Prenatal detection of fetal RhD DNA sequences in transcervical samples

pathogenetic processes in ovarian carcinogenesis. It provides specific testable hypotheses that can be explored in future epidemiological and laboratory research. Glinda S Cooper Epidemiology Branch A3-05, PO Box 12233, Research

National Institute of Environmental Health Sciences,

Triangle Park, NC 27709, USA

Surveillance, Epidemiology, and End Results: Incidence and mortality data, 1973-77. Washington DC: United States Government Printing Office, NIH pub no 81-2330, 1981. Hanai A. Trends and differentials in ovarian cancer: incidence, mortality and survival experience. APMIS Suppl 1990; 12: 1-20. Whittemore AS, Harris R, Itnyre J, Halpern J. Collaborative Ovarian Cancer Group. Characteristics relating to ovarian cancer risk: collaborative analysis of 12 case-control studies; I, methods. Am J Epidemiol 1992; 136: 1175-83. Whittemore AS, Harris R, Itnyre J. Collaborative Ovarian Cancer Group. Characteristics relating to ovarian cancer risk: collaborative analysis of 12 case-control studies; II, invasive epithelial ovarian cancers in white women. Am J Epidemiol 1992; 136: 1184-203. Negri E, Franceschi S, Tzonou A, et al. Pooled analysis of 3 European case-control studies: I. Reproductive factors and risk of epithelial ovarian cancer. Int J Cancer 1991; 49: 50-56.

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Polio eradication SIR-Preston (Oct 22, p 1163), responding to Patriarca’s Sept 3 commentary, focuses on the longstanding scientific debate about which poliomyelitis vaccine is better. We believe the oral (OPV) versus inactivated (IPV) poliomyelitis vaccine debate obscures the central issue of the two outbreaks. The use of OPV is not the cause of the poliomyelitis outbreak in Namibia. The outbreak resulted from two factors: the widespread circulation of wild poliovirus in Angola, where immunisation coverage is less than 40%, and rapid urbanisation after Namibia’s independence with the accumulation of clusters of unimmunised children in the periurban slums. It is hard to imagine that the use of IPV in either Angola or Namibia could have prevented the outbreak. Similarly, the Netherlands outbreak resulted from religious objections to immunisation, and the continued circulation of wild poliovirus on the Indian subcontinent. The Namibia and Netherlands outbreaks, as well as those identified by Patriarca, point to failure to vaccinate rather than vaccine failure as the principal cause of poliomyelitis in the world today. In this situation, no change in vaccine, vaccination schedule, or vaccine formulation will stop

poliomyelitis. As long as wild poliovirus circulates in countries with large populations of underimmunised children, every country in the world remains at risk of poliomyelitis importations. After the eradication of poliomyelitis from the western hemisphere, WHO and its partners remain committed to the goal of interrupting wild poliovirus transmission globally. WHO’s strategies are clear and unchanged: OPV is the only vaccine recommended for poliomyelitis eradication.’ We believe that the energy spent in debating the choice of vaccine would be more productive if channelled into advocacy and fundraising for the global poliomyelitis eradication initiative. The strategies are proven. Let us finish the job. *Nicholas A Ward, Harry F Hull on Immunisation, Global Programme for Vaccines and Immunization, World Health Organization, 1211 Geneva 27, Switzerland

Expanded Programme

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HF, Ward NA, Hull BP, Milstein JB, de Quadros C. Paralytic poliomyelitis: seasoned strategies, disappearing disease. Lancet 1994; Hull

343: 1331-37.

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Prenatal detection of fetal RhD DNA sequences in transcervical samples SIR-Several attempts have been made to perform prenatal diagnostic tests by non-invasive or semi-invasive techniques. One approach, first suggested by Shettles in 1971, is to collect fetal (trophoblastic) cells shed into the endocervical canal during pregnancy.’ The presence of trophoblasts in transcervical cell (TCC) samples, retrieved between 6 and 13 weeks of pregnancy, has been confirmed by use of the PCR assay to amplify chromosome Y DNA sequences in cells derived from male conceptuses1,2 or small tandem repeats (STR) specific for chromosome 21.3 Fluorescence in-situ

hybridisation (FISH) and probes for chromosomes X, Y, 21, and 18 have also been successfully employed to show fetal cells in TCC samples and chromosomal abnormalities such as trisomies 21 and 18.1,4 We now report results of detecting RhD DNA sequences by PCR amplification in TCC samples collected from RhD-negative pregnant women. Endocervical cells were retrieved from 6 mothers (table), at about 10 weeks’ gestation, by aspiration of cervical mucus before chorionic villus sampling (CVS). In another 6 women TCC samples were obtained by lavage1,3,4 between 6 and 13 weeks’ gestation before termination of pregnancy. All samples were collected after receiving consent from the mothers and with permission of our ethics committee. Two sets of primers specific for the 3’ untranslated region of RhD and exon 4 of RhCE (5’-TACCACATGAACCTGAGGCA-3’ and 5’-CATGGCAGACAAACTGGGTA-3’) were employed in a duplex PCR to analyse the TCC samples together with maternal peripheral blood, CVS, or fetal (placental) samples.** In 4 cases (1, 2, 3, and 4 in table), in which samples had been collected by aspiration before CVS, results of the RhD test by PCR on TCCs were compared with serological RhD typing done on cord blood at the time of delivery. In all other cases RhD phenotypes of the fetuses were established by analysis of CVS or fetal (placental) tissues.

TCC samples were collected by aspiration (A) or lavage (L) from serologically RhDnegative mothers TCC, CVS, and maternal blood samples were analysed by PCR for presence of RhD gene; in 4 cases (1-4) fetal RhD phenotype was tested serologically in cord blood samples (ND=not done). Full concordance was observed between RhD typing in TCC and CVS or newborn samples in 10 of 12 samples 6 TCC and CVS samples were also analysed for X and Y DNA sequences by PCR and by FISH; Y+ indicates presence of Y-specific sequence or XY cell. The same samples were also tested for STR markers derived from paternal genes*. Unmform=maternal and fetal phenotypes were identical.

Table : Detection of RhD sequences in transcervical cell

samples

As shown in the table, all mothers serologically RhD when investigated by PCR of the RhD gene. Good correlation was observed between the PCR RhD results obtained with TCC samples and those done on fetal or newborn specimens, with 2 exceptions. In case 3, the TCC sample retrieved by aspiration was RhD negative, whereas the neonate was serologically RhD positive. In case 11, PCR tests of the TCC and CVS samples were discordant probably because too few fetal cells were present and ongoing investigations suggest that negative results can be recorded if a TCC sample contains less than 2-5% trophoblastic cellular elements. The 6 TCC samples collected by lavage and the corresponding fetal tissues were also tested by PCR for detection of chromosomes X and Y. 1,4 As shown in the table, in all instances the sex of the fetus was correctly diagnosed with use of TCC samples. FISH analysis, with probes derived from chromosomes X and Y, confirmed the presence of fetal cells in 2 samples that were found to contain trophoblasts with Y fluorescence signals. The 6 TCC samples retrieved by lavage were also tested to detect chromosome 21 specific STR by quantitative fluorescence PCR amplifications.3 STR DNA sequences, present in fetal tissues and inherited from the father, could be detected in 4 of 6 TCC samples tested (table). Our results, while confirming the presence of fetal cells in TCC samples, document the possibility of doing prenatal diagnosis of the Rh phenotype of the conceptuses by a semiinvasive procedure. Work is in progress to fully evaluate this approach on a large number of RhD-negative mothers before CVS, and compare these results with those obtained on cord blood at time of delivery with serological analysis.

negative were also RhD negative amplification for the presence

This work was

association in this population is far from the smallest detectable relative risk from all connective tissues disease might be 12-5, the corresponding figure for specific connective tissue disease is much greater-for systemic sclerosis it is 19-5. Moreover, this calculation assumes zero latency between implantation and disease. However, case reports of systemic sclerosis releated to silicone breast implant, as well as studies of this disorder in silica-exposed occupational cohorts, suggest a median latent period of 10 years or more.3,4 If, as in studies of cancer with long latency, patient follow-up were delayed to begin only after the latent period had elapsed, the womenyears experienced by this population would be reduced absence of

considerably. We caution readers against assuming safety on the basis of such studies; in 1973 another Mayo Clinic study, with a sample size (818 women) and a study design very similar to that of the 1994 study,’ also showed no association between exposure and disease.’ However, in that case the disease endpoint was genital tract cancer and the exposure was

diethylstilboestrol. Shanna H Swan, Suzanne S Teuber, *M Eric Gershwin University of California, Berkeley, California; and *Division of Rheumatology/Allergy, Clinical Immunology, School of Medicine, University of California, Davis, CA 95616, USA

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partly supported by the Dunhill Medical Trust Foundation. 5

*M Adinolfi, J Sherlock, T Kemp, B Carritt, P Soothill, J Kingdom, C Rodeck Department of Obstetrics and Gynaecology, and *MRC Human Biochemical Genetics Unit, University College London, London NW1 2HE, UK 1 Adinolfi M, Soothill P, Rodeck C. A simple alternative to aminocentesis? Prenat Diagn 1994; 14: 231-33. 2 Griffith-Jones MD, Miller D, Lilford RJ, Scott J. Detection of fetal DNA in trans-cervical swabs from first trimester pregnancies by gene

amplification:

a new route to

prenatal diagnosis? Br J Gynaecol 1992;

99: 508-11. 3 Adinolfi M, Sherlock J, Soothill P, Rodeck C. Molecular evidence of fetal derived chromosome 21 markers (STRs) in transcervical samples. Prenat Diagn (in press). 4 Adinolfi M, Davies A, Sharif S, Soothill P, Rodeck C. Detection of trisomy 18 and Y-derived sequences in fetal nucleated cells obtained by transcervical flushing. Lancet 1993; 342: 403-04. 5 Lo Y-MD, Bowell PJ, Sellinger M, et al. Prenatal determination of fetal RhD status by analysis of peripheral blood of rhesus negative mothers. Lancet 1993; 341: 1147-48.

Silicone implant controversy SIR-Bridges (Nov 26, p 1451) is to be commended for pointing out that the silicone breast implant controversy is far from resolved. With respect to the Mayo Clinic Study,’ he correctly notes that for all connective tissue diseases combined, the smallest relative risk detectable in this cohort of 749 implanted women followed for an average of 7-8 years is at least 12-5. This is probably an underestimate of the relative risk that this study has the power to rule out. For illustrative purposes we focus our discussion on systemic sclerosis, a disease that has been frequently associated with silicone breast implants.2,3 In this population, only 1 case of systemic sclerosis was diagnosed and that was an unexposed woman. However, the

an

reassuring. Although

Gabriel SE, O’Fallon MO. Risk of connective-tissue diseases and other disorders after breast implantation. N Engl J Med 1994; 330: 1697-702. Varga J, Schumacher HR, Jiminez SA. Systemic sclerosis after augmentation with silicone implants. Ann Intern Med 1989; 111: 377-83. Spiera H, Kerr LD. Scleroderma following silicone implantation; a cumulative experience of 11 cases. J Rheumatol 1993; 20: 958-61. Haustein UF, Ziegler V. Silica-induced scleroderma. J Am Acad Derm

1990; 22: 3. Lanier AP, Noller KL, Elveback LR, Kurland LT. Cancer and stilbestrol: a follow-up of 1719 persons exposed to estrogens in and born 1943-1959. Mayo Clinic Proc 1973; 48: 793-99.

Is breast disease?

cancer a

progressive

or

utero

systemic

SiR-Views of the natural history of breast cancer differ, which might have important repercussions for treatment. Is it "a progressive disease whose development can be arrested by early diagnosis and treatment", as Duffy and Tabar (Oct 29, p 1236) suggest, or is it a systemic disease from the onset whose local/regional treatment is ineffective, as Devitt suggests (Sept 10, p 734)? It is generally accepted that invasive carcinoma has developed from in-situ carcinoma when invasion through the basement membrane is evident. At what stage does this transition occur, what triggers it, and what is the time-span between invasion and metastasis? We suggest that there is no progression time-span between invasion and metastasis, and that these events are variants of the same process, arising from ductal carcinoma in situ

(DCIS).’ Data from the Swedish Two County Mammography Trial, referred to by Duffy and Tabar, can be explained in another way. In this trial 123 DCISs were removed from women in the invited group and 20 from the control group at first screening. These numbers are small considering that DCIS is very common, especially in women aged 40-49 and can be as high as 39%.2 Mammographic screening can detect large late-stage DCIS, some of which might not yet have progressed to the invasive metastatic state. Surgical removal of these tumours from the invited group should contribute much to a decrease in mortality since many of these tumours will progress in the controls. In the Ostergotland component of the trial there were 119 deaths in the invited group and 319