveys has been illustrated latelyby two groups in the UK who used new methods todetect the virus.4.5 Young et al4 and Tidy et al-5

investigated the prevalence of HPV 16 in cervicalcancer biopsy specimens and in cervical scrapes fromwomen with normal Papanicolaou smears by use ofthe polymerase chain reaction (PCR) to amplifyspecific HPV DNA sequences. PCR is many timesmore sensitive than Southern blot or dot blot methodsand is theoretically capable of detecting a single virusparticle in 106 cells. In a series of carefully controlledexperiments designed to exclude the possibility ofcontamination with extraneous DNA, these workersshowed that HPV 16 DNA is present in 100% ofcancer biopsy specimens and in 80% of scrapes fromwomen with normal cervices, indicating that the rateof infection with HPV 16 in the general population isvery high indeed. Preliminary reports from centres inthe USA confirm the specificity and sensitivity of thisapproach.6,7The results of Young and Tidy highlight the

fact that earlier epidemiological studies with lesssensitive methods of virus detection consistentlyunderestimated the frequency of HPV 16 in thenormal cervix. The discrepancy can be attributed tothe very low copy number in the cervical scrapes used

3. Schiffman M. Seventh International Papillomavirus Workshop, May 16-20, 1988,Sophia Antipolis, France.

4. Young LS, Bevan IS, Johnson MA, et al. The polymerase chain reaction: a newepidemiological tool for investigating cervical human papillomavirus infectionBr Med J 1989; 298: 14-18

5. Tidy JA, Parry GCN, Ward P, et al. High rate of human papillomavirus type 16infection in cytologically normal cervices Lancet 1989; i: 434.

6. Manos MM, Wright DK, Lewis AJ, Wolinsky S, Broker TR, Ting Y. Detection andtyping of genital HPVs using PCR amplification with consensus primers. EighthInternational Papillomavirus Workshop, March 11-18, 1989, Taos, New Mexico(abstr).

7. Beckmann AM, Chu J, Miller BA, Christiansen AE, McDougall JK, Galloway DA.Evaluation of PCR for detection of HPV DNA in clinical specimens EighthInternational Papillomavirus Workshop, March 11-18, 1989, Taos, New Mexico(abstr)

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for population studies compared with the biopsyspecimens used for HPV 16 assay of cancerous tissue.Although the significance of these findings is

unknown, there are parallels with other presumptiveoncogenic viruses. Thus, infection rates of EpsteinBarr and hepatitis B viruses are high in the normalpopulation as well as in patients with their associatedmalignancies, indicating that other events are involvedin transformation. 8 Moreover, prospective studies

claiming that progression of cervical intraepithelialneoplasia (CIN) is more likely to occur in womeninfected with HPV 16 12 will need to be repeated in thelight of these findings, since women who were foundto be HPV 16 negative by in-situ filter hybridisationor slot blot techniques may well be found to harbourthe virus when investigated by the highly sensitivePCR. Similarly, reports that female partners of maleswith genital warts are at increased risk of CIN must beevaluated critically for the sensitivity of the HPVDNA detection technique used.9 In New Mexico,Quint et al reported that PCR can be used to detectHPV in male urine, so screening the male populationbecomes a practical proposition.1OMany more questions need to be answered before

we can understand how HPV effects transformation.An aspect of HPV 16 infection that strongly suggestsan oncogenic role for the virus is that viral DNA isoften integrated into the cellular genome in invasivelesions.2 Integration does not appear to be specific forthe host chromosome in vivo, although in two cervicalcancer cell lines integration has been mapped tochromosome 8 in the region of the myc gene."However, in every case virus integration results inelimination of the E2 gene, and this gene indirectlygoverns expression of the viral genes E6 and E7 thatencode for the transforming functions of the virus.2,12We do not know what proportion (if any) of the HPV16 DNA in the normal population is integrated andPCR techniques need to be developed to obtain thisinformation.The role of cofactors in the carcinogenic process

needs to be clarified. Smoking increases the risk ofcervical cancer and there is scope for measuringcovalently bound adducts in cervical DNA and

correlating them with the smoking habits of thepatient, dysplastic changes in the epithelium, andvirus integration patterns. At the New Mexico

meeting, Tidy et al also reported a subtype of HPV 16(HPV 16b) that may be less oncogenic than prototype

8. Editorial. Human papillomaviruses and cervical cancer: a fresh look at the evidence.Lancet 1987; i. 725-26.

9. Campion MJ, McCance DJ, Mitchell HS, Jenkins D, Singer A, Oriels JD. Subclinicalpenile human papillomavirus infection and dysplasia in consorts of women withcervical neoplasia. Genitourin Med 1988; 64: 90-99.

10. Quint W, Melchers W, Schift R, Stolz E, Linderman J. Detection of HPV in urinesamples of male patients by the polymerase chain reaction. Eighth InternationalPapillomavirus Workshop, March 11-18, 1989, Taos, New Mexico (abstr).

11. Durst M, Croce CM, Gissmann L, Schwarz E, Huebner K. Papillomavirus sequencesintegrate near cellular oncogenes in some cervical carcinomas. Proc Natl Acad SciUSA 1987; 84: 1070-74.

12. Ward P, Coleman DV, Malcolm ADB. Regulatory mechanisms of the

papillomaviruses Trends Genet 1989; 5: 97-99.

HPV 16 (HPV 16a)." Initial analysis of the PCRproducts in 210 women by agarose gel electrophoresisindicates that subtype HPV 16b is found

predominantly in women with normal cervices. If thisobservation is confirmed with a larger number ofsamples, subtype-specific HPV DNA screening withPCR may be a valuable addition to cytologicalexamination of cervical smears, and could be

important for the interpretation of the large case-control and prospective studies that have been

designed to determine the cancer risk associated withHPV infection. Whatever the outcome, the

application of PCR to the study of viral oncogenesishas opened up new areas for investigation in humanpapillomavirus research that will advance our

understanding of the relation between these virusesand anogenital cancer.

Metastatic Fundamentals

WHAT causes tumour cells to metastasise and howdo they do it? A Ciba Foundation symposium on thesubject1 provides valuable insight into some of thenewer approaches to this hugely complex problem.Two principal themes emerged the role of adhesiveglycoproteins and the characterisation of metastaticphenotypes in terms of modern molecular biology.The importance of adhesive glycoproteins in the

development, organisation, and maintenance ofnormal tissues is increasingly evident. Separatecategories of cell adhesion molecules, cell junctionalmolecules, and substrate adhesion molecules havebeen described. Best known are the substrate adhesionmolecules fibronectin and laminin. Fibronectin is

present in many interstitial tissues and mediates theattachment of numerous cell types; laminin isrestricted to the epithelial basal lamina and to nerves,muscles, and vessels. Several of the cell adhesion andjunctional adhesion molecules found in embryologicaltissues have been described in various experimentaland human tumours, many of which also express largenumbers of high-affmity surface receptors forsubstrate adhesion molecules. The cell-cell and cell-substrate adhesive systems in malignant tumoursmust be labile and able to change rapidly dependingon the phase of activity of the tumour cell-eg, duringdetachment from the main tumour mass, invasion

through local stroma, penetration into and escapefrom lymphatic or vascular systems, and eventualestablishment and reorganisation as a metastaticdeposit at some distant site.

Interaction between malignant tumours andfibronectin and laminin appears to be extremely

13. Tidy JA, Vousden KH, Farrell PJ. Infection with a subtype of HPV 16 is linked tocervical neoplasia. Eighth International Papillomavirus Workshop, March 11-18,1989, Taos, New Mexico (abstr).

1. Metastasis. Ciba Foundation symposium 141. Chichester: John Wiley & Sons, 1988.