144
GKACEY ET AL
syndrome’ in man is unknown. They would suggest, however, that there is an important and previously ignored mucosal lesion in this syndrome. Its relevance to specific digestive and absorptive defects such as peptidase activities in relation to hypoproteinaemia”. 18, require investigation in clinical and experimental situations. Acknowledgement
VOL.
5,
NO.
2
6 . GK4CEY. M.. BLRKP, V.. OSHlK. A.. BARKI.N, J.andGl.ASGtIw. E. F.11971): Bacteria. bile salts and intestinal monosaccharide malabsorption. Gur. 12, 7
8. 9.
10
11.
We are grateful to Mrs. A. Malajczuk for her expert technical assistance.
12
References
13.
I . DRA~AR B.. S.. S ~ i h k M.. ~ . and M r L t o ~ .G. M. (1969): Studies on the intestinal flora. P a n I . The bacterial flora of the gastrointestinal tract In healthy and achlorhydric perions. Gasiroenterologv, 56. 71. 2 GRACFY. M (1971) Intestinal abqorption in the ‘contaminnted small-howl ryndrome’. Cut. 12, 403. 3 CHI.NIY, F. E., BCRKI,V.. CLARK,M. L and SESIOK, I R. (IY70). Intestinal fatty acld absorption and estenfication from lumind micellar solutions containing deoxycholic acid. Proc. Sac. exp. Bwl. ( N . Y . ) ,133, 212. 4 SCHIONIBY.H 11973): The mechanism oS Vitamin B , , malabsorption in blind-loop syndrome, Scand. J . Casiroeni.. 8, 97. 5 AMENT,M ,E., SHIMOUA. S. S., SACSU?RS,D.R. and R u m . C. E. (1972): Pathogenesis of steatorrhea in three cases of small inteqtinal staqir \yn, 72X drome. G n m o e n t e r o l o ~ y63,
14 15
16. 17.
683. Ciu.cfY. IM.. P A P A ~ ) I U I T RJ I. O and I . B o w i K. G (1974). IJltra-structural changes m the small intCstincs of rats with self-filling blind-loops. Gu\tn,miemlogy. 61. 646. CAMERON. D . (3 . WATWV.G. M and WITTS.L. I. (1949) The experimental production of macrocytic anaemia by operation, on the inieitinal tract. Blood, 4, 803. MiLLfu. D. and CRASF. R. K . (1961)- The digestwe function of the epithelium of the amall intestine Iii) Localisation of disaccharide hydrolysir in the isolated brush border portion of intestinal epithelial cells. Biochim. hrophvs Acrn. ( A m s t . )52, 293 Hoir. I. H. and M I L ~ . L R D., (1962): The Iocalimtion of phosphomonoesterases and aminopeptidase in brush borden isolated from intestinal epithelial cells. Blochim biophhs. Acra (Am.\t i S8. 239 P t ~ub. i T J (1972) Subcellular fractionation of the enrerocrte with special reference to peptic hydrolases m Peptide rrunyorr in haripria and mummolinn p i . K. Elliott and M. O’Connor (Eds ) Elsevier. Excerpta Medica. North Holland DAHlOVIFl, A (1964): Method for assay of intestinal diaaccharidaser. Analyi. Bmrhrm., 7. 18 BURKE.V.. KtnnY. K. R. and A ~ D L R S OC N . M. (1965): The relationship ofdietary lactose t o refractory diarrhoea in infancy. Ausi. pnediat. J.. I, 147. HKGGITT,A. ST. I. and Nixoz. D . A. (1957) Use oS glucose oxidasc. peroxidase and 0-dianisidine in determination of blood and urinary glucose, Lancer, 2, 368 LOWRY. 0 H . R O S I H R O ~N ~ ~J.. ~ IFanu. I. A. L and R A N U A ~ R. ~I.,11951) Protein measurement with the Folin phenol reagent, J bml Chcm., 193,265 FI~HMAN, W. H . and GHOSII,N. K. (1967): lsoenzymes of human alkaline phosphatase. Advanc. clm. Chem.. 10, 255 CWKI:, W. T., Cox. E. V., FONE.D. I., MFYYEI.~., M. I. and GAUUIE,R . (1963): The clinical and metabolic significance of jejunal diverticula, Gut, 4, 115.
18
N i ~ ~ i i , F . . A ~ . ; T ( . l i t lC.. . A .WllROlRS. R B . M O I I I Y , 1) L.andROiIll1. C C (1967) Protein malnutrition after partial gastrectomy. Quori J Mud.,
36,469.
Aust. N.Z. J. Med. (1975), 5,pp. 144 147 CASE REPORT
Phenytoin Sensitivity in a Case of Phenytoin-Associated Hodgkin’s Disease* Tania C. Sorrellt and Ian J. Forbes: From the Adelaide University Department of Medicine, The Queen Elizabeth Hospital, Woodville, South Australia
Summary: The case of a patient who developed Hodgkin’s disease three years after commencement of therapy with phenytoin is presented. Humoral and *Supported by a grant from the National Health and Medical Research Council of Australia tPost-graduate Research Scholar :Reader in Medicine Correspondence: Dr. T. C . Sorrell, Department of Medicine, The Queen Elizabeth Hospital, Woodville, South Australia 501 1 Accepted for publication: 17 October, 1974
cellular immunological capacity were significantly depressed. Phenytoin caused a striking increase in DNA synthesis when lymphocytes were cultured in the presence of this drug, in contrast to significant inhibition in the lymphocytes of control subjects. These findings are consistent with the hypothesis that both chronic antigenic stimulation and immunosuppression by phenytoin are involved in the induction of lymphoma.
APRIL
1975
PHENYTOIN SENSITIVITY AND HODGKIN'S DISEASE
The incidence of lymphoma occurring in patients treated with phenytoin is unknown. A retrospective necropsy study of 85 persons dying with malignant lymphoma revealed four on long term therapy with phenytoin. The expected incidence is one epileptic patient for 200 cases of lymphoma. It was therefore deduced that there is a tenfold increase of malignant lymphoma in epileptics.' The lymph node abnormalities associated with phenytoin therapy have been divided into four categories.2 The first is a benign pleomorphic hyperplasia with preservation of lymph node architecture, regressing on withdrawal of phenytoin. The second is a reticulum cell hyperplasia resembling malignant lymphoma, again regressing on drug withdrawal. The third category is histologically indistinguishable from the second, but the lymphoma recurs after the cessation of therapy, and causes death of the patient. The fourth category is indistinguishable from malignant lymphoma. Seven cases in Group 4 were reported by Hyman and Sommer?, four with Reed-Sternberg cells characteristic of Hodgkin's disease. We are aware of 16 more reported cases, six of Hodgkin's disease', 4 * 5 , ', nine of non-Hodgkin's lymphoma6 and one of multiple m y e l ~ r n a . ~ Another case of Hodgkin's disease associated with phenytoin therapy is reported in this paper. Immunological studies revealed abnormalities which may be related to the pathogenesis of lymphoma developing during therapy with phenytoin. Case Report
The patient, a 26 year old male, presented with a three month history of enlarged left cervical nodes. He had been treated with phenytoin, 100 mg three times daily. for three years, for post-traumatic grand ma1 epilepsy. There was no history of weight loss, pruritis or night sweats at presentation. No other abnormalities were found on physical examination. In particular, there was no fever, rash or hepatosplenomegaly. The blood pictureshoweda haemoglobinof 15.2g/100ml and a white cell count of 11,600 cells per microlitre. with a normal differential count. The serum alkaline phosphatase. lactic dehydrogenase, urea, calcium, total protein, transaminase, copper and iron values were normal. The erythrocyte sedimentation rate was 5 mm in the first hour. Biopsy of the enlarged lymph nodes revealed loss of normal architecture with replacement by lymphocytes
145
arranged in rounded follicles, moderate numbers of abnormal reticulum cells, classical Reed-Sternberg cells, eosinophils, and occasional plasma cells. These features were consistent with Hodgkin's disease, mixed cellularity type.5 The chest radiograph was normal. The specific staging procedures, abdominal lymphangiography, bone marrow biopsy, laparotomy with splenectomy and biopsy of liver and an abdominal lymph node all yielded negative results. The patient was therefore considered to have Stage IA disease. He received megavoltage therapy to lymph nodes of the mantle area in a dosage of 45M) rads. Immunological studies were performed ten months later. Measurement of Immunological Status Cellular immunological function was assessed by the capacity to manifest delayed hypersensitivity reactions, and the capacity of the lymphocytes to transform in culture with phytohaemagglutinin.8 PHA-stimulated DNA synthesis was measured by a modification of the method of Junge et Triplicate cultures contained heparinised blood, 0 .2 ml, foetal calf serum (Commonwealth Serum Laboratories, Aust.) or autologous serum, 0.4 ml, PHA, (Wellcome), 0.02 ml. and HEPES buffered medium 199, such that the total culture volume was 4 . 0 ml. Tritiated thymidine (3H-methyl thymidine, 2 5 microcuries, specific activity 500 millicuries per millimol, Radiochemical Centre, Amersham) was added to cultures after incubation for 92 hours at 37'C. The amount of 3H-thymidine incorporated into trichloroacetic acid-precipitable DNA was measured in cultures terminated four hours after the addition of tritiated thymidine. DNA synthesis was also measured in freshly drawn, nonstimulated, blood cell cultures, after incubation with 'H-thymidine for four hours at 37'C. No delayed hypersensitivity reactions developed to candida, streptococcal and mumps antigens.8 By morphological criteria, only 30% of the lymphocytes transformed in culture with phytohaemagglutinin (normal more than 50%>usually more than 70%). PHA-induced DNA synthesis was greatly depressed in v i m , both in cultures containing autologous serum (600 disintegrations per minute (dpm)/ culture, normal range 1 9 . 0 x lo3 to 152 x lo3 dpm/culture). and in cultures containing foetal calf serum (3180 dpm/ culture; normal range 15.0 x lo3 to 130 x lo3 dpm/ culture). DNA synthesis in unstimulated circulating lymphocytes was normal (460 dpm/culture; normal range 155 to 965 dpm/culture). Humoral immunological function was assessed by measurement of immunoglobulin levels, the C3 component of complement and specific antibody responses after challenge. Immunoglobulin levels were in the normal range (IgG, 950 mg/100 ml, normal 600-1900 mg/100 ml, IgA, 110 mg/ 100 ml, normal 70-380 mg/lOO ml, IgM, 80 mg/100 ml, normal 50-370 mg/lOO ml). The C3 component of complement was 164 mg/100 ml, (normal 70-225 mg/lOO ml). Specific antibody (titre at least I : 640) was present two weeks after immunization with S. typhi. No antibody was detectable two weeks post-immunization with Tetanus toxoid, i.e. there was evidence of a partial deficiency of humoral immune function.' The average of three circulating lymphocyte counts was 450jmicrolitre. This is substantially below the normal range of 1500 to 3000/microlitre. These data indicate significant depression of cellular and humoral immunological function.
146
SORRELL AND FORBES
Influence of Phenytoin on D N A Synthesis by Lymphocytes in Culrure The effect of phenytoin on lymphocyte DNA synthesis was measured in unstimulated whole blood cultures incubated with different concentrations of phenytoin. Triplicate cultures were incubated for four or five days. Phenytoin for intravenous use (Parke Davis: Michigan), 250 mg, was dissolved in a sterile solution of propylene glycol, 40%, ethyl alcohol, lo%, distilled water, 50%. adjusted to pH 12 with sodium hydroxide. Defined concentrations of drug were added to cultures in a volume of 0.01 ml. Control cultures from each subject were established with drug solvent. and with no solvent. A striking increase in DNA synthesis occurred in lymphocytes of the patient when cultured for four or five days in the presence of phenytoin (Fig. 1). Phenytoin inhibited DNA synthesis in cultures of three healthy control subjects, six patients treated with phenytoin, but without evidence of lymph node enlargement or other side effects, and in two patients with Hodgkin's disease treated by radiotherapy twelve months before study. Lymphocyte counts were normal in the control subjects, except the patients with Hodgkin's disease, in whom counts were low (1090 and 430 cells/microlitre respectively). Discussion
The aetiology and pathogenesis of Hodgkin's disease and the lymphomas remain obscure. The association between human lymphoid tissue malignancy and therapy with the anticonvulsant drug phenytoin sodium has provided one approach to the study of these malignancies in man. Studies in mice have suggested that the combination of chronic antigenic stimulation and partial immunosuppression by phenytoin may lead to the premature development of lymphoid tissue malignancies in a strain of mice predisposed to lymphomas. l o This proposed mechanism of induction of lymphomas was supported by studies in which other agents were used for immunosuppression and antigenic stimulation, for example when non-oncogenic viruses, Freund's adjuvant or HeLacells were used as antigens and azathioprine was used as the immunosuppressant.l' Immunological defects are common in patients treated with phenytoin.'*, 1 3 , 14, l 5 The phenytoin-treated patients failed to manifest delayed hypersensitivity reactions to common antigens, and to make antibody to S. typhi and Tetanus toxoid. Serum levels of IgG, IgA, and IgM, and DNA synthesis were also low. Investigation of our patient who developed Hodgkin's disease three years after commencement of treatment with phenytoin for epilepsy,
PtIENIIOIN
VOL.
mgi,,)
PIIENITOlN
I
5,
ygim1
NO.
2
I
F I G U R E 1 Lymphocyte DNA synthesis in phenytoincontaining cultures from the patient who developed lymphoma while on phenytoin (dotted lines) are compared with the average curves of 11 control subjects (solid lines) Cultures were processed at 96 hours (A) and 120 hours ( B )
demonstrated inhibition of parameters of humoral and cellular immunological capacity. The patient however. had received radiotherapy, which commonly results in significant immunological depression. Phenytoin inhibited DNA synthesis by lymphocytes from normal subjects, non-lymphomatous patients treated with phenytoin, and patients with Hodgkin's disease not associated with phenytoin therapy. In the case of our patient with lymphoma, phenytoin caused a striking stimulation of DNA synthesis. This is interpreted as being a manifestation of hypersensitivity, similar to that seen in some patients with drug hypersensitivities.l 6 It is presumed that phenytoin stimulated DNA synthesis in B cells since the failure of stimulation by PHA probably indicates T cell dysfunction." Not all subjects with drug hypersensitivity develop lymphoma. Similarly, the lymphocytes of patients with clinical features of phenytoin sensitivity but without lymphoma are not always stimulated by phenytoin in vitro. MacKinney and Booker studied eight patients with clinical features of phenytoin sensitivity. Four had skin rashes, another had the StevensJohnson syndrome, two had lupus erythematosus syndrome, and one had fever, splenomegaly, leukopenia and atypical lymphocytes in the circulation. Phenytoin stimulated lymphocyte DNA synthesis only in the case of the patient with Stevens-Johnson syndrome."
APRIL
1975
The findings of the present study support the hypothesis that both chronic antigenic stimulation and immunosuppression are involved in the induction of human lymphoma.
6 7
U. 9. 10.
Acknowledgements
We wish to thank Mr. G . K. Mann for excellent technical assistance.
References I. A \ T n O s Y , I. I. (1970): Malignant lyniphorna associated with hydantoin drugs, Arch. Nsurol. 22, 450. 2 C.AW). R. A.. NPAI.. J. A. and Cohnnn. F. G . (19681. Hydantoin-induced pseudo-paeudolymphoma. Ann. intern. Med. 69, 557. 3. HYMAS. G. A. and SOMXER5, S.C. (1966): The development of Hvdgkin's disease and lymphoma during anticonvulsant therapy. Blood 28, 416. 4 H A R H I N G I OW N .. , BROWN. E.. Pt.iEl>SFR. 'I , KlSahF, J.. LOFB, V , !dCr%LiISTtR. PARKER, 8 . . s A i L S l + l U . s. and Tosrfsoh, P. (1962).
w..
5
147
PHI.:NYTOJN SENSITIVITY A N D HODGKIN'S DlSEASE
Clinico-pathologic conference Lymphoma or drug reaction occurring during hydantoin therdpy for epilepsy, Amer. J Med 3% 286 flnowN. J. M. (1971): Drug-assnciared lymphadenopathiec with special reference to the Reed-Sternberg cell, Med. J Auar. 1. 375.
11.
R A r w u o , A. and Tnrl L, E. (IY711: Malignant lymphogranulomatoais and anticunvulsant therdpy. Arra. med. .scund. 189, 131. C ~ s n i r o M. ~ . H. and L t ~ s P F U R u . D (1971) Le sostanze di idantoma come posrrbdi cause del hnfoma maligno, Minervu mrd. 62, 2185. FORBFS. 1. J (1971) Measurement of nnmunological function in clinical medicine. Ausi. ?v.Z.J Med. 1, IhO. JbhGt. u., IfOtKSTKA, I., WUl.ft. L. and DFINHARDT. F (1970).Microtechnique for quantitative evaluation of in virro lymphocyte transformation, Chn cxp. Immunol 7, 431 KRWFR, G R. F and H A R R ~ D S , (1972)- Is phenytoin carcinogenic" Lancer 1, 323. KRUI'GFR,G. R F., M A I M ~ R I "R, . A and BFRARD.C. W (1971) Malignant lymphomas and plsrmauytosis in mice under prolonged immunosuppression and persistent antigenic stimulation. Tririnspluniurron 11,
nx.
12. S0nnri.L. T. C..FOHBES, 1. J.. B u n x s r . F. R and RISTHRIETH, R. H C. (1971) Depression of immunological function in patients treated with phcnytoin sodium (sodium diphenylhydantoin). Luncei 2, 1233 13 GROB,P . J . and HLROI~D, G. E (1972). Jmmunological abnormalities and h y d a n t o m , Bril med. 3 2, 561. 14 SonnFi.1. T. C and Funnrs. 1. 1 (1975) Clm err Immunol (Accepted for publication ) 15 Lirc. N. R (1968): Lymphocyte Srimulurton North Holland Publishing Co., Amsterdam. 237 16. HOILAYD,P and MAIJER.A M (1965): Diphenylhydantnin-induced hypersensitivity reaction. J . Pedmr. 66, 322 17 MacKi%sFu. A A and B O O K L R , H E. (1972): Diphenylhydantoin eiTectr on human lymphocyter in b i f m and in w v o An hypnrhesi\ to explain some drug reactions. Arch inlem. Med 129, 988 18 GREAVES, M.F..OWEN,J. J . T . , R a r r , M C.(1973):TandflLymphocytesOrigms. pruperrres and rules in immunp reyonsrs. Exerpta Medica, Amsterdam. American Elselvier Publishing Co.. Inc.. New York. p. 87.
_____~~_
Aust. N.Z. J. Med. (1975),5,pp. 147-154 ~
.-
CASE REPORT
Emphysema in Papua New Guinea-A
Pathological Study
Robin Cooke' and Ian Toogoodi.
From the Department of Pathology, Royal Brisbane Hospital
Summary: The authors examined 47 lungs obtained at post mortem in Papua New Guinea. These were inflated with formalin, fixed under pressure, sliced and examined for emphysema using a "pointcounting" method. There was no emphysema before the age of 30 years. The pathological types encountered were 'Anatomical Pathologist, Royal Brisbane Hospital, formerly Senior Specialist Pathologist, Papua New Guinea. tRegistrar, Adelaide Children's Hospital. Correspondence: Dr. R. A. Cooke, Department of Pathology, Royal Brisbane Hospital, Herston Road, Herston, Clueensland 4029 Accepted for publication: 10 October, 1974
similar to those in the United Kingdom. In patients over 50 years of age there appeared to be little difference between the amount of emphysema present in Papua New Guinea and in the United Kingdom. Environmental air pollution seemed to be relatively unimportant in the pathogenesis. Repeated lower respiratory tract infections may be more important. Emphysema appeared to be more prevalent in lowland than in highland dwellers. The findings of this pathological study supported the clinical and epidemiological studies carried out concurrently, but independently by others.
GKACEY ET AL
syndrome’ in man is unknown. They would suggest, however, that there is an important and previously ignored mucosal lesion in this syndrome. Its relevance to specific digestive and absorptive defects such as peptidase activities in relation to hypoproteinaemia”. 18, require investigation in clinical and experimental situations. Acknowledgement
VOL.
5,
NO.
2
6 . GK4CEY. M.. BLRKP, V.. OSHlK. A.. BARKI.N, J.andGl.ASGtIw. E. F.11971): Bacteria. bile salts and intestinal monosaccharide malabsorption. Gur. 12, 7
8. 9.
10
11.
We are grateful to Mrs. A. Malajczuk for her expert technical assistance.
12
References
13.
I . DRA~AR B.. S.. S ~ i h k M.. ~ . and M r L t o ~ .G. M. (1969): Studies on the intestinal flora. P a n I . The bacterial flora of the gastrointestinal tract In healthy and achlorhydric perions. Gasiroenterologv, 56. 71. 2 GRACFY. M (1971) Intestinal abqorption in the ‘contaminnted small-howl ryndrome’. Cut. 12, 403. 3 CHI.NIY, F. E., BCRKI,V.. CLARK,M. L and SESIOK, I R. (IY70). Intestinal fatty acld absorption and estenfication from lumind micellar solutions containing deoxycholic acid. Proc. Sac. exp. Bwl. ( N . Y . ) ,133, 212. 4 SCHIONIBY.H 11973): The mechanism oS Vitamin B , , malabsorption in blind-loop syndrome, Scand. J . Casiroeni.. 8, 97. 5 AMENT,M ,E., SHIMOUA. S. S., SACSU?RS,D.R. and R u m . C. E. (1972): Pathogenesis of steatorrhea in three cases of small inteqtinal staqir \yn, 72X drome. G n m o e n t e r o l o ~ y63,
14 15
16. 17.
683. Ciu.cfY. IM.. P A P A ~ ) I U I T RJ I. O and I . B o w i K. G (1974). IJltra-structural changes m the small intCstincs of rats with self-filling blind-loops. Gu\tn,miemlogy. 61. 646. CAMERON. D . (3 . WATWV.G. M and WITTS.L. I. (1949) The experimental production of macrocytic anaemia by operation, on the inieitinal tract. Blood, 4, 803. MiLLfu. D. and CRASF. R. K . (1961)- The digestwe function of the epithelium of the amall intestine Iii) Localisation of disaccharide hydrolysir in the isolated brush border portion of intestinal epithelial cells. Biochim. hrophvs Acrn. ( A m s t . )52, 293 Hoir. I. H. and M I L ~ . L R D., (1962): The Iocalimtion of phosphomonoesterases and aminopeptidase in brush borden isolated from intestinal epithelial cells. Blochim biophhs. Acra (Am.\t i S8. 239 P t ~ub. i T J (1972) Subcellular fractionation of the enrerocrte with special reference to peptic hydrolases m Peptide rrunyorr in haripria and mummolinn p i . K. Elliott and M. O’Connor (Eds ) Elsevier. Excerpta Medica. North Holland DAHlOVIFl, A (1964): Method for assay of intestinal diaaccharidaser. Analyi. Bmrhrm., 7. 18 BURKE.V.. KtnnY. K. R. and A ~ D L R S OC N . M. (1965): The relationship ofdietary lactose t o refractory diarrhoea in infancy. Ausi. pnediat. J.. I, 147. HKGGITT,A. ST. I. and Nixoz. D . A. (1957) Use oS glucose oxidasc. peroxidase and 0-dianisidine in determination of blood and urinary glucose, Lancer, 2, 368 LOWRY. 0 H . R O S I H R O ~N ~ ~J.. ~ IFanu. I. A. L and R A N U A ~ R. ~I.,11951) Protein measurement with the Folin phenol reagent, J bml Chcm., 193,265 FI~HMAN, W. H . and GHOSII,N. K. (1967): lsoenzymes of human alkaline phosphatase. Advanc. clm. Chem.. 10, 255 CWKI:, W. T., Cox. E. V., FONE.D. I., MFYYEI.~., M. I. and GAUUIE,R . (1963): The clinical and metabolic significance of jejunal diverticula, Gut, 4, 115.
18
N i ~ ~ i i , F . . A ~ . ; T ( . l i t lC.. . A .WllROlRS. R B . M O I I I Y , 1) L.andROiIll1. C C (1967) Protein malnutrition after partial gastrectomy. Quori J Mud.,
36,469.
Aust. N.Z. J. Med. (1975), 5,pp. 144 147 CASE REPORT
Phenytoin Sensitivity in a Case of Phenytoin-Associated Hodgkin’s Disease* Tania C. Sorrellt and Ian J. Forbes: From the Adelaide University Department of Medicine, The Queen Elizabeth Hospital, Woodville, South Australia
Summary: The case of a patient who developed Hodgkin’s disease three years after commencement of therapy with phenytoin is presented. Humoral and *Supported by a grant from the National Health and Medical Research Council of Australia tPost-graduate Research Scholar :Reader in Medicine Correspondence: Dr. T. C . Sorrell, Department of Medicine, The Queen Elizabeth Hospital, Woodville, South Australia 501 1 Accepted for publication: 17 October, 1974
cellular immunological capacity were significantly depressed. Phenytoin caused a striking increase in DNA synthesis when lymphocytes were cultured in the presence of this drug, in contrast to significant inhibition in the lymphocytes of control subjects. These findings are consistent with the hypothesis that both chronic antigenic stimulation and immunosuppression by phenytoin are involved in the induction of lymphoma.
APRIL
1975
PHENYTOIN SENSITIVITY AND HODGKIN'S DISEASE
The incidence of lymphoma occurring in patients treated with phenytoin is unknown. A retrospective necropsy study of 85 persons dying with malignant lymphoma revealed four on long term therapy with phenytoin. The expected incidence is one epileptic patient for 200 cases of lymphoma. It was therefore deduced that there is a tenfold increase of malignant lymphoma in epileptics.' The lymph node abnormalities associated with phenytoin therapy have been divided into four categories.2 The first is a benign pleomorphic hyperplasia with preservation of lymph node architecture, regressing on withdrawal of phenytoin. The second is a reticulum cell hyperplasia resembling malignant lymphoma, again regressing on drug withdrawal. The third category is histologically indistinguishable from the second, but the lymphoma recurs after the cessation of therapy, and causes death of the patient. The fourth category is indistinguishable from malignant lymphoma. Seven cases in Group 4 were reported by Hyman and Sommer?, four with Reed-Sternberg cells characteristic of Hodgkin's disease. We are aware of 16 more reported cases, six of Hodgkin's disease', 4 * 5 , ', nine of non-Hodgkin's lymphoma6 and one of multiple m y e l ~ r n a . ~ Another case of Hodgkin's disease associated with phenytoin therapy is reported in this paper. Immunological studies revealed abnormalities which may be related to the pathogenesis of lymphoma developing during therapy with phenytoin. Case Report
The patient, a 26 year old male, presented with a three month history of enlarged left cervical nodes. He had been treated with phenytoin, 100 mg three times daily. for three years, for post-traumatic grand ma1 epilepsy. There was no history of weight loss, pruritis or night sweats at presentation. No other abnormalities were found on physical examination. In particular, there was no fever, rash or hepatosplenomegaly. The blood pictureshoweda haemoglobinof 15.2g/100ml and a white cell count of 11,600 cells per microlitre. with a normal differential count. The serum alkaline phosphatase. lactic dehydrogenase, urea, calcium, total protein, transaminase, copper and iron values were normal. The erythrocyte sedimentation rate was 5 mm in the first hour. Biopsy of the enlarged lymph nodes revealed loss of normal architecture with replacement by lymphocytes
145
arranged in rounded follicles, moderate numbers of abnormal reticulum cells, classical Reed-Sternberg cells, eosinophils, and occasional plasma cells. These features were consistent with Hodgkin's disease, mixed cellularity type.5 The chest radiograph was normal. The specific staging procedures, abdominal lymphangiography, bone marrow biopsy, laparotomy with splenectomy and biopsy of liver and an abdominal lymph node all yielded negative results. The patient was therefore considered to have Stage IA disease. He received megavoltage therapy to lymph nodes of the mantle area in a dosage of 45M) rads. Immunological studies were performed ten months later. Measurement of Immunological Status Cellular immunological function was assessed by the capacity to manifest delayed hypersensitivity reactions, and the capacity of the lymphocytes to transform in culture with phytohaemagglutinin.8 PHA-stimulated DNA synthesis was measured by a modification of the method of Junge et Triplicate cultures contained heparinised blood, 0 .2 ml, foetal calf serum (Commonwealth Serum Laboratories, Aust.) or autologous serum, 0.4 ml, PHA, (Wellcome), 0.02 ml. and HEPES buffered medium 199, such that the total culture volume was 4 . 0 ml. Tritiated thymidine (3H-methyl thymidine, 2 5 microcuries, specific activity 500 millicuries per millimol, Radiochemical Centre, Amersham) was added to cultures after incubation for 92 hours at 37'C. The amount of 3H-thymidine incorporated into trichloroacetic acid-precipitable DNA was measured in cultures terminated four hours after the addition of tritiated thymidine. DNA synthesis was also measured in freshly drawn, nonstimulated, blood cell cultures, after incubation with 'H-thymidine for four hours at 37'C. No delayed hypersensitivity reactions developed to candida, streptococcal and mumps antigens.8 By morphological criteria, only 30% of the lymphocytes transformed in culture with phytohaemagglutinin (normal more than 50%>usually more than 70%). PHA-induced DNA synthesis was greatly depressed in v i m , both in cultures containing autologous serum (600 disintegrations per minute (dpm)/ culture, normal range 1 9 . 0 x lo3 to 152 x lo3 dpm/culture). and in cultures containing foetal calf serum (3180 dpm/ culture; normal range 15.0 x lo3 to 130 x lo3 dpm/ culture). DNA synthesis in unstimulated circulating lymphocytes was normal (460 dpm/culture; normal range 155 to 965 dpm/culture). Humoral immunological function was assessed by measurement of immunoglobulin levels, the C3 component of complement and specific antibody responses after challenge. Immunoglobulin levels were in the normal range (IgG, 950 mg/100 ml, normal 600-1900 mg/100 ml, IgA, 110 mg/ 100 ml, normal 70-380 mg/lOO ml, IgM, 80 mg/100 ml, normal 50-370 mg/lOO ml). The C3 component of complement was 164 mg/100 ml, (normal 70-225 mg/lOO ml). Specific antibody (titre at least I : 640) was present two weeks after immunization with S. typhi. No antibody was detectable two weeks post-immunization with Tetanus toxoid, i.e. there was evidence of a partial deficiency of humoral immune function.' The average of three circulating lymphocyte counts was 450jmicrolitre. This is substantially below the normal range of 1500 to 3000/microlitre. These data indicate significant depression of cellular and humoral immunological function.
146
SORRELL AND FORBES
Influence of Phenytoin on D N A Synthesis by Lymphocytes in Culrure The effect of phenytoin on lymphocyte DNA synthesis was measured in unstimulated whole blood cultures incubated with different concentrations of phenytoin. Triplicate cultures were incubated for four or five days. Phenytoin for intravenous use (Parke Davis: Michigan), 250 mg, was dissolved in a sterile solution of propylene glycol, 40%, ethyl alcohol, lo%, distilled water, 50%. adjusted to pH 12 with sodium hydroxide. Defined concentrations of drug were added to cultures in a volume of 0.01 ml. Control cultures from each subject were established with drug solvent. and with no solvent. A striking increase in DNA synthesis occurred in lymphocytes of the patient when cultured for four or five days in the presence of phenytoin (Fig. 1). Phenytoin inhibited DNA synthesis in cultures of three healthy control subjects, six patients treated with phenytoin, but without evidence of lymph node enlargement or other side effects, and in two patients with Hodgkin's disease treated by radiotherapy twelve months before study. Lymphocyte counts were normal in the control subjects, except the patients with Hodgkin's disease, in whom counts were low (1090 and 430 cells/microlitre respectively). Discussion
The aetiology and pathogenesis of Hodgkin's disease and the lymphomas remain obscure. The association between human lymphoid tissue malignancy and therapy with the anticonvulsant drug phenytoin sodium has provided one approach to the study of these malignancies in man. Studies in mice have suggested that the combination of chronic antigenic stimulation and partial immunosuppression by phenytoin may lead to the premature development of lymphoid tissue malignancies in a strain of mice predisposed to lymphomas. l o This proposed mechanism of induction of lymphomas was supported by studies in which other agents were used for immunosuppression and antigenic stimulation, for example when non-oncogenic viruses, Freund's adjuvant or HeLacells were used as antigens and azathioprine was used as the immunosuppressant.l' Immunological defects are common in patients treated with phenytoin.'*, 1 3 , 14, l 5 The phenytoin-treated patients failed to manifest delayed hypersensitivity reactions to common antigens, and to make antibody to S. typhi and Tetanus toxoid. Serum levels of IgG, IgA, and IgM, and DNA synthesis were also low. Investigation of our patient who developed Hodgkin's disease three years after commencement of treatment with phenytoin for epilepsy,
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F I G U R E 1 Lymphocyte DNA synthesis in phenytoincontaining cultures from the patient who developed lymphoma while on phenytoin (dotted lines) are compared with the average curves of 11 control subjects (solid lines) Cultures were processed at 96 hours (A) and 120 hours ( B )
demonstrated inhibition of parameters of humoral and cellular immunological capacity. The patient however. had received radiotherapy, which commonly results in significant immunological depression. Phenytoin inhibited DNA synthesis by lymphocytes from normal subjects, non-lymphomatous patients treated with phenytoin, and patients with Hodgkin's disease not associated with phenytoin therapy. In the case of our patient with lymphoma, phenytoin caused a striking stimulation of DNA synthesis. This is interpreted as being a manifestation of hypersensitivity, similar to that seen in some patients with drug hypersensitivities.l 6 It is presumed that phenytoin stimulated DNA synthesis in B cells since the failure of stimulation by PHA probably indicates T cell dysfunction." Not all subjects with drug hypersensitivity develop lymphoma. Similarly, the lymphocytes of patients with clinical features of phenytoin sensitivity but without lymphoma are not always stimulated by phenytoin in vitro. MacKinney and Booker studied eight patients with clinical features of phenytoin sensitivity. Four had skin rashes, another had the StevensJohnson syndrome, two had lupus erythematosus syndrome, and one had fever, splenomegaly, leukopenia and atypical lymphocytes in the circulation. Phenytoin stimulated lymphocyte DNA synthesis only in the case of the patient with Stevens-Johnson syndrome."
APRIL
1975
The findings of the present study support the hypothesis that both chronic antigenic stimulation and immunosuppression are involved in the induction of human lymphoma.
6 7
U. 9. 10.
Acknowledgements
We wish to thank Mr. G . K. Mann for excellent technical assistance.
References I. A \ T n O s Y , I. I. (1970): Malignant lyniphorna associated with hydantoin drugs, Arch. Nsurol. 22, 450. 2 C.AW). R. A.. NPAI.. J. A. and Cohnnn. F. G . (19681. Hydantoin-induced pseudo-paeudolymphoma. Ann. intern. Med. 69, 557. 3. HYMAS. G. A. and SOMXER5, S.C. (1966): The development of Hvdgkin's disease and lymphoma during anticonvulsant therapy. Blood 28, 416. 4 H A R H I N G I OW N .. , BROWN. E.. Pt.iEl>SFR. 'I , KlSahF, J.. LOFB, V , !dCr%LiISTtR. PARKER, 8 . . s A i L S l + l U . s. and Tosrfsoh, P. (1962).
w..
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Clinico-pathologic conference Lymphoma or drug reaction occurring during hydantoin therdpy for epilepsy, Amer. J Med 3% 286 flnowN. J. M. (1971): Drug-assnciared lymphadenopathiec with special reference to the Reed-Sternberg cell, Med. J Auar. 1. 375.
11.
R A r w u o , A. and Tnrl L, E. (IY711: Malignant lymphogranulomatoais and anticunvulsant therdpy. Arra. med. .scund. 189, 131. C ~ s n i r o M. ~ . H. and L t ~ s P F U R u . D (1971) Le sostanze di idantoma come posrrbdi cause del hnfoma maligno, Minervu mrd. 62, 2185. FORBFS. 1. J (1971) Measurement of nnmunological function in clinical medicine. Ausi. ?v.Z.J Med. 1, IhO. JbhGt. u., IfOtKSTKA, I., WUl.ft. L. and DFINHARDT. F (1970).Microtechnique for quantitative evaluation of in virro lymphocyte transformation, Chn cxp. Immunol 7, 431 KRWFR, G R. F and H A R R ~ D S , (1972)- Is phenytoin carcinogenic" Lancer 1, 323. KRUI'GFR,G. R F., M A I M ~ R I "R, . A and BFRARD.C. W (1971) Malignant lymphomas and plsrmauytosis in mice under prolonged immunosuppression and persistent antigenic stimulation. Tririnspluniurron 11,
nx.
12. S0nnri.L. T. C..FOHBES, 1. J.. B u n x s r . F. R and RISTHRIETH, R. H C. (1971) Depression of immunological function in patients treated with phcnytoin sodium (sodium diphenylhydantoin). Luncei 2, 1233 13 GROB,P . J . and HLROI~D, G. E (1972). Jmmunological abnormalities and h y d a n t o m , Bril med. 3 2, 561. 14 SonnFi.1. T. C and Funnrs. 1. 1 (1975) Clm err Immunol (Accepted for publication ) 15 Lirc. N. R (1968): Lymphocyte Srimulurton North Holland Publishing Co., Amsterdam. 237 16. HOILAYD,P and MAIJER.A M (1965): Diphenylhydantnin-induced hypersensitivity reaction. J . Pedmr. 66, 322 17 MacKi%sFu. A A and B O O K L R , H E. (1972): Diphenylhydantoin eiTectr on human lymphocyter in b i f m and in w v o An hypnrhesi\ to explain some drug reactions. Arch inlem. Med 129, 988 18 GREAVES, M.F..OWEN,J. J . T . , R a r r , M C.(1973):TandflLymphocytesOrigms. pruperrres and rules in immunp reyonsrs. Exerpta Medica, Amsterdam. American Elselvier Publishing Co.. Inc.. New York. p. 87.
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Aust. N.Z. J. Med. (1975),5,pp. 147-154 ~
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CASE REPORT
Emphysema in Papua New Guinea-A
Pathological Study
Robin Cooke' and Ian Toogoodi.
From the Department of Pathology, Royal Brisbane Hospital
Summary: The authors examined 47 lungs obtained at post mortem in Papua New Guinea. These were inflated with formalin, fixed under pressure, sliced and examined for emphysema using a "pointcounting" method. There was no emphysema before the age of 30 years. The pathological types encountered were 'Anatomical Pathologist, Royal Brisbane Hospital, formerly Senior Specialist Pathologist, Papua New Guinea. tRegistrar, Adelaide Children's Hospital. Correspondence: Dr. R. A. Cooke, Department of Pathology, Royal Brisbane Hospital, Herston Road, Herston, Clueensland 4029 Accepted for publication: 10 October, 1974
similar to those in the United Kingdom. In patients over 50 years of age there appeared to be little difference between the amount of emphysema present in Papua New Guinea and in the United Kingdom. Environmental air pollution seemed to be relatively unimportant in the pathogenesis. Repeated lower respiratory tract infections may be more important. Emphysema appeared to be more prevalent in lowland than in highland dwellers. The findings of this pathological study supported the clinical and epidemiological studies carried out concurrently, but independently by others.
