DIAGNOSIS OF RENAL ALLOGRAFT REJECTION BY ANALYSIS OF FINE-NEEDLE ASPIRATION BIOPSY SPECIMENS WITH IMMUNOSTAINS AND SIMPLE CYTOLOGY

Saturday 20 September

DIAGNOSIS OF RENAL ALLOGRAFT REJECTION BY ANALYSIS OF FINE-NEEDLE ASPIRATION BIOPSY SPECIMENS WITH IMMUNOSTAINS AND SIMPLE CYTOLOGY G. ALEX BISHOP BRUCE M. HALL WAUGH JENNIFER JEANETTE PHILIPS S. HORVATH GEOFFREY G. DUGGIN JOHN R. A. G. Ross SHEIL JAMES JOHNSON DAVID J. TILLER

Department of Renal Medicine, Royal Prince Alfred Hospital, Camperdown, NSW 2050, Australia Fine-needle aspiration biopsy specimens of renal transplants were analysed by means of commercially available monoclonal antibodies and an immunoperoxidase stain. Three cellular features associated with acute cellular rejection were identified—heavy infiltrates of activated T cells or large mononuclear cells strongly expressing HLA-DR antigens, and HLA-DR expression by renal tubular cells. A combination of semiquantitative scores for these features correctly identified rejection in 32 of 34 cases, with no false positives in cases of cyclosporin nephrotoxicity or stable graft function.

Summary

Introduction IN renal transplantation early and accurate diagnosis of rejection presents great difficulties. Deterioration in renal function may be due not only to rejection but also to drug nephrotoxicity, vascular or urinary obstruction, or infection. Furthermore, in patients with post-transplant renal failure, rejection may supervene without any signs to alert the clinician. 1,2 The ultimate diagnostic test for rejection is core renal biopsy but this carries a substantial morbidity and thus cannot be done repeatedly.2,3 Fine-needle aspiration biopsy (FNAB), by contrast, allows safe and frequent sampling. 4,5 In the analysis ofFNAB specimens described by Hayry and Von Willebrand,4,5 a differential count of leucocyte populations in the aspirate is compared with that in blood-the corrected increment.4,5 The diagnosis of rejection depends upon identification of a few inflammatory cells such as lymphoblasts, macrophages, and plasma cells that are indicative of rejection, but only a very small fraction of the aspiration sample is examined. We report here a study in which FNAB specimens were analysed with monoclonal antibodies and an indirect immunoperoxidase stain to identify cellular changes unique to immune inflammation that have been identified in core

1986

biopsy specimens and in experimental models of rejection. By means of simple cytology combined with immunostains, a large proportion of the aspirated cells could renal

be screened for these features. Three features that are associated with rejection and are not present in blood at the time of rejection were looked for—(1) a mononuclear cell infiltrate that includes a large number of activated and lymphoblastoid T cells;6-s (2) most of these infiltrating mononuclear cells expressing HLA-DR antigens, because they are activated T cells, monocytes, or macrophages, while only a minority of circulating mononuclear cells express HLA-DR;9,10 (3) renal tubular cells expressing large amounts of HLA-DR antigens during rejection, compared with the slight or absent expression of HLA-DR antigens by renal tubular cells in grafts without rejection.9,lo

Materials and Methods Clinical FNAB specimens were obtained from renal transplant patients as described by Hayry.4,5 Briefly, a 22 G spinal needle was used to aspirate into medium consisting of RPMI 1640 (Flow Laboratories, NSW, Australia) plus 1 mmol/1 HEPES, 0-1% human serum albumin, and heparin 25 U/ml. When in the graft, the needle was gently passed up and down through 1-15cm, two samples being taken from different parts and mixed. One-third was used for conventional cytological analysis4,s and two-thirds was used for immunoperoxidase staining. 155 specimens were obtained from 37 patients during their initial hospital stay, 1-8 weeks after transplantation. Of these, 46 could not be analysed because the sample was inadequate (see below). Of the 109 aspirates analysed, 70 were from 24 patients treated with cyclosporin (1-13 samples per patient) and 39 were from 11 patients treated with azathioprine/prednisone (1-8 samples per patient). Rejection episodes in both groups were treated with methylprednisolone for three days as described elsewhere.2 The rejection status of the graft at the time of each biopsy was determined according to clinical criteria. In functioning grafts, rejection was diagnosed when a > 25 % rise in serum creatinine not due to urinary or vascular obstruction or nephrotoxic drugs was accompanied by one or more ofthefollowingsigns: oliguria, fever, and graft swelling or tenderness. Of the 34 episodes of rejection, 10 occurred while the patient had post-transplant oliguria and all these were confirmed by core renal biopsy performed within three days of the FNAB and scored by previously described criteria.These core biopsies were performed every seven to ten days during post-transplant oliguria. In the remaining 24 rejection episodes, core renal biopsy was done within three days in only 14 but the diagnosis of rejection was confirmed in all of these. Cyclosporin nephrotoxicity was diagnosed if a core biopsy specimen showed diffuse or stippled interstitial fibrosis or vascular changes (as described by Mihatsch 1) or, in the absence of a core biopsy, if the patient did not respond to antirejection therapy 8508

646 but did respond to a reduction in the doseofcyclosporin. According to these criteria FNAB samples were categorised into four groups: "rejection", from one day before diagnosis to one day after cessation of rejection treatment; "quiescent", no evidence of rejection; "prerejection", from five days before rejection to two days before rejection ; "post-rejection", from two days aftertreatmentto four days after treatment, with an accompanying fall in serum creatinine.

Irnmunoperoxidase Staining FNAB samples were centrifuged for 5 min at 300 g and the supernatant was aspirated. All of the cell pellet was resuspended in a minimum of 200 III of Earle’s medium (Gibco, New York, USA) plus 10% AB serum with more medium added if necessary to given an even layer of cells in cytospin preparations. 50 1 specimens were centrifuged onto microscope slides for 5 min at 100 in a Cytospin 2 (Shandon Southern Products, Runcom, UK) then air-dried for at least 60 min. Slides were fixed for 30 s in buffered formol acetone 12 at 4°C, then washed in two changes of distilled water and once in phosphate buffered saline (PBS). The area around the cells was carefully blotted dry and 25 III of mouse monoclonal antibody was added to each slide. Antibodies used were T 11(Coulter Electronics, Florida, USA) reactive with T lymphocytes,1 in 40 dilution; 13 and12 (Coulter) reactive with HLA-DR monomorphic antigen, 1 in 80 dilution.14 For a control an anti-influenza neuraminidase mouse monoclonal antibody, 19 not reactive with any human antigens, was used atain 20 dilution. After 30 min at 37°C in a humid atmosphere, each slide was rinsed with 5 ml ofPB S then washed in a stirred bath of PBS for 5 min and blotted dry. Slides were incubated with rabbit antiserum to mouse immunoglobulin (Dako, Copenhagen, Denmark) at 1 in 200 dilution then swine antiserum to rabbit immunoglobulin (Dako) at 1 in 25 dilution and finally rabbit peroxidase anti-peroxidase complexes (Dako) at 1 in 50 dilution. Each incubation was for 15 min at 37°C and was followed by rinsing, washing, and drying. All antibodies were diluted in 5 % AB serum in PBS plus 15 mmol/1 sodium azide (Sigma). Slides were incubated with substrate solution consisting of 1 mg/ml diaminobenzidine tetrahydrochloride (Sigma), 0’01% hydrogen peroxide, and 0-3% sodium azide in 0-05 mol/l "tris" buffer pH 7-6. After 10 min slides were washed in tapwater and counterstained in Harris haematoxylin for 1 min. After rinsing in tapwater, slides were dehydrated and mounted in ’Eukitt’ (Carl Zeiss Pty Ltd) and examined by light microscopy. Additional stains were done on some specimens to identify the composition of the large HLA-DR + mononuclear cells. These were: T4 (Coulter), reactive with the CD4 antigen ofT helper// inducer cells,1 in 40 dilution; 15 T8 (Coulter), reactive with the CD8 antigen of cytotoxic/suppressor T lymphocytes, 1 in 80 dilution;16 OKMI (Ortho Diagnostics, New Jersey, USA), reactive with monocytes and granulocytes,1 in 160 dilution; 17 and F8 (Australian Monoclonal Developments, Artarmon, Australia) reactive with vascular endothelium, 1 in 40 dilution. IS

Scoring of FNAB The whole cytospin area was assessed for each slide and the semiquantitative score for each cell type was determined by counting all cells in the cytospin area (table I). 46 of155 FNAB samples that had fewer than 10 tubular cell clusters were regarded as inadequate and were excluded from analysis.

Results The three features examined were identified by a combination of immunoperoxidase stains and simple cytological criteria. T lymphoblasts were identified as T cells by the T 11 monoclonal antibody and as lymphoblasts if their diameter was equal to or greater than that of polymorphonuclear leucocytes. This criterion was simple because the cells can be readily compared with the polymorphs present in FNAB samples because of blood contamination. Fig la shows a typical T lymphoblast. Tubular cells were identified by their morphology, as described by Von Willebrand and Hayry.4Tubular HLA-

TABLE I-SCORING INDEX*

*All cells in a cytospin preparation are counted and absolute numbers ofT 11 + lymphoblasts and HLA-DR + mononuclear cells are counted. The number of HLA-DR + tubular cell clusters are expressed as a percentage of the total number of tubular cell clusters.

DR staining was seen either as a diffuse or granularstain of the whole cell or less often as membrane staining observed especially in clusters of tubular cells. It was stronger in intensity than the faint cytoplasmic staining sometimes seen in biopsy specimens from normal kidney. Fig lb shows a cluster of tubular cells from a patient with rejection; these cells have strong expression of HLA-DR. FNAB taken during rejection often showed an infiltrate of large mononuclear cells that stained strongly for HLA-DR antigen (fig lc). Most of these strongly HLA-DR positive mononuclear cells were identified morphologically as activated monocytes or macrophages. They were rarely seen in FNAB samples from kidneys that were not undergoing rejection, although in patients with acute tubular necrosis the specimens often showed large numbers of smaller, moderately HLA-DR positive mononuclear cells. The large HLA-DR + mononuclear cells in FNAB samples taken during rejection were further characterised with monoclonal antibodies. A variable proportion stained with OKMI or OKT4, but none reacted with Til. The staining patterns suggest that many of these cells are of the monocyte macrophage lineage, since both OKMI and T4 stain these

cells.20,21 A scoring system was devised for quantitation of each of the three rejection indicators (table I). Each rejection indicator was scored - to + + + and the score was compared with the patient’s rejection state (table II). With the T lymphoblast score all 44 FNAB specimens in the quiescent group had scores of - or + ; of 34 graded as rejection, 30 scored + + or + + + andonly4were +.Ifascoreof + + or greater is taken to indicate rejection, there are no false positives in quiescent cases

andonly4falsenegatives. These 4 were patients who had

received at least two days of corticosteroid treatment for the rejection episode, two of them in each treatment group. 23 of the 34 FNAB samples from patients with rejection had + + or + + + expression of HLA-DR antigens compared with 6 of 44 in the quiescent group (p < 0001, table II). 16 of the 18 samples from azathioprine-treated patients were positive, compared with 7 from 16 cyclosporintreated patients. Only 6 of 44 samples from patients with no active rejection had HLA-DR + tubular cells; these 6 had all had rejection episodes six to ten days previously. In the immediate pre and post rejection period about one-third of biopsy specimens showed HLA-DR + tubular cells. Mononuclear HLA-DR + cells were also more common (p < 0-01) in patients with active rejection than in those with no rejection. If a score of + + or greater for mononuclear HLA-DR was taken as positive, then only 4 of 14 quiescent cases were falsely positive. These were in the first week posttransplant-a period in which a monocytic infiltrate is commonly associated with recovery from ischaemic

damage.4,5 When all three of the indicators are used, the best indicator of rejection in a FNAB specimen is a + + or + + + score for T lymphoblasts or a + + or + + + for both HLA-DR +

647 tubular cells and mononuclear cells. Assessed in this way, 32 of the 34 FNAB specimens from grafts with rejection were correctly identified as showing rejection whereas none of the 44 specimens from quiescent grafts were graded as showing rejection. In the pre-rejection period 6 of 12 samples were scored as showing rejection and in the post-rejection period 4 of 19.

Cyclosporin versus Conventional Immunosuppression 15 FNAB specimens were taken from cyclosporin-treated patients and 19 from azathioprine-treated patients during rejection episodes. The ranked data were assessed by BrandtSnedecor contingency table analysis. A T lymphoblast infiltrate was present in all but 2 specimens in each treatment group, and

+ + or + + + in cases without

was never

rejection. Azathioprine-treated patients showed a significantly (p=005) greater expression of HLA-DR on tubular cells during rejection than did the cyclosporin group. There was no difference between the groups in the proportion with T lymphoblast infiltrate or with a HLA-DR + mononuclear cell infiltrate. In the FNAB samples from quiescent grafts, the cyclosporin patients had less tubular HLA-DR expression than the azathioprine patients (p 0-05), but there was no difference in the numbers of T lymphoblasts or mononuclear HLA-DR + cells. In the whole group of FNAB samples, azathioprine patients had far more HLA-DR + tubular cells than did cyclosporin patients (p 00005). =

=

Sequential Changes Sequential FNAB scores were available on several patients. These could indicate the course of rejection-either resolution (fig 2) or persistence requiring repeated further treatment (fig 3). Rejection recurred within ten days in all 4 patients who had T 11 + lymphoblasts in their aspiration samples on completion of rejection treatment compared with 2 of 15 who did not. Persistence of HLA-DR + mononuclear cells or HLA-DR + tubular cells did not correlate with recurrence of rejection in the short term. In sequential studies, HLA-DR + tubular cells were not detected before the first

rejection episode but thereafter they could be detected for up days even in the absence of other clinical or FNAB markers of rejection (see fig 2). to ten

Fig 1-Immunoperoxidase jecting allografts.

stains of FNAB

specimens

from

re-

Stained cells appear dark grey. (A) T lymphocytes are identified by staining with Tll. The smaller T lymphocyte is a normal resting cell whereas the larger T lymphocyte (arrowed) is an activated T lymphoblast. (B) Tubular cells expressing HLA-DR are identified by staining with I2. The cluster of tubular cells (large arrow) is stained while an adjacent cluster (small arrow) is unstained. (C) Large mononuclear cell showing strong expression of HLA-DR identified by staining with 120

Comparison with Core Biopsy Specimens 33 of 36 core biopsy specimens from 24 patients z4 per patient) could be assessed according to previously describedl,11 morphological criteria. Moderate or severe interstitial cellular rejection was found in 8 on cyclosporin and 6 on azathioprine; nil or minimal focal interstitial cellular infiltrate was found in 7 on cyclosporin and 4 on azathioprine; and signs of cyclosporin nephrotoxicity alone were present in

TABLE II-CLINICAL STATE OF ALLOGRAFT VERSUS ASPIRATE SCORE

Cy = cyclosporin; aza = azathioprine.

648

Fig 3-Sequential monitoring of a renal allograft patient with immunoperoxidase staining of FNAB. Rejection marker scores continue but fall after the second.

WEEKS POST TRANSPLANT

Fig 2-Sequential monitoring of a renal allograft patient with immunoperoxidase staining of FNAB specimens. High initial score for rejection markers falls after immunosuppressive therapy. 8. Table illshows how the findings on FNAB samples taken either on the day of core biopsy or one day before core biopsy correlated with those in the core biopsy specimen. All but one case of moderate/severe rejection were associated with T lymphoblast infiltrate, and HLA-DR + tubular expression and HLA-DR + mononuclear cell infiltrate were also found. Cases showing cyclosporin nephrotoxicity and nil or mild cellular infiltrate had no T lymphoblast infiltrates and never had more than one score of + + or + + + for HLA-DR + tubular cells or HLA-DR + mononuclear cells. The mild focal cellular infiltrate in core biopsy specimens from

cyclosporin patients during clinically quiescent periods was not associated with a positive T lymphoblast score on the aspiration specimens. Comparison with Conventional Cytology of FNAB 31 FNAB samples from patients with rejection were assessed by conventional cytology. 15 had a corrected increment of > 2-5(diagnostic of rejection), 6 were negative, and 10 were not analysed because they were inadequate. In all these 31 samples rejection was diagnosed from the

to rise

after first anti-rejection treatment

immunostained FNAB specimen. 30 FNAB samples were from patients without rejection. 13 of these had a corrected increment <2-5. Immunostaining produced only 1 false positive, in a patient who had no T lymphoblast infiltrate but had a mononuclear cell infiltrate; a further 16 FNAB samples from quiescent grafts were not suitable for analysis. Discussion Of the three characteristics of acute cellular rejection, the specific was activated T cells-not surprisingly, since these are the effectors of rejection22 and are found in large numbers when core biopsy specimens of renal allograft rejection are examined by both conventional and immunopathological stains.’,’,8,10 The failure to identify these cells in some instances of rejection may reflect the rapid disappearance of these cells after the iriitiation of rejection treatment (in all these cases the patients had received at least two days of treatment). Activated T lymphocytes are independent of the type of immunosuppressive therapy used, as evidenced by immunoperoxidase staining of core biopsy , specimens21 and our comparison of T lymphocyte infiltrates in the two treatment groups. The presence of large HLA-DR + mononuclear cells and the expression of HLA DR + by renal tubular cells are probably secondary to the T cell effector response of the rejection process. The HLA-DR + mononuclear cells include activated T cells, B cells, and monocytes and macrophages. Even during rejection these cells usually make up < 15% of peripheral blood mononuclear cells, thus any blood contamination will make only a small contribution to most

TABLE III-CORE RENAL BIOPSY FINDINGS VERSUS ASPIRATE SCORE

649 the cells in the aspiration biopsy specimen. Furthermore, with cytological analysis it was possible to identify a large number of the HLA-DR + mononuclear cells in rejecting allografts as either lymphoblasts or macrophages, which are

morphologically distinct from the HLA-DR + cells ofblood, samples taken at the time of rejection. The identity of the HLA-DR + mononuclear cells remains to be firmly established. They do not react with T 11 antibody, or with a monoclonal antibody to human factor VIIIantigen. They do show partial reactivity with OKM1 and T4. OKM1 reacts with all peripheral blood mononuclear cells but may not react with most of the activated macrophages present in rejecting renal allografts.8 The CD4 antigen identified by T4 also appears in the cytoplasm of activated macrophages but is not present on normal resting monocytes.20,21 Thus the staining and cytology of these cells suggest they are activated even in

macrophages. The expression

.

,

of HLA-DR on renal tubules has been shown to coincide with acute cellular rejection, but often takes days to subside after successful rejection treatment.9,23 Similar increases in class IImajorhistocompatibility antigens on cells that normally do not express them have been seen in other tissues during rejection responses 24-26 and in T-cellmediated autoimmune diseases27,28 HLA-DR expression is probably induced by gamma interferon released by infiltrating activated T cells.29,30 In core biopsy specimens showing mild to moderate rejection, cyclosporin treatment is associated with less expression of HLA-DR on renal tubular cells than is azathioprine treatment and this may be due to the capacity of cyclosporin to inhibit the release of gamma interferon from activated T cells.31 This would explain the lower frequency of HLA-DR + tubular cells in cyclosporin treated patients with rejection. For these reasons HLA-DR expression is a less efficient marker of rejection than T

lymphoblasts. The assessment of fme-needle aspiration samples by conventional cytological techniques requires much skill. The corrected increment depends upon identification of only a few lymphoblasts or macrophages in a differential count of 200 white cells.4,5 Our results with this technique gave good correlation but we found the immunopathology approach to be simpler and more often correct. This may be because the cytospin slides treated with immunoperoxidase allow assessment of the critical cells indicative of rejection in 500 - 3000 white cells and screen a much larger proportion of the aspiration sample. Immunopathological analysis of lymphocyte subsets in FNAB samples has been attempted with immunofluorescent labels and flow cytometry or with immunoperoxidase stains and CytOlogy21,32 but these did not prove satisfactory methods for diagnosis of rejection, probably because there is not a great difference in the ratio of CD4:CD8 cells in blood and graft infiltrate. 10 One potential drawback of the immunostaining technique is that the results might be affected by cyclosporin therapy. This was not so. If the stained FNAB specimen shows no evidence of rejection, nephrotoxicity or another cause of deterioration of renal function should be suspected. Activated T lymphoblasts seem to be the hallmark of rejection and large numbers of HLA-DR + mononuclear cells and/or HLA-DR + expression by renal tubular cells provide supporting evidence. The method we described here is simple, does not require the cytological skills demanded by the method of Hayry and Von Willebrand,4,5 and seems highly accurate. The development of monoclonal antibodies that directly identify alloactivated T cells and activated macrophages could allow further simplification; but,

meanwhile, this technique employing commercially available monoclonal antibodies information.

can

provide important diagnostic

This work was supported by the National Health and Medical Research Council of Australia, The Australia Kidney Foundation, and the Lions Clubs of District 102N2. We thank Carole Fraser for her assistance in performing aspirates and Karen Hynes for typing the script.

Correspondence

should be addressed to G. A. B., Department of Renal Alfred Hospital, Missenden Road, Camperdown,

Medicine, Royal Prince NSW 2050, Australia.

REFERENCES 1. Famsworth

A, Hall BM, Ng ABP, et al. Renal biopsy morphology in renal transplantation. A comparative study of the light-microscopic appearances of biopsies from patients treated with cyclosporin A or azathioprine prednisone and antilymphocyte globulin. Am J Surg Pathol 1984; 8: 243-52. 2. Hall BM, Tiller DJ, Horvath JS. Treatment of renal transplant rejection. Cyclosporin A versus conventional treatment with azathioprine, prednisone and antithymocyte immunoglobulin in primary cadaver renal transplantation. MedJ Aust. 1985; 142: 179-85. 3. Corson JM. The pathologist and the kidney transplant. Pathol Annu 1977; 7: 251-92. 4. Hayry P, Von Willebrand E. Practical guidelines for fine needle aspiration biopsy of human renal allografts. Ann Clin Res 1981; 13: 288-306. 5. Hayry P, Von Willebrand E, Ahonen J, Eklund B, Lautenschlager I. Monitoring of organ allograft rejection by transplantation aspiration cytology. Ann Clin Res 1981; 13: 264-87. 6. Hayry P, Von Willebrand E. Transplant aspiration cytology. Transplantation 1984; 38: 7-11. 7. Strom TB, Tilney NL, Paradysz JM, Bancewicz J, Carpenter CB. Cellular components of allograft rejection: Identity, specificity and cytotoxic function of cells infiltrating acutely rejecting allografts. Immunology 1977; 118: 2020-26. 8. Hall BM, Bishop GA, Farnsworth A, et al. Identification of the cellular subpopulations infiltrating rejecting cadaver renal allografts. T4 cells are the predominant T cell subset. Transplantation 1984; 37: 564-70. 9. Hall BM, Bishop GA, Duggin GG, Horvath JS, Philips J, Tiller DJ. Increased expression of HLA-DR antigens on renal tubular cells in renal transplants: relevance to the rejection response. Lancet 1984; ii: 247-51. 10. Bishop GA, Hall BM, Duggin GG, Horvath JS, Sheil AGR, Tiller DJ. Immunopathology of renal allograft rejection analysed with monoclonal antibodies to mononuclear cell markers. Kidney Int 1986; 29: 708-17. 11.Mihatsch MJ,Thiel G, Spechtin HP, et al. Morphological findings in kidney transplants after treatment with cyclosporine. Transplant Proc 1983; 15 (suppl) 65. 12. Yam LT, Li CY, Crosby WH. Cytochemical identification of monocytes and granulocytes. Am J Clin Pathol 1971; 55: 283-90. 13. Kamoun M, Martin PJ, Hanson JA, Brown MA, Suadak AW, Nowinski RC: Identification of human T lymphocyte surface protein associated with the E rosette receptor J Exp Med 1981; 153: 207-12. 14. Nadler LM, Stashenko P, Hardy R, Pesando JM, Yunis FJ, EJ Schlossman SF. Monoclonal antibodies defining serologically distinct HLA-D/DR related Ia-like antigens in man. Human Immunol 1981; 2: 77-90. 15. Reinherz EL, Kung PC, Goldstein G, Schlossman SF. A separation of functional subsets of human T cells by a monoclonal antibody. Proc Natl Acad Sci USA 1979; 76: 4061-65. 16. Reinherz EL, Kung PC, Goldstein G, Levey RH, Schlossman SF. Discrete stages of human intrathymic differentiation: analysis of normal thymocytes and leukemic lymphoblasts of T cell lineage. Proc Natl Acad Sci USA 1980; 77: 1588-92. 17. Breard J, Reinherz EL, Kung PC, Goldstein G, Schlossman SF. Amonoclonal antibody reactive with human peripheral blood monocytes. J Immunol 1980; 124: 1943-48. 18. Francis SE, Joshua DE, Emer T, Kronenberg H. Monoclonal antibodies to human FVIIIR:Ag and FVIIIC. Pathology 1985; 17: 579-85. 19. Holmes KT,Hampson AW,Raison RL, Webster RJ, O’ Sullivan WJ, Mountford CE. A comparison of two antineuraminidase antibodies by complement activation. Eur J Immunol 1982; 12: 523-26. 20. Wood GS, Warner NL, Warnke RA. Anti-leu-3 T4 antibodies react with cells or monocyte/macrophage and Langerhans lineage. J Immunol 1983; 131: 212-16. 21. Wood RFM, Thompson JF, Carter NP. Changes in lymphocyte subpopulations in

blood and kidney after renal transplantation. In: Morris PJ,Tilney NL, eds. Progress transplantation. Edinburgh. Churchill Livingstone, 1984; 148-85. BM, Dorsch S, Roser BJ. The cellular basis of allograft rejection in vivo. 1. The cellular requirements for first-set rejection of heart grafts. J Exp Med 1978; 149:

in 22. Hall

878-89. 23. Hayry P, Von Willebrand E, Ahouon J, Eklund B. Do well-to-do and repeatedly rejecting renal allografts express the transplantation antigens similarly on their surface> Scand J Nephrol 1981; 64: 52-55. 24. de Waal RMW, Bogman MJJ, Maass CN, Corneilssen LMH, Tax WJM, Koene RAP. Variable expression Ia antigens on the vascular endothelium of mouse skin allografts. Nature 1983; 303: 426-29. 25. Milton AD, Fabre JW. Massive induction of donor-type class I and class II MHC antigen m rejecting cardiac allografts in the rat. J Exp Med 1984, 161: 98. 26. Benson EM, Colvm RB, Russell PS. Induction of Ia antigens in murine renal transplants. J Immunol 1985; 132: 7. 27. Hanafusa T, Pujol-Borrell R, Chiovato L, Russell RCG, Doniach D, Bottazzo GF. Aberrant expression of HLA-DR antigen on thyrocytes in Graves’ disease: Relevance for autoimmunity. Lancet 1983; ii: 1111-15 28. Lambert IA, Suitters AJ, Chisholm PM. Expression of Ia antigen on epidermal keratinocytes in graft-versus-host disease. Nature 1981; 293: 149-51. 29. Pober JS, Gimbrone MA, Cotran RS et al. Ia expression by vascular endothelium is inducible by activated T cells and by human gamma interferon. J Exp Med 1983; 157: 1339-53.

650

ORAL CONTRACEPTIVE USE AND BREAST CANCER IN YOUNG WOMEN A Joint National Case-control

Study in Sweden and

Norway OLAV MEIRIK1 EILIV LUND2 HANS-OLOV ADAMI3 REINHOLD BERGSTRÖM4 PER BERGSJÖ6 THORALF CHRISTOFFERSEN5

Departments of Social Medicine,1 Surgery,3 and Statistics,4 University of Uppsala, Sweden; Clinical Trial Branch, Norwegian Radium Hospital, Oslo, Norway;2 Department of Pharmacology, University of Oslo,5 Department of Obstetrics and Gynaecology, University of Bergen, Norway6

possible association between oral contraceptive (OC) use and the risk of breast cancer developing before the age of 45 was investigated by means of a population based case-control study in Sweden and Norway. Information was obtained by personal interview from 422 (89.2%) of all eligible patients with a newly diagnosed breast cancer from May, 1984, to May, 1985, and from 722 (80.6%) of all contacted age-matched controls. A multivariate analysis, which accounted for several possible confounding factors, revealed a significant (p = 0.03) association between total duration of OC use and breast cancer risk. The relative risk (RR) of Summary

breast

The

after 12 or more years of OC use was 2.2 OC use for more than 7 years before first (1.2-4.0). full-term pregnancy entailed an increased breast cancer risk (RR=2.0 [1.0-4.2]) which was of borderline significance. When total duration of use was considered, the risk of breast cancer was virtually unrelated to age at first OC use and latency from first use. The results suggest that long-term use of OCs may increase the risk of breast cancer in young cancer

women.

Introduction IN the autumn of 1983 reports from Los Angeles, USA,1 and Oxford, UK,2 of an association between the use of oral contraceptives (OCs) and breast cancer in young women caused serious concern in Sweden and Norway, as elsewhere. OCs were introduced in Sweden in 1964 and in Norway in 1967. Fertility surveys in these two countries had revealed that 77% of Swedish women and 42% of Norwegian women were ever users of OCS.3,4 Among women aged 20-24, such contraceptives were used by 36 % in Sweden in 1981 and by 22% in Norway in 1977. National health authorities in both countries strongly supported an investigation within the two countries. In the spring of 1984 a case-control study was initiated with the aim of elucidating any association between use of OCs and breast cancer in young women. The objective was to cover all cases of incident breast cancer in 1 year in young women in Norway and Sweden.

Methods The

design, data collection, and analysis of this joint study were

coordinated.

30.

Because

of

minor

differences

in

national

Bishop GA, Hall BM, Suranyi MG, Tiller DJ, Horvath JS, Duggin GG. Expression of

HLA antigens on renal tubular cells in culture 1. MLC supematants and gamma interferon increase both class I and class II HLA antigens. Transplantation (in press) 31. Groenewegen G, Baurman WA, Jeunhomme GMAA, Van der Linden CJ. Effect of cyclosponne on MHC-class II antigen expression on artenal and venous endothelium m vitro. Transplantation 1985; 40: 21-24. 32. Von Willebrand E. Fine needle aspiration cytology of renal transplants: Background and present application. Transplant Proc 1985; 17: 2071-74

characteristics, the methodology is described separately for Sweden and Norway. Sweden Cases.-In Sweden notification of all newly diagnosed cases of is mandatory. Clinicians and pathologists report separately to the six regional cancer registries, which together cover all Sweden. For the purpose of the present study copies of the notification forms for all cases of cancer of the breast diagnosed in women born in 1939 or later were obtained from the registries. Eligibility criteria were: (1) newly diagnosed, histologically confirmed, invasive carcinoma of the breast diagnosed from May, 1984, to May, 1985 inclusive; (2) residence in Sweden on Jan 1, 1960; (3) less than 45 years of age at diagnosis; and (4) no history of previous malignant disease. All women under 40 years of age at diagnosis and every second woman, chosen at random, between 40 and 44 years of age were eligible for the study. Altogether reports for 248 women under 40 years of age and 282 women aged 40 -44 years were received. 5 women had a history of previous malignancy and 36 women were not resident in Sweden in 1960. Thus 489 women, 223 less than 40 years old and 266 aged 40 - 44 met the eligibility criteria. Of the latter group of 266, 136 were randomly assigned to the study. 30 women (8-4%) declined to participate, in 3 cases the physician refused participation, 2 women had a psychiatric disorder, and 7 died before interview. Thus, 88-3% of the eligible women were included, 196 under 40 years old and 121 aged 40 -44. cancer

Controls.-For each woman (case) who agreed to participate, 1 control was chosen from a continuously updated population register covering all Sweden and held by the National Central Bureau of Statistics. Criteria for inclusion as a control were that the woman should have no history of previous malignant disease, she should have been resident in Sweden in 1960 and born in the same year and month ( t 1) as the case woman, and she should be resident in the same county. A second control was chosen for each case less than 40 years of age at diagnosis from a continuously updated fertility registry covering all Swedish women giving birth in 1960 or later. In addition to the criteria given above she should have given birth for the first time at the same age ( f 2 months) as the case woman. The reason for choosing this second control series was that age at first birth is possibly an important confounder in this context. (Because of initial misclassification, no second control was chosen for 1 case woman under 40 years old.) For every set of controls three additional control sets were selected for potential replacement in the event’that a control woman should refuse to participate or prove not to be eligible. 2 controls were replaced because they were found to have a history of breast cancer. For 69 controls a reserve control was interviewed, since the first control selected refused to participate. 11 control women were not reached and 9 had moved to another county or abroad. Thus out of 601 potentially eligible controls contacted or sought, 85-2% (or 88-1% of those contacted) were included in this series. Interview.-The cases and controls received an introductory letter and a brief description of the aim and scope of the study. In one region a physician’s approval had to be granted before the introductory letter was mailed. After about one week the women were telephoned by an interviewer and asked if they would participate. The cases were interviewed three to twelve months after diagnosis. The cases and controls were interviewed personally by specially trained professional female interviewers employed by the Swedish National Central Bureau of Statistics. The same interviewer interviewed pairs and triplets of case and control(s). As a rule the interview was conducted in the woman’s home. For 12 controls (2-3%) an interview over the telephone had to be accepted. The interview followed a detailed schedule that focused on the social background and the reproductive and contraceptive histories. As an aid for recalling the contraceptive history a calendar was used in which life events such as menarche, cohabitation, marriage, divorce, childbirths, lactation, and abortions were recorded. The contraceptive history was then recalled relative to life events. To facilitate accurate recall of the name of the various OC brands, the interviewers had a binder with photographs of the forty-six different packages of OCs used in Sweden from 1964 to 1984 and the dates of their approval for marketing and their withdrawal.