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Neuraminidase and Regression of Solid Tumours
523
induced fibrosarcoma in mice, SIMMONS and his colleagues have investigated early and late challenge with v.c.N.-treated cells,13,14 Early challenge (within the first 10 days) either prevented or impaired growth of the initial tumour graft. Late challenge, delayed until the untreated fibrosarcoma cells were well established, gave striking resultsthe tumours stopped growing and about a third of them regressed and disappeared permanently (most of these animals remaining immune to subsequent challenge). Tumour growth resumed in the remainder after an interval of about 35 days. One limiting factor was the size of the tumour, no regressions being recorded in lesions greater than 1 cm. in diameter. On the other hand, regression of smaller tumours was sometimes achieved as late as 30 days after their These results were specific for initial grafting. neuraminidase and also for the tumour, no crossreactions being obtained with a second fibrosarcoma induced with 3-methylcholanthrene in the same strain of mice.
cally
THE LANCET Neuraminidase and Regression of Solid Tumours
general agreement among clinicians and experimentalists that immunotherapy has only a limited role in the management of malignant disease, and is likely to be effective only when most neoplastic tissue has been eliminated by other means.1-3 This precondition is perhaps most nearly achieved in acute lymphoblastic leukaemia, where the value of adjuvant immunotherapy is at present under investigation.4,5 The prospect of immune mechanisms playing a part in the treatment of well-established neoplasms has always seemed remote, but preliminary evidence is beginning to emerge which suggests that immunotherapy can sometimes be effective against solid THERE is
tumours.
The main observations stem from experimental neoplasms in mice, treated in vitro with Vibrio cholera neuraminidase (v.c.N.). This enzyme is directed against sialic (N-acetylneuraminic) acid, which is one of the main surface constituents of cell membranes; it acts by splitting the glycosidic linkages between sialic-acid residues and mucopolysaccharides. Tumour cells treated with v.c.N. become more immunogenic 6-12; recipient animals are rendered immune and the growth of tumour inocula is partly or completely inhibited. There are few successful " takes ", and tumours that do appear develop slowly and are prone to regress. These effects are not seen if V.C.N. is first inactivated by heating, nor if v.c.N.-treated tumour cells are incubated with sialic acid before injection-confirmation that the changes brought about by V.C.N. are indeed specific for the enzymic action of neuraminidase. Granted that V.C.N. renders tumour cells more immunogenic, what happens when v.c.N.-treated cells are given to animals already inoculated with untreated cells of the same tumour ? Information on this point is now available.l3_5 Using a chemi1. 2. 3.
4.
5. 6. 7. 8. 9. 10. 11. 12.
13.
14. 15.
Fairley, G. H. Br. med. J. 1969, ii, 467. Fairley, G. H. Proc. R. Soc. Med. 1971, 64, 1044. Alexander, P. ibid. p. 1042. Mathé, G., Amiel, J. L., Schwarzenberg, L., Schneider, M., Cattan, A., Schlumberger, J. R., Hayat, M., de Vassal, F. Lancet, 1969, i, 697. Mathé, G. Br. med. J. 1970, iv, 487. Currie, G. A., Bagshawe, K. D. Lancet, 1967, i, 708. Currie, G. A. ibid. 1967, ii, 1336. Sanford, B. H. Transplantation, 1967, 5, 1273. Bagshawe, K. D., Currie, G. A. Nature, 1968, 218, 1254. Currie, G. A., Bagshawe, K. D. Br. J. Cancer, 1968, 22, 843. Currie, G. A., Bagshawe, K. D. ibid. 1969, 23, 141. Bekesi, J. G., St.-Arneault, G., Holland, J. F. Cancer Res. 1971, 31, 2130. Simmons, R. L., Rios, A., Lundgren, G., Ray, P. K., McKhann, C. F., Haywood, G. R. Surgery, St. Louis, 1971, 70, 38. Simmons, R. L., Rios, A. Science, 1971, 174, 591. Simmons, R. L., Rios, A., Ray, P. K., Lundgren, R. J. natn. Cancer Inst. 1971, 47, 1087.
It thus appears that tumours can regress totally and permanently after immunisation with modified tumour tissue. The incidence of such regressions is clearly small, and attempts have been made to boost it. Previous clinical 16 and experimental 17,18 observations indicate that injection of B.C.G. into tumour nodules sometimes brings about their regression,
presumably
through
non-specific
mechanisms.
SIMMONS and Rios 14 have now examined the consequences of combined challenge with v.c.N.-treated fibrosarcoma cells and B.C.G.: using several different schedules of injection, they found that the mixed challenge increased the incidence of regression to about half. B.C.G. alone was ineffective, given into the tumour or outside it. The greater efficacy of the combined challenge was not accompanied by any relaxation of specificity, no cross-reaction being In these observed with a second fibrosarcoma. experiments, growth of the challenging tumour cells was completely suppressed, partly by v.c.N. and partly by incubating the cells with mitomycin C before inoculation. The exact mode of action of v.c.N. is still uncertain. The enzyme does not appear to damage cells directly (except after prolonged incubation 12) and it seems to act solely by increasing cellular immunogenicity. This activity is not confined to tumours and has been described in relation to both embryonic tissues and normal lymphoid cells.6,19-21 Predictably, increased immunogenicity can only be expressed in vivo in 16. 17. 18. 19.
20. 21.
Morton, D. L., Eilber, F. R., Malmgren, R. A., Wood, W. C. Surgery, St. Louis, 1970, 68, 158. Zbar, B., Tanaka, T. Science, 1971, 172, 271. Zbar, B., Bernstein, I. D., Rapp, H. J. J. natn. Cancer Inst. 1971, 46, 831. Simmons, R. L., Lipschultz, M. L., Rios, A., Ray, P. K. Nature New Biol. 1971, 231, 111. Simmons, R. L., Rios, A., Ray, P. K. ibid. p. 179. Ray, P. K., Simmons, R. L. Proc. Soc. exp. Biol. Med. 1971, 138, 600.
524
immunologically competent hosts; v.c.N.-treated tumour cells behave like untreated cells when injected into immunosuppressed animals 7,10,11,13,15 (an observation which illustrates that proliferation of v.c.N.-treated cells is unimpaired). The main discussion centres on the basis for this increased immunogenicity. It was originally thought that v.c.N. unmasked specific antigens on the cell surface,6 but this view has been criticised on the ground that normal lymphoid cells treated with v.c.N. do not show the increased uptake of allospecific antibody in vitro which would follow the exposure of fresh reactive sites.21 Attempts to unmask H2 antigens in mouse trophoblast with v.c.N. have also been unsuccessful.19 It is, however, likely that weak antigens (such as the e antigen found in thymic lymphocytes) may well be uncovered by V.C.N.22 SmoNS and his group favour a broader view and suggest that increases V.C.N. immunogenicity non-specifically.14,20,21 Treatment with V.C.N. removes sialicacid residues (a possible source of steric hindrance) and reduces the negative charge; the membrane becomes more deformable and transport of aminoacids across it is altered. Such cells are rendered more susceptible to phagocytosis and to complementmediated lysis. Unmasking of antigen is now seen as one probable consequence of exposure to v.c.N., and several others have to be considered-notably, more effective recognition of antigen and more effective interaction between antigen-bearing and antigen-responsive cells. There is little need to stress the preliminary nature of this work, nor the difficult questions that are raised. Its main interest lies in the demonstration that solid tumours can, in certain circumstances, regress as, a result of immunotherapy-a finding which may provoke new interest in what has hitherto seemed an unpromising field of inquiry.
CHLORAL HYDRATE AND ORAL ANTICOAGULANTS ORAL anticoagulants of the coumarin-indandione group interact with many other compounds, including a wide variety of sedative and hypnotic drugs .23 At
least two basic mechanisms may underlie these interactions. The first is displacement, by the other drug, of the anticoagulant from its binding sites on plasmaproteins. This mechanism is important in the case of compounds such as anticoagulants, where a high proportion of the circulating drug is bound to plasmaprotein after absorption. Displacement of only a part of this bound inactive fraction by other avid proteinbinders such as phenylbutazone, salicylates, and sulphonamides may result in a large increase in the active unbound fraction, and hence an increase in its anticoagulant activity. The second mechanism in22. 23.
Schlesinger, M., Amos, B. D. Transplant. Prescott, L. F. Lancet, 1969, ii, 1239.
Proc. 1971,
3, 895.
volves changing the rate at which the anticoagulant drug is metabolised by the enzymes of the liver endoplasmic reticulum. Some drugs, including phenobarbitone and phenytoin, induce these enzymes, leading to an increase in rate of metabolism of the anticoagulant and so reducing its therapeutic action. Others, such as disulfiram, 24 inhibit hepatic metabolising enzymes, leading to a decrease in rate of breakdown of the anticoagulant and so to an increase in its pharmacological effect.25
When an interaction between an oral anticoagulant drug and another compound is noted, one or both of these mechanisms must be considered. Cucinell et al.26 described a patient in whom chloral hydrate had apparently reduced the anticoagulant effect of bishydroxycoumarin, so that when the hypnotic drug was withdrawn the same dose of the anticoagulant produced a fatal haemorrhage. Dichloralphenazone (’Welldorm ’) is a popular hypnotic drug, being a complex of chloral hydrate and phenazone (antipyrine). Breckenridge et al. 27 found that these two constituents had different effects on the disposition of warfarin. Chloral hydrate produced a fall in steady-state plasma-warfarin concentrations in five individuals because the anticoagulant was displaced from its protein-binding sites by trichloroacetic acid, a major metabolite of chloral hydrate, but there was no change in anticoagulant control. Administration of phenazone also caused a fall in steady-state plasma-warfarin and reduced its plasma half-life, with increased urinary excretion of warfarin metabolites consistent with hepatic enzyme induction. Sellers and Koch-Weser 21 also demonstrated that trichloroacetic acid displaced warfarin from its binding sites on plasma-albumin, leading to a temporary increase in plasma-levels of unbound warfarin and a decrease in its plasma halflife. In contrast to Cucinell26 and Breckenridge, 27 Sellers and Koch-Weser found that this increase in unbound drug was associated with an enhanced anticoagulant effect. Further evidence for such an effect is provided by a report from the Boston Collaborative Drug Surveillance Program 29 in which patients anticoagulated in hospital were divided into three groups receiving continuous, occasional, or no chloral hydrate during the first few days of warfarin therapy. Those receiving continuous chloral-hydrate therapy required significantly less warfarin during the induction phase of anticoagulation than those receiving none, while patients given only occasional chloral hydrate required an intermediate dose. Apparently, therefore, the interaction between these drugs, though not consistent in its effects, is of clinical importance, and care is needed when an oral-anticoagulation regimen is being planned for patients receiving chloral hydrate alone or in combination with other drugs. 24. 25. 26. 27.
28.
Vesell, E. S., Passananti, G. T., Lee, C. H. Clin. Pharmac. Ther. 1971, 12, 785. Rothstein, E. J. Am. med. Ass. 1968, 206, 1574. Cucinell, S. A., Odessky, L., Weiss, M., Dayton, P. G. ibid. 1966, 197, 366. Breckenridge, A., Orme, M. L’E., Thorgeirsson, S., Davies, D. S., Brooks, R. V. Clin. Sci. 1971, 40, 351. Sellers, E. M., Koch-Weser, J. New Engl. J. Med. 1970, 283,
827. 29. Boston Collaborative 286, 53.
Drug Surveillance Program. ibid. 1972,
induced fibrosarcoma in mice, SIMMONS and his colleagues have investigated early and late challenge with v.c.N.-treated cells,13,14 Early challenge (within the first 10 days) either prevented or impaired growth of the initial tumour graft. Late challenge, delayed until the untreated fibrosarcoma cells were well established, gave striking resultsthe tumours stopped growing and about a third of them regressed and disappeared permanently (most of these animals remaining immune to subsequent challenge). Tumour growth resumed in the remainder after an interval of about 35 days. One limiting factor was the size of the tumour, no regressions being recorded in lesions greater than 1 cm. in diameter. On the other hand, regression of smaller tumours was sometimes achieved as late as 30 days after their These results were specific for initial grafting. neuraminidase and also for the tumour, no crossreactions being obtained with a second fibrosarcoma induced with 3-methylcholanthrene in the same strain of mice.
cally
THE LANCET Neuraminidase and Regression of Solid Tumours
general agreement among clinicians and experimentalists that immunotherapy has only a limited role in the management of malignant disease, and is likely to be effective only when most neoplastic tissue has been eliminated by other means.1-3 This precondition is perhaps most nearly achieved in acute lymphoblastic leukaemia, where the value of adjuvant immunotherapy is at present under investigation.4,5 The prospect of immune mechanisms playing a part in the treatment of well-established neoplasms has always seemed remote, but preliminary evidence is beginning to emerge which suggests that immunotherapy can sometimes be effective against solid THERE is
tumours.
The main observations stem from experimental neoplasms in mice, treated in vitro with Vibrio cholera neuraminidase (v.c.N.). This enzyme is directed against sialic (N-acetylneuraminic) acid, which is one of the main surface constituents of cell membranes; it acts by splitting the glycosidic linkages between sialic-acid residues and mucopolysaccharides. Tumour cells treated with v.c.N. become more immunogenic 6-12; recipient animals are rendered immune and the growth of tumour inocula is partly or completely inhibited. There are few successful " takes ", and tumours that do appear develop slowly and are prone to regress. These effects are not seen if V.C.N. is first inactivated by heating, nor if v.c.N.-treated tumour cells are incubated with sialic acid before injection-confirmation that the changes brought about by V.C.N. are indeed specific for the enzymic action of neuraminidase. Granted that V.C.N. renders tumour cells more immunogenic, what happens when v.c.N.-treated cells are given to animals already inoculated with untreated cells of the same tumour ? Information on this point is now available.l3_5 Using a chemi1. 2. 3.
4.
5. 6. 7. 8. 9. 10. 11. 12.
13.
14. 15.
Fairley, G. H. Br. med. J. 1969, ii, 467. Fairley, G. H. Proc. R. Soc. Med. 1971, 64, 1044. Alexander, P. ibid. p. 1042. Mathé, G., Amiel, J. L., Schwarzenberg, L., Schneider, M., Cattan, A., Schlumberger, J. R., Hayat, M., de Vassal, F. Lancet, 1969, i, 697. Mathé, G. Br. med. J. 1970, iv, 487. Currie, G. A., Bagshawe, K. D. Lancet, 1967, i, 708. Currie, G. A. ibid. 1967, ii, 1336. Sanford, B. H. Transplantation, 1967, 5, 1273. Bagshawe, K. D., Currie, G. A. Nature, 1968, 218, 1254. Currie, G. A., Bagshawe, K. D. Br. J. Cancer, 1968, 22, 843. Currie, G. A., Bagshawe, K. D. ibid. 1969, 23, 141. Bekesi, J. G., St.-Arneault, G., Holland, J. F. Cancer Res. 1971, 31, 2130. Simmons, R. L., Rios, A., Lundgren, G., Ray, P. K., McKhann, C. F., Haywood, G. R. Surgery, St. Louis, 1971, 70, 38. Simmons, R. L., Rios, A. Science, 1971, 174, 591. Simmons, R. L., Rios, A., Ray, P. K., Lundgren, R. J. natn. Cancer Inst. 1971, 47, 1087.
It thus appears that tumours can regress totally and permanently after immunisation with modified tumour tissue. The incidence of such regressions is clearly small, and attempts have been made to boost it. Previous clinical 16 and experimental 17,18 observations indicate that injection of B.C.G. into tumour nodules sometimes brings about their regression,
presumably
through
non-specific
mechanisms.
SIMMONS and Rios 14 have now examined the consequences of combined challenge with v.c.N.-treated fibrosarcoma cells and B.C.G.: using several different schedules of injection, they found that the mixed challenge increased the incidence of regression to about half. B.C.G. alone was ineffective, given into the tumour or outside it. The greater efficacy of the combined challenge was not accompanied by any relaxation of specificity, no cross-reaction being In these observed with a second fibrosarcoma. experiments, growth of the challenging tumour cells was completely suppressed, partly by v.c.N. and partly by incubating the cells with mitomycin C before inoculation. The exact mode of action of v.c.N. is still uncertain. The enzyme does not appear to damage cells directly (except after prolonged incubation 12) and it seems to act solely by increasing cellular immunogenicity. This activity is not confined to tumours and has been described in relation to both embryonic tissues and normal lymphoid cells.6,19-21 Predictably, increased immunogenicity can only be expressed in vivo in 16. 17. 18. 19.
20. 21.
Morton, D. L., Eilber, F. R., Malmgren, R. A., Wood, W. C. Surgery, St. Louis, 1970, 68, 158. Zbar, B., Tanaka, T. Science, 1971, 172, 271. Zbar, B., Bernstein, I. D., Rapp, H. J. J. natn. Cancer Inst. 1971, 46, 831. Simmons, R. L., Lipschultz, M. L., Rios, A., Ray, P. K. Nature New Biol. 1971, 231, 111. Simmons, R. L., Rios, A., Ray, P. K. ibid. p. 179. Ray, P. K., Simmons, R. L. Proc. Soc. exp. Biol. Med. 1971, 138, 600.
524
immunologically competent hosts; v.c.N.-treated tumour cells behave like untreated cells when injected into immunosuppressed animals 7,10,11,13,15 (an observation which illustrates that proliferation of v.c.N.-treated cells is unimpaired). The main discussion centres on the basis for this increased immunogenicity. It was originally thought that v.c.N. unmasked specific antigens on the cell surface,6 but this view has been criticised on the ground that normal lymphoid cells treated with v.c.N. do not show the increased uptake of allospecific antibody in vitro which would follow the exposure of fresh reactive sites.21 Attempts to unmask H2 antigens in mouse trophoblast with v.c.N. have also been unsuccessful.19 It is, however, likely that weak antigens (such as the e antigen found in thymic lymphocytes) may well be uncovered by V.C.N.22 SmoNS and his group favour a broader view and suggest that increases V.C.N. immunogenicity non-specifically.14,20,21 Treatment with V.C.N. removes sialicacid residues (a possible source of steric hindrance) and reduces the negative charge; the membrane becomes more deformable and transport of aminoacids across it is altered. Such cells are rendered more susceptible to phagocytosis and to complementmediated lysis. Unmasking of antigen is now seen as one probable consequence of exposure to v.c.N., and several others have to be considered-notably, more effective recognition of antigen and more effective interaction between antigen-bearing and antigen-responsive cells. There is little need to stress the preliminary nature of this work, nor the difficult questions that are raised. Its main interest lies in the demonstration that solid tumours can, in certain circumstances, regress as, a result of immunotherapy-a finding which may provoke new interest in what has hitherto seemed an unpromising field of inquiry.
CHLORAL HYDRATE AND ORAL ANTICOAGULANTS ORAL anticoagulants of the coumarin-indandione group interact with many other compounds, including a wide variety of sedative and hypnotic drugs .23 At
least two basic mechanisms may underlie these interactions. The first is displacement, by the other drug, of the anticoagulant from its binding sites on plasmaproteins. This mechanism is important in the case of compounds such as anticoagulants, where a high proportion of the circulating drug is bound to plasmaprotein after absorption. Displacement of only a part of this bound inactive fraction by other avid proteinbinders such as phenylbutazone, salicylates, and sulphonamides may result in a large increase in the active unbound fraction, and hence an increase in its anticoagulant activity. The second mechanism in22. 23.
Schlesinger, M., Amos, B. D. Transplant. Prescott, L. F. Lancet, 1969, ii, 1239.
Proc. 1971,
3, 895.
volves changing the rate at which the anticoagulant drug is metabolised by the enzymes of the liver endoplasmic reticulum. Some drugs, including phenobarbitone and phenytoin, induce these enzymes, leading to an increase in rate of metabolism of the anticoagulant and so reducing its therapeutic action. Others, such as disulfiram, 24 inhibit hepatic metabolising enzymes, leading to a decrease in rate of breakdown of the anticoagulant and so to an increase in its pharmacological effect.25
When an interaction between an oral anticoagulant drug and another compound is noted, one or both of these mechanisms must be considered. Cucinell et al.26 described a patient in whom chloral hydrate had apparently reduced the anticoagulant effect of bishydroxycoumarin, so that when the hypnotic drug was withdrawn the same dose of the anticoagulant produced a fatal haemorrhage. Dichloralphenazone (’Welldorm ’) is a popular hypnotic drug, being a complex of chloral hydrate and phenazone (antipyrine). Breckenridge et al. 27 found that these two constituents had different effects on the disposition of warfarin. Chloral hydrate produced a fall in steady-state plasma-warfarin concentrations in five individuals because the anticoagulant was displaced from its protein-binding sites by trichloroacetic acid, a major metabolite of chloral hydrate, but there was no change in anticoagulant control. Administration of phenazone also caused a fall in steady-state plasma-warfarin and reduced its plasma half-life, with increased urinary excretion of warfarin metabolites consistent with hepatic enzyme induction. Sellers and Koch-Weser 21 also demonstrated that trichloroacetic acid displaced warfarin from its binding sites on plasma-albumin, leading to a temporary increase in plasma-levels of unbound warfarin and a decrease in its plasma halflife. In contrast to Cucinell26 and Breckenridge, 27 Sellers and Koch-Weser found that this increase in unbound drug was associated with an enhanced anticoagulant effect. Further evidence for such an effect is provided by a report from the Boston Collaborative Drug Surveillance Program 29 in which patients anticoagulated in hospital were divided into three groups receiving continuous, occasional, or no chloral hydrate during the first few days of warfarin therapy. Those receiving continuous chloral-hydrate therapy required significantly less warfarin during the induction phase of anticoagulation than those receiving none, while patients given only occasional chloral hydrate required an intermediate dose. Apparently, therefore, the interaction between these drugs, though not consistent in its effects, is of clinical importance, and care is needed when an oral-anticoagulation regimen is being planned for patients receiving chloral hydrate alone or in combination with other drugs. 24. 25. 26. 27.
28.
Vesell, E. S., Passananti, G. T., Lee, C. H. Clin. Pharmac. Ther. 1971, 12, 785. Rothstein, E. J. Am. med. Ass. 1968, 206, 1574. Cucinell, S. A., Odessky, L., Weiss, M., Dayton, P. G. ibid. 1966, 197, 366. Breckenridge, A., Orme, M. L’E., Thorgeirsson, S., Davies, D. S., Brooks, R. V. Clin. Sci. 1971, 40, 351. Sellers, E. M., Koch-Weser, J. New Engl. J. Med. 1970, 283,
827. 29. Boston Collaborative 286, 53.
Drug Surveillance Program. ibid. 1972,