SERUM-ALPHA1-FETOPROTEIN IN PATIENTS WITH TESTICULAR TUMOURS

433 Discussion Of the 41 mothers of haemophiliacs in simplex families ; seems that 39 were probably carriers of haemophilia on :he basis of tests carried out in our laboratory. HallaneJ showed that if mutation were balanced by selec:I,ii some hxmophiliacs must be the sons of homozygous normal mothers the defect having arisen by mutation in ;he mother’s germ-cell. Haldane3 called this proportion sand showed that :-

whereis the mutation-rate

in ova, v is mutation-rate sperm cells, and f is the fitness. Fitness represents the number of children born to the affected persons compared to the number born to normal males at the same period in time. If normal males on average have 2 children and haemophiliacs have 1 child, then their fitness would be a or 0-5. In the past the average age at death of haemophiliacs was 16-20 years and many affected males did not live long enough to have children. In 1947 Haldane3calculated that the fitness of hoemom

philiacs was 0-28. Since 1947 the treatment of haemophiliacs has improved substantially but insufficient time has elapsed for this improved treatment to have affected fitness.

Of the present series of mothers of haemophiliacs 34+ 11 see table i) are known to be heterozygous from the family history and 39 of the 41 mothers in simplex families are probably heterozygous and thus :-

Mdv=29.3[i. This

means that the mutation-rate during spermatois genesis likely to be 30 times the rate in oogenesis. It is true that the mutation-rate is not now balanced by selection since the proportion of haemophiliacs in the community is increasing. Nevertheless it seems likely that the mutation rate in oogenesis is lower than that Juring spermatogenesis. This conclusion agrees with the suggestion of Haldane3 but not with Barrai et al. and

Kosower et al.8 However, these workers did not take :nto account the results of reliable tests to detect the carrier state. Our results also do not agree with those of Ratnoff and Jones6 who found fewer abnormal results by tests similar to ours for the mothers of single cases of hemophilia than for obligatory carriers. Ratnoff and Jones’ found fewer than expected abnormal results for he daughters of carriers who had no haemophilic sons. Results similiar to ours have been reported by Franke :t aL9 for the sex-linked recessive Lesch-Nyhan disease. k this condition no affected males have survived to proiuce children and the fitness is thus 0. Analysis of tests r the carrier state showed that of 27 mothers of single - 3ses only 4 appeared to be normal from laboratory ::5t5. Study of sisters of carriers and of first cousins the maternal aunt was a carrier showed more

number of heterozygous females. Franke et al. suggest that some of the tests used may have given false-positive results but no evidence is given to support this supposition. They also suggest that the selection may have been biased in favour of selecting families with more than one affected boy. They note that "there appears to be a true deficiency of new mutants among males...". There may have been some bias in our series in the selection for testing of women having a 1 in 2 chance of being carriers of haemophilia. The women whose samples we have tested include many who have come to the laboratory asking to have a blood-test to detect the carrier state. Women in haemophilic families who have themselves experienced abnormal bleeding are likely to be those with the lowest levels of factor VIII and these women will be the most likely to consult the haemophilia specialist. Haemostatically normal women who have normal factor VIII levels are less likely to come for testing. A bias of this sort could account for the high proportion of abnormal results which we found for the daughters of obligatory carriers of haemophilia. Whatever the explanation of our findings, it seems to us that mothers of single cases of haemophilia in simplex families should for practical purposes all be regarded as carriers of haemophilia. It is not easy to see how bias in the selection of patients for study can have produced such an apparently low incidence of true sporadic cases arising by mutation during oogenesis. We thank Dr Martin Bobrow for much help and advice during the preparation of the manuscript. Requests for reprints should be addressed to R. B., Research Laboratory, Oxford Haemophilia Centre, Churchill Hospital, Heading-

than the

expected

ton, Oxford OX3

7LJ. REFERENCES

Andreassen, M. Hæmophilia i Danmark. Copenhagen, 1943. Ramgren, O. Acta med. scand., 1962, suppl. 379. 3. Haldane, J. B. S. Ann. Eugen. 1947, 13, 262. 4. Cavalli-Sforza, L. L., Bodmer, W. F. The Genetics of Human Populations. San Francisco, 1971. 5. Rizza, C. R., Rhymes, I. L., Austen, D. E. G., Kernoff, P. B. A., Aroni, S. A. Br. J. Hœmat. 1975, 30, 447. 6. Ratnoff, O. D., Jones, P. K. Am. J. Clin. Path. 1976, 65, 129. 7. Barrai, I., Cann, H. M., Cavalli-Sforza, L. L., de Nicola, P. Am. J. hum. Genet. 1968, 20, 175. 8. Kosower, N., Christiansen, R., Morton, N. E. ibid. 1962, 14, 159. 9. Francke, V., Felsenstein, J., Garter, S. M., Migeon, B. R., Dancis, J., Seegmiller, J. E., Bakay, F., Nyhan, W. L. ibid. 1976, 28, 123. 1. 2.

SERUM-ALPHA1-FETOPROTEIN IN PATIENTS WITH TESTICULAR TUMOURS A. H. ORR J. KOHN Supra-Regional Protein Reference Unit. Putney Hospital and Queen Mary’s Hospital, Roehampton, London SW15 5PN T.

M. BENTALL J. MCELWAIN M. J. PECKHAM Testicular Tumour Unit, Royal Marsden Hospital, Sutton,

Surrey

Summary

Serum-alpha1-fetoprotein (A.F.P.)

was

assayed in 88 men with testicular teratomas and 34 with seminomas. 61% of teratoma patients with clinically evident disease had serum-A.F.P. greater than 25 ng/ml, whilst it is highly unlikely that any patient with a pure seminoma had a titre above this level.

434

concentrations correlated with response to radiotherapy and chemotherapy and predicted relapse in

A.F.P.

patients. Serum-A.F.P. should be measured in all patients with testicular tumours. some

tomy before referral to our unit, and information about titres before orchidectomy was available for only 1 of the;Serum was assayed for A.F.P. before radiotherapy or chel1therapy was given, during treatment, and regularly after tre2,, ment had stopped.

Introduction RAISED

Methods

serum-alphacfetoprotein (A.F.P.)

was

first

demonstrated in patients with gonadal tumours by Abelev et al. in 1967 in 10 out of 47 patients with testicular teratomas. 15-60% of adults and about 70-80% of children with teratomas have raised serum-A.F.p.2-9 Patients with seminomas, practically without exception, have not had raised serum-A.F.p.,2 1° and significantly increased serum-A.F.p. concentrations10 11have not been found in children with other tumours.3 The detection of raised serum-A.F.p. in patients with testicular tumours is influenced by the sensitivity of the technique used for its demonstration.2 9 12 For meaningful results a sensitive and reliable quantitative assay of A.F.P., such as radioimmunoassay (R.1.A.) coupled with sequential sampling, is essential. This will give an estimate of A.F.P. "positivity" in patients with testicular tumours plus an indication of the importance of A.F.P. estimations in the overall management of their disease. We have assessed the value of A.F.P. assay in the management of a group of men with tumours of the testis attending the Royal Marsden Hospital in a prospective study which included sequential blood sampling for A.F.P. at short intervals (1-3 months). A.F.P. concentrations were correlated with the clinical and radiological status of the patients and with their treatment by radiotherapy and chemotherapy. Patients men with testicular tumours and 34 with seminomas studied. The disease staging system was as follows: Stage /.- Tumour confined to testis (negative lympho-

88

were

gram). Stage 11.-Tumour confined to testis and abdominal lymphnodes (positive lymphogram). Stage 7/7.—Supradiaphragmatic nodal disease (mediastinal and/or supraclavicular). Stage IV.-Extralymphatic spread (liver, lung, bone, brain, skin, &c.). Histological grading of the tumour was that of the Testicu-

A.F.P.

Assay sample of venous blood was taken. Serum-u was assayed by two techniques-screening counter-current m munoelectrophoresisl-’ and R.I.A. The R.I.A. was a slight modi A 10 ml

fication of the methods of Purves and Purves" and Nishi anc Hirai. 17 Samples were diluted with borate buffer pH 8.53 me taining ethylenediamine tetra-acetic acid in 0.2% bovine serum albumin. Standards and samples were incubated at 4O( with fixed amounts of labelled A.F.P. Bound and free fractions were separated after 48 hours’ incubation by means of polyethylene glycol at a final concentration of 12½%.18 After centrifugation at 2500 g/20 min (at 4°C) the superna tant was decanted and the radioactivity of the precipitate M counted in a Wallac counter. 25--400 ng/ml A.F.P. was th; assay range routinely used. Published reports on the normal range of serum-A.F.p, van considerably from 1 to 20 nglm1.19-21 In our study 25 ng/m was chosen as an arbitrary upper limit of "normal" values. W report only those patients who had serum-A.F.p. estimated b, R.I.A. as well as by the screening technique.

Results

Frequency of Raised Serum-A.F.P. in Teratoma To assess the frequency of raised serum-A.F.p. in patients with known tumour only stage n, III, and IV patients can be considered since the stage-i patients had had an orchidectomy and had no clinically detectable residual disease at the time of blood sampling. Th frequency of raised serum-A.F.P. in patients with detectable disease is shown in table I and correlated with hismand with stage of disease. Overall, about

logical grade TABLE

I-FREQUENCY

OF RAISED SERUM-A.F.P. IN TERATOMA PATIENT

lar Tumour Panel of Great Britain. 13 Details of treatment with radiotherapy and chemotherapy have been described else-

where. 14 In the teratoma group, 27 patients were in stage 1, 7 were stage 11, 4 stage tii, and 50 stage iv. Of the seminoma patients, 14 were stage i, 10 were stage n, 1 was stage ui, and 6 were stage iv. 1 had a primary mediastinal seminoma, 1 had a seminoma in one testis and a teratoma in the second testis, and one was unstaged. All the patients had undergone orchidec-

two-thirds of

with known residual disease pos. had serum-A.F.p. above 25 ng/ml. There was no difference in the frequency of raised serum-A F between the two major histological types of tumour-in

patients

orchidectomy

TABLE II-SERUM-A.F.P. IN STAGE-I TESTICULAR TERATOMA

*Second primary tumour, +ve titre -ve after surgery, no tSmgle titre of 28 ng/ml. Patient remains disease free. :j:Smgle titre of 38 ng/ml. Patient remains disease free.

relapse since.

435

"rmediate and undifferentiated-nor was there a sigticant difference between patients with stage-ii and - tage-m and stage-iv disease. Of the 34 stage i and ii patients, 9 had initially raised titres (27%). In the stage-i group of 27 patients who had no detectable tumour at the time of blood sampling, table 11) 6 patients had raised titres initially, but only of these have subsequently relapsed. However, 2 of 4 patients in whom a rise in A.F.P. subsequently developed did relapse, in both cases in the supraclavicular lymphnodes. These 2 patients both had several abnormal A.F.P. measurements, whereas the 2 patients who did not relapse each had only one slightly abnormal reading. In the stage-i group only 1 patient with persistently "negative" titres has relapsed. Only 7 stage-n patients have been studied (table III). TABLE III-SERUM-A.F.P. IN STAGE-II TESTICULAR TERATOMA

3 weeks after orchidectomy. He was a stage-1 case and the A.F.P. titre fell to within normal limits before abdominal radiotherapy was given. He remains well three years later. It is probable that the tumour contained an undetected teratomatous element, and that the titre of 230 ng/ml represented residual A.F.P. from this source. The- 3rd patient had 2 tumours-one in each testis, both diagnosed as seminomas. His A.F.P. titre rose to 6300 ng/ml when mediastinal deposits developed. Surgical exploration revealed the presence of unsuspected teratomatous metastases.

Discussion The consensus of opinion seems to be that A.F.P. synthesis by a tumour represents a derepression of the original response for the production of the protein.3 22 23 Theories postulating A.F.P. synthesis by vitelline cells of yolk-sac origin 1 24 25 are convincing, and recent stu-

dieS26-29 support this view. Raised serum-A.F.p. was found in many patients with tumours in which the yolksac element predominated. The normal levels of A.F.P. in

proportion of cases of teratoma could be due absence of yolk-sac elements in these tumours. a

to

Stationary or even falling serum-A.F.p. concentration,

E,D,=no evidence of disease.

in

cases

in which the

explained by progression 3 patients who had initially raised titres all relapsed; 4 who had normal titres remain disease-free. One patient had an initially normal A.F.P. titre which subsequently became raised after abdominal-node irradiation. He relapsed with a tumour in the left side of his

neck. After treatment with chemotherapy and radiotherapy the A.F.P. titre fell to normal again. 34 out of 54 patients in stages in and iv had raised titres of A.F.P. (63%). In 7 of the 34 patients (21%) a rising A.F.P. titre was the first evidence of relapse after treatment had apparently eradicated all evident disease. In 11 of the 34 patients with raised A.F.P. titres, treatment reduced serum-A.F.p. to below 25 ng/ml. No pre-

viously A.F.P.-positive patient

whose

titre did not clinical remis-

A.F.P.

fall to normal levels achieved a complete sion of his disease, although 3 of the 11 patients (27%) whose A.F.P. titres fell from greater than 25 ng/ml to less than 25 ng/ml on achieving a complete remission, did not produce abnormal A.F.P. concentrations in a subsequent relapse. Thus, although about a third of patients with known disease never had A.F.P. titres higher than 25 ng/ml, no patient with a persistently raised A.F.P. titre failed to show clinical evidence of active tumour either at the time of the first raised titre or subsequently. In other words, although there were false-negative correlations btween A.F.P. titre and the presence of tumour, there sere no false-positive results in this series.

F’equency of Raised Serum A.F.P.

in Seminoma

Patients Of the 34 seminoma patients only 3 had A.F.P. titres above 2S ng/ml at any time. 1 patient had a single titre of 440 ng/ml 2 years after curative treatment. It fell ontaneously to less than 25 ng/ml without treatment and he remained well one year later. It seems probable ::,at the initial high titre was due to clerical error. The second patient had a single raised titre of 230 ng/ml

tumour

progresses, could be

of disease

to a

degree of de-difsynthesise drugs on

ferentiation where the cell is longer able to A.F.P., and also by the effect of cytotoxic

A.F.P.-producing yolk-sac elements. This study shows that R.i.A. is a good way of detecting A.F.P. in patients with testicular tumours and that serum-A.F.p. has clinical relevance in diagnosis, staging, and prognosis. In about two-thirds of teratoma patients, A.F.P. titre will be elevated, although the presence or absence of a positive titre does not distinguish histological subtypes of teratomas. However, a positively raised A.F.P. titre will distinguish between seminoma and teratoma since persistently abnormal titres of A.F.P. are not found (or at least only very rarely found) in patients with pure seminomas. A raised A.F.P. titre in a patient thought to have a seminoma should alert the clinician to the very high chance that a teratomatous element is present in the tumour. This is important in the choice of appropriate treatment. 14 In patients with stage-i testicular teratoma treated by orchidectomy and irradiation, approximately 20% subsequently relapse, predominantly outside the abdomen (neck nodes and lung). 14 In stage-i patients treated with either inadequate irradiation or orchidectomy alone, there is evidence that occult abdominal-node metastases are present despite a negative lymphogram.30 The finding in this study that 6 of 27 stage-! teratoma patients had raised serum-A.F.p. post orchidectomy and that in 4 patients who remain free from disease, the titre fell after nodal irradiation suggests that metastases were, in fact, present. The finding, however, that 39% of stage n, III, and iv patients with unequivocal evidence of metastases did not have raised titres means that false-negative results can occur. In the stage i and II group only 1 patient has relapsed despite persistently normal titres. Thus patients with apparently early-stage disease who have raised titres of A.F.P. should be monitored extremely closely, particularly with regard to supraclavicular node and pulmonary metastases. Furthermore, a rising titre after

436

apparently successful therapy is strongly suggestive of relapse. In our experience about a fifth of patients who subsequently relapse will have abnormal titres of before there is other evidence of disease A activity. high titre after initial therapy is a strong indication for thorough investigations, again with particular serum-A.F.p.

lung and supradiaphragmatic nodal metasregard tases. Finally, a normal titre does not necessarily exclude the presence of tumour, although these observations suggest that when the initial titre is high and falls to normal levels after radiotherapy this may indicate that nodal metastases have been eradicated. However, we found that 27% of tumours produced abnormal amounts of A.F.P. at one time and failed to do so at another, so that a normal A.F.P. titre in a patient in whom the serum-A.F.p. was raised before treatment does not exclude disease activity. There can be no doubt that measurement of the serum-A.F.p. is clinically useful in the management of patients with testicular tumours. A raised serum-A.F.p. titre in a patient after orchidectomy, confirmed by repeated examination, should be regarded as definite proof of residual tumour so long as there is no obvious liver disease. It is unfortunate that in this series only 1 patient had a pre-orchidectomy A.F.P. titre estimated. Clearly this should now be part of the clinical management of any patient in whom a testicular tumour is sus-

PROGNOSTIC SIGNIFICANCE OF IN-VITRO COMPLEMENT FIXATION IN LIVER BIOPSY SPECIMENS FROM PATIENTS WITH ACUTE VIRAL HEPATITIS TYPE B

Department of Gastroenterology, Ospedale Mauriziano Umberto I, Corso Turati 46, 10128 Turin, Italy

prognostic significance of in-vitro complement fixation (V.C.F.) by hepatitis-B core antigen/antibody immunocomplexes in hepatitis-B surface antigen (HBsAg) positive liver biopsy specimens was prospectively evaluated in 47 patients presenting with acute viral hepatitis type B. 34 of 37 V.C.F.-negative patients made an uneventful recovery and became HBsAg negative; in all patients with a V.C.F.-positive test chronic hepatitis and persistent antigenæmia developed. The V.C.F. test is a simple and reliable prognostic indicator of persistent infection and of progression of apparently acute hepatitis to a chronic liver disorder.

Introduction

IN:VITRO complement fixation (v.c.F.), presumably by core antigen (HBcAg) immune complexes,

hepatitis-B

We thank the many surgeons and radiotherapists who have referred to the testicular tumour unit. The histology was reviewed in every case by Prof. N. F. C. Gowing or Prof. A. M. Neville, both members of the Testicular Tumour Panel of Great Britain. Radiological investigations were done by Dr J. S. Macdonald. Mrs D. E. Austin provided valuable help with the data, and secretarial help was given by Mrs Jean Lewis.

was

Requests for reprints should be addressed to T.J.McE. REFERENCES

Abelev, G. I., Assercritova, I, V., Kraevsky, N. A., Perova, S. D., Perevodchikova, N. I. Cancer, 1967, 2, 551. 2. Abelev, G. I. Cancer Res. 1971, 14, 295. 3. Masopust, J., Kithier, K., Radl, J., Koutecky, J., Kotal, L. Int. J. Cancer, 1968, 3, 364. 4. Hull, E. W., Moertel, C. G., Carbone, P. P. Clin. Res. 1969, 17, 403. 5. Adinolfi, A., Adinolfi, M., Lessof, M. H. J. med. Genet. 1975, 12, 138. 6. Mawas, C., Kohen, M., Lemerle, J., Buffe, D., Schweisguth, O., Burtin, P. Int. J. Cancer, 1969, 4, 76. 7. Lamerz, R., Fateh Moghadam, A. Klin. Wschr. 1975, 53, 147. 8. Bourgeaux, G., Martin, F., Aupecle, P., Guerrin, J. Presse méd. 1971, 79, 1.

1589. D. A., Abelev, G. I., Levina, D. M., Marienbach, E. V., Martochkina, G. A., Laskina, A. V., Solovjeva, E. A. Int. J. Cancer, 1973, 11, 586. 10. Buffe, D. GANN Monogr. Cancer Res. 1973, 14, 117. 11. Mawas, C., Buffe, D., Schweisguth, O., Burtin, P. Rev. Eur. clin. Biol. 1971, 16, 430. 12. Alpert, E., Hersberg, R., Schur, P. H., Isselbacher, K. J. Gastroenterology, 1971, 61, 137. 13. Collins, D. H., Pubh, R. C. B. Br. J. Urol. 1964, 36, suppl. 2. 14. Peckham, M. J., McElwain, T. J. Clins Endocr. Metab. 1975, 4, 665. 15. Kohn, J., Kahan, M. J. immun. Meth. 1976, 11, 303. 16. Purves, L. R., Purves, M. S. Afr. med. J. 1972, 46, 1290. 17. Nishi, S., Hirai, H. GANN Monogr. Cancer Res. 1973, 14, 79. 18. Leek, A. E., Ruoss, C. F., Kitau, M. J., Chard, T. Br. J. Obstet. Gynaec. 1975, 82, 669. 19. Masseyef, R. Path. Bil. 1972, 20, 703. 20. Ruoslahti, E., Seppala, M. Nature, New Biol. 1972, 235, 161. 21. Hirai, H., Nishi, S., Watabe, H. J. nucl. Med. 1973, 13, 783. 22. Monod, J., Jacob, F. Symp. quant. Biol. 1961, 26, 193. 23. Omenn, G. S. Ann. intern. Med. 1970, 72, 136. 24. Ballas, M. Ann. clin. Lab. Sci. 1974, 4, 267. 25. Tsuchida, Y., Endo, Y., Urano, Y., Ishida, M. Ann. N.Y. Acad. Sci. 1975, 9.

Elgort,

1, 221. 26. Talerman, A., Haije, W. G. Cancer, 1974, 34, 1722. 27. Grigor, K., Neville, A. M. Personal communication. G. Acta path. microbiol. scand. 1975, 83, 573. 29. Palmer, P. E., Safaii, H., Wolfe, H. J. Am. J. clin. Path. 1976, 65, 575. 30. Tyrell, C. J., Peckham, M. J. Unpublished.

28.

Norgaard Pedersen, B., Albrechtsen, R., Teilum,

The

Summary

pected. patients

F. BONINO G. VERME

M. RIZZETTO S. DIANA

to

detected in the liver biopsy specimens of patients with chronic hepatitis-B surface antigen (HBsAg) positive liver disease, whereas it was not observed in hepatitis, or in healthy carriers, in acute self-limited HBsAg-negative liver disorders. 12 Since it is often impossible to predict the chronic evolution of apparently acute hepatitis on biochemical and histological grounds, we studied the prognostic significance of v.c.F. detection by prospectively evaluating 47 patients with no evidence of previous liver disease, presenting clinically with acute type-B viral hepatitis. The results of the V.C.F. test in the biopsy specimens taken after admission to hospital and the appearance of symptoms were correlated with the histological and clinical data at follow-up 7-12 months afterwards, Persistence of antigenaemia and the development of chronic liver disease were strikingly more common in the

v.c.F.-positive patients. Patients and Methods 47 patients with no evidence of previous liver disease, pre senting with an episode of acute HBsAg-positive hepatitis, were studied. Patients with a history of jaundice or hepatin or with abnormal serum-protein concentrations were exclude The clinical diagnosis of acute type-B hepatitis was basedoc the

recent onset

of symptoms, abnormal concentrationsc:

serum-transaminases, and detection of HBsAg by radio munoassay (R.I.A.). Liver biopsy specimens were obtainr.’ within 30 days of admission to hospital, usually between tbw 4th and 9th days. Steroids or azathioprine were not given any patient. After leaving hospital all patients were followed up for to a

year

at

two-monthly intervals by physical examination;

terviews; determinations of serum bilirubin, transaminas and protein concentrations; and detection of circuit HBsAg by R.I.A. A second biopsy specimen was obtained from all patiend who still had symptoms 7-12 months after the first bior-