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METHYL-CHOLANTHRENE-INDUCED SARCOMATA IN MICE AFTER IMMUNISATION WITH CORYNEBACTERIUM PARVUM PLUS SYNGENEIC SUBCELLULAR MEMBRANE FRACTIONS
1397
developing H.z. rather than a "slow" varicella virus infection (producing Hodgkin’s disease ?). Institute of Cancer Research and Division of Hæmatology,
Radiumstationen, Nörrebrogade 44, DK-8000 Aarhus C, Denmark.
E. B. THORLING K. THORLING.
CHEMOTHERAPY OF HODGKIN’S DISEASE SIR,-A report from this institute described 24 children under 15 who were treated between 1967 and 1971 with the M.o.p.p. chemotherapy (nitrogen mustard, vincristine, prednisone, procarbazine) six-course regimen.1 7 of the 24 presented with localised tumours (stages I and 11), and the remainder had generalised disease (stages III and IV), all achieved complete remission initially. At the time of the first report (1972), 7 patients had relapsed. Because of the well-documented success of chemotherapeutic regimens in the treatment of advanced Hodgkin’s disease, some investigators propose the use of chemotherapy in earlier stages.2,3 However, there have been disturbing reports of second cancers in patients with Hodgkin’s disease receiving both radiotherapy and chemotherapy. 4-6 Since our original series contains the only published data regarding M.o.p.p. chemotherapy of early-stage Hodgkin’s disease, continued follow-up of these patients is of interest. The outcome in all patients successfully followed up to FOLLOW-UP OF 24 UGANDAN CHILDREN WITH TREATED WITH M.O.P.P. CHEMOTHERAPY
HODGKIN’S DISEASE
(1967-1971)
is shown in the table. 2 patients (nos. 2 and 6) could not be traced and when last seen were in complete remission at 12 and 18 months, respectively. 1 patient (no. 7) relapsed in addition to the 7 noted in the original series. The median remission duration in patients who relapsed was 13 months from the start 01 therapy (range 8-38 months). 3 patients have died: 1 after multiple subsequent relapses (no. 20), 1 of encephalitis while in remission (no. 22), and 1 of an unrelated illness while in a second remission (no. 23). The surviving 6 patients who relapsed have had long second remissions reinduced by M.o.p.p. therapy.13 patients (4 stage 1-11, 8 stage ill, 1 stage iv) remain in their initial unmaintained remissions for a median of 72 + months (range 35-78 months) from the start of therapy. The overall median survival for the entire series (excluding patients lost to follow-up) is more than 60 months. 12 patients in this series presented with stage I-IIIA disease and would ordinarily have received radiotherapy as part or all of their initial treatment. 2 were lost to follow-up, and 1 relapsed. The remaining 9 have sustained initial remissions following the start of M.o.p.p. chemotherapy for 35-78 months. Late complications possibly attributable to chemotherapy were limited to the development of moderate-tosevere gynxcomastia in 7 of prepubertal and early pubertal boys. This observation is unexplained and is under investigation. None of the patients has yet reached marital or reproductive status, and none has developed a second
July, 1974,
neoplasm. This report does
not
substantiate doubts about the
efficacy of chemotherapy alone in curing early stages of Hodgkin’s disease. While the optimum use of chemotherapy and radiotherapy await the outcome of controlled trials, it is of interest that M.o.p.p. chemotherapy alone results in sustained remission in the majority of early-stage patients and offers excellent control for advanced disease.
Uganda Cancer Institute, Makerere University Medical School, Kampala, Uganda.
CHARLES L. M. OLWENY EDWARD KATANGOLE MBIDDE JOSEPHINE NKWOCHA IAN MAGRATH JOHN L. ZIEGLER.
METHYL-CHOLANTHRENE-INDUCED SARCOMATA IN MICE AFTER IMMUNISATION WITH CORYNEBACTERIUM PARVUM PLUS SYNGENEIC SUBCELLULAR MEMBRANE FRACTIONS
SIR,-We wish to report the result of preliminary experidesigned to test an hypothesis that implicates mitochondrial D.N.A. in malignant transformation.1 It has been suggested that some conditions, including those arising during chemical carcinogenesis, may eventually lead to nuclear uptake of mitochondrial D.N.A., thus effecting ments
*
Rye classification. t L.P. = lymphocyte predominant.
M.C. = mixed cellularity.
L.D. =
lymphocyte depleted. N.S. = nodular sclerosis. t Died in remission. 1. 2. 3.
4.
5. 6.
Ziegler, J. L., Bluming, A. Z., Fass, L., Magrath, I. T., Templeton, A. C. Lancet, 1972, ii, 679. DeVita, V. T., Serpick, A. A., Carbone, P. P. Ann. intern. Med. 1970, 73, 881. Moore, M. R., Bull, J. M., Jones, S. E., Rosenberg, S. A., Kaplan, H. S. ibid. 1972, 77, 1. Arseneau, J. L., Sponzo, R. W., Levin, D. L., Schnipper, L. E., Bonner, H., Young, R. C., Canellos, G. P., Johnson, R. E., DeVita, V. T. New Engl. J. Med. 1972, 287, 1119. Castro, G. A. M., Church, A., Pechet, L., Snyder, L. M. ibid. 1973, 23, 103. Canellos, G. P., DeVita, V. T., Arseneau, J. C., Johnson, R. Recent Results Cancer Res. (in the press).
transformation of the nuclear genome.2,3 If the new nuclear genes were now expressed, they would code for a limited number of hydrophobic proteins,4 normally restricted to the inner mitochondrial membrane but now synthesised on cytoplasmic ribosomes and possibly incorporated into the plasma membrane of the cell. Such a change in the cell-surface would, it is suggested, endow the new line of transformed cells with its malignant characteristics and would also introduce into it new antigenic determinants. To test this hypothesis we attempted to immunise mice against chemical carcinogenesis, using as antigen submito1. 2. 3.
Baum, H. Lancet, 1973, ii, 738. Hadler, H. I., Daniel, B. G. Cancer Res. 1973, 33, 117. Schumacher, H. R., Szekely, I. E., Fisher, D. R. Lancet, 1973, ii,
4.
Borst, P. Ann. Rev. Biochem. 1972, 41,
1207. 333.
1398 microsomes.
Furthermore, the protection afforded by
adjuvant alone, although significant, was not absolute and was assessed on the basis of subjective examination of the animals. It should also be emphasised that the basis of any protection initially afforded by mitochondria (or of any detrimental effect of microsomes) may lie in factors (e.g., changes in metabolism of the carcinogen) quite unrelated to the hypothesis that prompted the experiments. Nevertheless, it does seem that the rather arbitrary procedure might have influenced the development of carcinogeninduced tumours, and we are therefore encouraged to pursue this work further, both for its obvious therapeutic implications and for the light it might throw on the origin of malignant transformation. Days Alter Methyl-Cholanthrene Treatment.
Numbers of mice with tumours at intervals following inoculation of 20-methylcholanthrene.
Department of Biochemistry, Chelsea College, University of London. Department of Surgery, Welsh National School of Medicine, Cardiff.
chondrial particles prepared by sonication of mitochondria obtained from livers of syngeneic animals. Microsomes from the same liver homogenate, and also subjected to sonication, were used as a control. Corynebacterium parvum was used as an immunological adjuvant in each case. C$H syngeneic male mice were used in all experiments. The mitochondrial and microsomal membrane fractions were prepared from the livers of 5 of these mice, freeze-dried, and stored at 4°C until required. The remaining mice were then divided into four groups with 10 to 13 mice in each. Group A was injected with 0.1 rnl. of sterile distilled water (S.D.W.) intraperitoneally (i.p.). Group B was injected with 0-1 ml. S.D.W., i.p., and 0-1 ml. C. parvum (Well come Research Laboratories) subcutaneously (s.c.). Group C was injected with 01 ml. of a suspension of the mitochondrial fraction i.P. plus 0-1 ml. C. parvum s.c. Group D was injected with 0’1 ml. of a suspension of the microsomal fraction i.p. plus 0-1 ml. C. parvum s.c. The treatment schedule for each group was repeated a week later at the time of inoculation with the carcinogen. Fibrosarcomata were induced by intramuscular injection of 0-1 ml. of olive oil containing a suspension of 1-0 mg. 20-methylcholanthrene into the right hind leg of each mouse.
The mice were checked twice a week for the presence of tumours, most of which developed between 90 and 125 days after injection of methylcholanthrene. The latent period until the development of an unequivocal tumour (checked by two observers) was recorded, after which the animal was killed. The experiment was repeated three times, the pattern of response being very similar in each case. The figure illustrates the time course of development of tumours for each of the four groups of animals, the results of the three separate experiments being pooled.
Immunisation with C. parvum alone resulted in an increased latency in the appearance of palpable sarcomata, by comparison with the unimmunised mice (=0002).8 Immunisation with C. parvum plus microsomes gave no such protection, implying that the microsomes were provoking an effect antagonistic to that of adjuvant alone. It may be significant in this connection that, in each experiment, the mice in this group developed a strikingly mangy appearance. The results in the mice immunised with adjuvant plus mitochondria followed a consistent but complex pattern. Initially there was an apparent delay in the appearance of tumours (even by comparison with the adjuvanttreated control group), followed by an acceleration in the rate of tumour appearance. The overall effect of this pattern was that this group, despite the encouraging initial trend, showed less latency than did the group treated with C. parvum alone. We must be cautious in interpreting these findings. The number of animals used was rather small, and the mitochondrial preparation was undoubtedly contaminated with other cellular components-possibly " detrimental " 5.
Cox,
D. R.
Jl R. statist. Soc. 1972, 34, 187.
H. BAUM. M. BAUM.
OTITIS MEDIA DUE TO AMPICILLINRESISTANT HÆMOPHILUS INFLUENZÆ
SIR,-Haemophilus influenza, a common respiratory pathogen, has consistently accounted for from 12-5% to 27% of cases of acute otitis media.’ Several cases of ampicillin-resistant H. influenzae producing baeteraemia and meningitis have been reported.2-4 We report here the isolation of ampicillin-resistant H. influenxce in a patient with purulent otitis media. A 4-year-old White boy with known hypogammaglobuluuemia and thymic dysfunction was admitted to Children’s Hospital National Medical Center with a diagnosis of chronic otitis for myringotomy and insertion of Reuter tubes under general aneesthesia. On admission he was found to have bilateral purulent otitis and enlarged adenoids. At operation a culture of the purulent discharge from the ears yielded a heavy growth of H. influenzz, type B. By the KirbyBauer disc-sensitivity method the organism was found to be resistant to ampicillin and susceptible to chloramphenicol, cephalothin, tetracycline, and carbenicillin. By the tube-dilution sensitivity test the minimal inhibitory concentration of ampicillin was found to be 6’25 (J.g. per ml.
In two reported cases of ampicillin-resistant H. influenzae meningitis otitis media had preceded the meningitis and had been treated with oral ampicillin. We are currently observing with considerable concern what appears to be an emerging pattern of ampicillin resistance in the Washington area. Recently, Khan et al. monitored the susceptibility of 60 fresh isolates of H. influenza, type B, from various sites and reported a disquieting 10% resistance to ampicillin.4 The resistance appears to be due to the production of beta-lactamase.44 This phenomenon undoubtedly reflects the intensive use of this drug during the past several years. These observations should be taken into consideration by physicians when prescribing treatment for children with acute otitis media.
Children’s Hospital, National Medical Center, 2125 Thirteenth Street, N.W., Washington, D.C. 20009, U.S.A.
GUIDO CONTRONI WILLIAM RODRIGUEZ ROBERT PUMPHREY SYDNEY Ross CORDELL DEANE WAHEED KHAN.
Howie, V. M., Ploussard, J. H., Lester, R. Pediatrics, Springfield, 1970, 45, 29. 2. Thomas, W. J., McReynolds, J. W., Mock, C. R., Bailey, D. C. Lancet, 1974, i, 313. 3. Center for Disease Control. Morbid. Mortal. Wkly Rep. March 2, 1974, 23. 4. Khan, W., Ross, S., Rodriguez, W., Controni, G., Saz, A. K. J. Am. med. Ass. 1974, 229, 298. 1.
developing H.z. rather than a "slow" varicella virus infection (producing Hodgkin’s disease ?). Institute of Cancer Research and Division of Hæmatology,
Radiumstationen, Nörrebrogade 44, DK-8000 Aarhus C, Denmark.
E. B. THORLING K. THORLING.
CHEMOTHERAPY OF HODGKIN’S DISEASE SIR,-A report from this institute described 24 children under 15 who were treated between 1967 and 1971 with the M.o.p.p. chemotherapy (nitrogen mustard, vincristine, prednisone, procarbazine) six-course regimen.1 7 of the 24 presented with localised tumours (stages I and 11), and the remainder had generalised disease (stages III and IV), all achieved complete remission initially. At the time of the first report (1972), 7 patients had relapsed. Because of the well-documented success of chemotherapeutic regimens in the treatment of advanced Hodgkin’s disease, some investigators propose the use of chemotherapy in earlier stages.2,3 However, there have been disturbing reports of second cancers in patients with Hodgkin’s disease receiving both radiotherapy and chemotherapy. 4-6 Since our original series contains the only published data regarding M.o.p.p. chemotherapy of early-stage Hodgkin’s disease, continued follow-up of these patients is of interest. The outcome in all patients successfully followed up to FOLLOW-UP OF 24 UGANDAN CHILDREN WITH TREATED WITH M.O.P.P. CHEMOTHERAPY
HODGKIN’S DISEASE
(1967-1971)
is shown in the table. 2 patients (nos. 2 and 6) could not be traced and when last seen were in complete remission at 12 and 18 months, respectively. 1 patient (no. 7) relapsed in addition to the 7 noted in the original series. The median remission duration in patients who relapsed was 13 months from the start 01 therapy (range 8-38 months). 3 patients have died: 1 after multiple subsequent relapses (no. 20), 1 of encephalitis while in remission (no. 22), and 1 of an unrelated illness while in a second remission (no. 23). The surviving 6 patients who relapsed have had long second remissions reinduced by M.o.p.p. therapy.13 patients (4 stage 1-11, 8 stage ill, 1 stage iv) remain in their initial unmaintained remissions for a median of 72 + months (range 35-78 months) from the start of therapy. The overall median survival for the entire series (excluding patients lost to follow-up) is more than 60 months. 12 patients in this series presented with stage I-IIIA disease and would ordinarily have received radiotherapy as part or all of their initial treatment. 2 were lost to follow-up, and 1 relapsed. The remaining 9 have sustained initial remissions following the start of M.o.p.p. chemotherapy for 35-78 months. Late complications possibly attributable to chemotherapy were limited to the development of moderate-tosevere gynxcomastia in 7 of prepubertal and early pubertal boys. This observation is unexplained and is under investigation. None of the patients has yet reached marital or reproductive status, and none has developed a second
July, 1974,
neoplasm. This report does
not
substantiate doubts about the
efficacy of chemotherapy alone in curing early stages of Hodgkin’s disease. While the optimum use of chemotherapy and radiotherapy await the outcome of controlled trials, it is of interest that M.o.p.p. chemotherapy alone results in sustained remission in the majority of early-stage patients and offers excellent control for advanced disease.
Uganda Cancer Institute, Makerere University Medical School, Kampala, Uganda.
CHARLES L. M. OLWENY EDWARD KATANGOLE MBIDDE JOSEPHINE NKWOCHA IAN MAGRATH JOHN L. ZIEGLER.
METHYL-CHOLANTHRENE-INDUCED SARCOMATA IN MICE AFTER IMMUNISATION WITH CORYNEBACTERIUM PARVUM PLUS SYNGENEIC SUBCELLULAR MEMBRANE FRACTIONS
SIR,-We wish to report the result of preliminary experidesigned to test an hypothesis that implicates mitochondrial D.N.A. in malignant transformation.1 It has been suggested that some conditions, including those arising during chemical carcinogenesis, may eventually lead to nuclear uptake of mitochondrial D.N.A., thus effecting ments
*
Rye classification. t L.P. = lymphocyte predominant.
M.C. = mixed cellularity.
L.D. =
lymphocyte depleted. N.S. = nodular sclerosis. t Died in remission. 1. 2. 3.
4.
5. 6.
Ziegler, J. L., Bluming, A. Z., Fass, L., Magrath, I. T., Templeton, A. C. Lancet, 1972, ii, 679. DeVita, V. T., Serpick, A. A., Carbone, P. P. Ann. intern. Med. 1970, 73, 881. Moore, M. R., Bull, J. M., Jones, S. E., Rosenberg, S. A., Kaplan, H. S. ibid. 1972, 77, 1. Arseneau, J. L., Sponzo, R. W., Levin, D. L., Schnipper, L. E., Bonner, H., Young, R. C., Canellos, G. P., Johnson, R. E., DeVita, V. T. New Engl. J. Med. 1972, 287, 1119. Castro, G. A. M., Church, A., Pechet, L., Snyder, L. M. ibid. 1973, 23, 103. Canellos, G. P., DeVita, V. T., Arseneau, J. C., Johnson, R. Recent Results Cancer Res. (in the press).
transformation of the nuclear genome.2,3 If the new nuclear genes were now expressed, they would code for a limited number of hydrophobic proteins,4 normally restricted to the inner mitochondrial membrane but now synthesised on cytoplasmic ribosomes and possibly incorporated into the plasma membrane of the cell. Such a change in the cell-surface would, it is suggested, endow the new line of transformed cells with its malignant characteristics and would also introduce into it new antigenic determinants. To test this hypothesis we attempted to immunise mice against chemical carcinogenesis, using as antigen submito1. 2. 3.
Baum, H. Lancet, 1973, ii, 738. Hadler, H. I., Daniel, B. G. Cancer Res. 1973, 33, 117. Schumacher, H. R., Szekely, I. E., Fisher, D. R. Lancet, 1973, ii,
4.
Borst, P. Ann. Rev. Biochem. 1972, 41,
1207. 333.
1398 microsomes.
Furthermore, the protection afforded by
adjuvant alone, although significant, was not absolute and was assessed on the basis of subjective examination of the animals. It should also be emphasised that the basis of any protection initially afforded by mitochondria (or of any detrimental effect of microsomes) may lie in factors (e.g., changes in metabolism of the carcinogen) quite unrelated to the hypothesis that prompted the experiments. Nevertheless, it does seem that the rather arbitrary procedure might have influenced the development of carcinogeninduced tumours, and we are therefore encouraged to pursue this work further, both for its obvious therapeutic implications and for the light it might throw on the origin of malignant transformation. Days Alter Methyl-Cholanthrene Treatment.
Numbers of mice with tumours at intervals following inoculation of 20-methylcholanthrene.
Department of Biochemistry, Chelsea College, University of London. Department of Surgery, Welsh National School of Medicine, Cardiff.
chondrial particles prepared by sonication of mitochondria obtained from livers of syngeneic animals. Microsomes from the same liver homogenate, and also subjected to sonication, were used as a control. Corynebacterium parvum was used as an immunological adjuvant in each case. C$H syngeneic male mice were used in all experiments. The mitochondrial and microsomal membrane fractions were prepared from the livers of 5 of these mice, freeze-dried, and stored at 4°C until required. The remaining mice were then divided into four groups with 10 to 13 mice in each. Group A was injected with 0.1 rnl. of sterile distilled water (S.D.W.) intraperitoneally (i.p.). Group B was injected with 0-1 ml. S.D.W., i.p., and 0-1 ml. C. parvum (Well come Research Laboratories) subcutaneously (s.c.). Group C was injected with 01 ml. of a suspension of the mitochondrial fraction i.P. plus 0-1 ml. C. parvum s.c. Group D was injected with 0’1 ml. of a suspension of the microsomal fraction i.p. plus 0-1 ml. C. parvum s.c. The treatment schedule for each group was repeated a week later at the time of inoculation with the carcinogen. Fibrosarcomata were induced by intramuscular injection of 0-1 ml. of olive oil containing a suspension of 1-0 mg. 20-methylcholanthrene into the right hind leg of each mouse.
The mice were checked twice a week for the presence of tumours, most of which developed between 90 and 125 days after injection of methylcholanthrene. The latent period until the development of an unequivocal tumour (checked by two observers) was recorded, after which the animal was killed. The experiment was repeated three times, the pattern of response being very similar in each case. The figure illustrates the time course of development of tumours for each of the four groups of animals, the results of the three separate experiments being pooled.
Immunisation with C. parvum alone resulted in an increased latency in the appearance of palpable sarcomata, by comparison with the unimmunised mice (=0002).8 Immunisation with C. parvum plus microsomes gave no such protection, implying that the microsomes were provoking an effect antagonistic to that of adjuvant alone. It may be significant in this connection that, in each experiment, the mice in this group developed a strikingly mangy appearance. The results in the mice immunised with adjuvant plus mitochondria followed a consistent but complex pattern. Initially there was an apparent delay in the appearance of tumours (even by comparison with the adjuvanttreated control group), followed by an acceleration in the rate of tumour appearance. The overall effect of this pattern was that this group, despite the encouraging initial trend, showed less latency than did the group treated with C. parvum alone. We must be cautious in interpreting these findings. The number of animals used was rather small, and the mitochondrial preparation was undoubtedly contaminated with other cellular components-possibly " detrimental " 5.
Cox,
D. R.
Jl R. statist. Soc. 1972, 34, 187.
H. BAUM. M. BAUM.
OTITIS MEDIA DUE TO AMPICILLINRESISTANT HÆMOPHILUS INFLUENZÆ
SIR,-Haemophilus influenza, a common respiratory pathogen, has consistently accounted for from 12-5% to 27% of cases of acute otitis media.’ Several cases of ampicillin-resistant H. influenzae producing baeteraemia and meningitis have been reported.2-4 We report here the isolation of ampicillin-resistant H. influenxce in a patient with purulent otitis media. A 4-year-old White boy with known hypogammaglobuluuemia and thymic dysfunction was admitted to Children’s Hospital National Medical Center with a diagnosis of chronic otitis for myringotomy and insertion of Reuter tubes under general aneesthesia. On admission he was found to have bilateral purulent otitis and enlarged adenoids. At operation a culture of the purulent discharge from the ears yielded a heavy growth of H. influenzz, type B. By the KirbyBauer disc-sensitivity method the organism was found to be resistant to ampicillin and susceptible to chloramphenicol, cephalothin, tetracycline, and carbenicillin. By the tube-dilution sensitivity test the minimal inhibitory concentration of ampicillin was found to be 6’25 (J.g. per ml.
In two reported cases of ampicillin-resistant H. influenzae meningitis otitis media had preceded the meningitis and had been treated with oral ampicillin. We are currently observing with considerable concern what appears to be an emerging pattern of ampicillin resistance in the Washington area. Recently, Khan et al. monitored the susceptibility of 60 fresh isolates of H. influenza, type B, from various sites and reported a disquieting 10% resistance to ampicillin.4 The resistance appears to be due to the production of beta-lactamase.44 This phenomenon undoubtedly reflects the intensive use of this drug during the past several years. These observations should be taken into consideration by physicians when prescribing treatment for children with acute otitis media.
Children’s Hospital, National Medical Center, 2125 Thirteenth Street, N.W., Washington, D.C. 20009, U.S.A.
GUIDO CONTRONI WILLIAM RODRIGUEZ ROBERT PUMPHREY SYDNEY Ross CORDELL DEANE WAHEED KHAN.
Howie, V. M., Ploussard, J. H., Lester, R. Pediatrics, Springfield, 1970, 45, 29. 2. Thomas, W. J., McReynolds, J. W., Mock, C. R., Bailey, D. C. Lancet, 1974, i, 313. 3. Center for Disease Control. Morbid. Mortal. Wkly Rep. March 2, 1974, 23. 4. Khan, W., Ross, S., Rodriguez, W., Controni, G., Saz, A. K. J. Am. med. Ass. 1974, 229, 298. 1.