- Email: [email protected]
PROLONGED SURVIVAL OF SKIN-GRAFTS FROM CANCER PATIENTS ON NORMAL RECIPIENTS
414
erythrocytes of some cases of polycythxmia vera, chronic myeloid leukxmia, and hereditary spherocytosis after splenectomy. 22 By contrast, the higher activity in mongol erythrocytes seems to be a consistent finding, and the difference between the mean levels of activity for mongols and normals is approximately 50°o,as might be expected if it
due
the dose-effect of a gene on chromothe absence of overlap between the results for normal and mongol subjects may distinguish these results from results previously published 8 12 for leucocyte enzymes in mongolism, where overlaps with results for normal subjects were considerable. The finding, with minor exceptions, of normal levels of activity for all the enzymes of the Embden-Meyerhof pathway except phosphohexokinase, and of normal values for all the other constituents of the mongol erythrocytes suggests that the increased phosphohexokinase activity is not merely a reflection of a general and non-specific derangement of erythrocyte metabolism. By contrast, the results of Mellman et al.12 may be regarded as indicating a non-specific disturbance of enzyme activity in leucocytes in mongolism. The activities of two enzymes-galactose1-phosphate uridyl transferase and glucose-6-phosphate dehydrogenase-have now been studied in both the erythrocytes and leucocytes of mongols. The results indicate that the activities of these enzymes do not vary in parallel in the two cell series. It has been shown 11 that the increased activity of galactose-1-phosphate uridyl transferase in the blood of mongols resides in the leucocytes and not in the erythrocytes. Similarly, Mellman et al.12 have found increased activity of glucose-6phosphate dehydrogenase in mongol leucocytes, and we have found normal activity in the erythrocytes. It is at present impossible to see any relation between the increased level of phosphohexokinase activity reported here and the other abnormalities described in mongol some
were
21.
A. G. BAIKIE
M.B.
to
tion of genes on autosomes available to us have been concerned with much simpler organisms where the one-gene one-enzyme relation may seem more likely. Nevertheless, the varied cytogenetic findings in effective trisomy for chromosome 21 in man and in partial deletion of that chromosome provide several potentially informative situations for the study of the possible genic control of
erythrocyte-phosphohexokinase activity. SUMMARY
statistically significant increase in the activity of the enzyme phosphohexokinase was found in the erythrocytes of mongols. Other enzymes of the Embden-Meyerhof glycolytic pathway, as well as glucose-6-phosphate dehydrogenase and glutathione reductase, displayed no abnormal activity. Of 21 mongols, only 1 had a level of phosphohexokinase activity within the distribution of our results in healthy controls. Chromosome studies showed this patient to be a normal mongol mosaic in whom the bone-marrow was composed predominantly of cells of normal chromosome constitution. The level of phosphohexokinase activity in mongol erythrocytes may be directly related to the action of genes on chromosome 21, and not merely a concomitant of cell function altered by A
other mechanisms. This study was financed partly by
a
grant from the National Health
Medical Research Council and in part by the Anti-Cancer Council 22. Loder. P
B , de Gruchy, G C. Unpublished.
Glasg.,
F.R.C.P.E.,
M.C.PATH.
P. BRONWEN LODER B.SC. Sydney. G. C. DE GRUCHY M.D. Melb., F.R.A.C.P.,
Furthermore,
erythrocytes.16-18a The possibility exists of complex multigenic control of the activity of a single enzyme in differentiated cells in man. At present, the only model studies for the localisa-
and
of Victoria, Australia. We are grateful to Dr. 0. Margaret Garson and Mrs. J. Brasch, B.sc., for the chromosome studies; to Miss G. Babarczy and Miss K. Esmonde, B.sc., for assistance with the erythrocyte studies; and to Dr. G. Gregory and Miss Sandra Weste, B.Sc., for statistical advice and analysis
University of Melbourne Department of Medicine, St. Vincent’s Hospital, Melbourne, Australia
M.R.C.P., M.C.P.A.
Children’s Cottages, Kew, Victoria, Australia
M.D.
D. B. PITT Melb., M.R.A.C.P.
PROLONGED SURVIVAL OF SKIN-GRAFTS FROM CANCER PATIENTS ON NORMAL
RECIPIENTS MANY studies on the antigenicity of cells from tumour tissue in animals and in man have revealed quantitative as well as qualitative differences. 1 There have been considerably fewer studies on antigenic alterations in normal tissues from cancer patients or from tumourbearing animals. We are at present investigating a series of twelve patients with recurrent cancer. The reactivity of the patients to foreign antigens is being studied by . means of skin-grafts, their response to bacterial antigens, and the reactions of their blood-lymphocytes in tissue culture. In addition we are investigating the antigens of normal tissues from these patients. Skin-grafting from cancer patients, studies on the reactivity of normal lymphocytes in cancer patients and of lymphocytes from
patients in healthy persons, and in-vitro tests for cytotoxic damage by a battery of known potent antisera are also in progress and will be reported in detail elsewhere. Some of the findings in skin-grafting have been so unexpected and striking as to warrant a preliminary cancer
report. METHODS
specimens of full-thickness skin (11 mm. in were taken from the upper arm of five of the twelve diameter) with advanced cancer and were applied to similarly patients prepared sites on six healthy volunteers. The technique used was that of Rapaport and Converse.3 The grafts were examined at short intervals, and graft survival was determined by gross observations. Criteria for graft rejection included cyanosis and oedema of the graft and the appearance of erythema and induration around the graft. The subsequent escharification of the graft confirmed the diagnosis of rejection. The distinctive appearance of the white graft permits ready recognition of this type of response. Biopsy specimens of the graft sites were taken at intervals and were fixed in phosphate-buffered formalin, and Feulgen and hxmatoxylin-and-eosin stains were made of 5[1. sections. Circular
RESULTS
Skin from
white male
with rhabdomyosarcoma, was transplanted to two normal healthy persons, one male and one female. Both grafts took well, and there were no signs of rejection of either graft 60 days and 34 days after grafting when the recipients moved away and contact with them was lost. A Feulgen-stained biopsy specimen at 29 days on the female recipient showed the graft to be male. Skin from case 2, a white female with carcinoma of the breast, was rejected at 3 days with a typical white-graft reaction (fig. 1). The recipient was a 23-year-old healthy 1. 2. 3.
case
1,
a
patient
Old, L. T., Boyse, E. A. Ann. Rev. Med. 1964, 15, 167. Day, E. D. The Immunochemistry of Cancer. New York, 1964. Rapaport, F. T., Converse, J. M. Ann. Surg. 1956, 143, 306.
415
2: white-graft action at 3 days.
Fig. 1-Case
white male who had
no
re-
Fig.
2-Case 5:
at 32
history
graft-survival
days.
of transfusion
or
previous
skin-grafting. Skin from case 3, a white female with mammary carcinoma, was still in situ with no signs of rejection when contact was lost with the male recipient after 40 days. Feulgen staining of a biopsy specimen at 33 days showed the graft to be female. Case 4 was a patient with carcinoma of the breast. Skin was placed on a healthy male and was rejected indolently between the 21st and 24th day. No biopsy specimen was taken. Case 5, also a patient with carcinoma of the breast, donated skin to a healthy male volunteer. Biopsy specimens were taken on the 22nd and 35th days. This graft was also rejected indolently between the 35th and 39th day. On the 22nd day, Feulgen-positive cells were present in the graft. Fig. 2 shows the appearance of this
the 32nd day. all grafts, on microscopy there was extensive infiltration with lymphocytes, but no hxmorrhage and surprisingly little damage to the epidermal cells. Excluding the very
graft
on
In
atypical
response
to
skin from patient 2, Fig. 3-Skin-grafts from cancer patthe median survivalients on healthy recipients. time of the remaining five grafts was over 38 days (fig. 3). This is over three times the average duration of grafts between unrelated individuals and considerably longer than the rejection-time of normal skin placed upon cancer patients in our series. DISCUSSION
Chance
compatibility between donor and recipient was in five of the six cases. Typing of lymphocytes with a battery of isoimmune sera indicated numerous antigenic differences in the five pairs tested. Impaired reactivity of the recipients was also unlikely, since all were in good health and several were skin-tested with bacterial antigens and showed excellent delayed hypersensitivity reactions at 48 hours. Further, an exchange of skin-grafts between two of the recipients after they had rejected skin-grafts from the patients resulted in white grafts. Diminished antigenicity of the skin itself seems possible, and the lymphocytes from cancer or chronically debilitated patients often fail to react in tests of cytotoxicity or agglutination with known potent isoantisera.4 unlikely
4
Amos, D. B., Zmijewski, C. M., Metzgar, R S. Unpublished
It is generally assumed that transplantation antigens are structural components,5 but this is unproven, and decreased production of antigen resulting from a general disturbance of metabolism is possible. Of other possibilities, the presence of an enhancing type of antibody on the tissues, or a generally increased resistance to attack by homograft-rejection mechanisms seem worth exploring. Experiments to determine the cause should be sought in laboratory animals in which the antigenic patterns of the tissues are better understood. The findings suggest a major change in the normal tissues of patients with advanced cancer. D. B. A. was supported by United States Public Health Service grants Gm-Al 10356 and A1-K6 18 399; and B. G. H. was supported by United States Public Health Service grant 3T1GM-946.
D. B. AMOS M.D.
Lond.
B. G. HATTLER Department of Microbiology and Immunology and Department of Surgery, Duke University Medical Center, Durham, North Carolina, U.S.A.
M.D.
Cornell
W. W. SHINGLETON M.D.
Bowman
Gray
Hypothesis STRONG TRANSPLANTATION ANTIGENS IN MAN in man is still highly it is arguable if the continued use of live human donors (apart from identical twins) is really justifiable until more progress has been made in the identification of transplantation antigens in man. 6These antigens are all unidentified as yet. Nevertheless, given a few reasonable assumptions, the recent report from the Kidney Transplant Registry8 suggests that serological identification of some-perhaps no more than four to seven-of the strongest antigens could lead More recent to a radical improvement in the results. data 9 10 suggest that the identification of even two or three would suffice.
RENAL
homotransplantation experimental: so much so that
ASSUMPTIONS
1. The strongest transplantation antigens are all determined by different alleles of a single unidentified histocompatibility locus, S (strong). This is an extrapolation from the factual situation in mice (H-2) and chickens (B)-the only species sufficiently investigated to provide adequate information on this point. The evidence that a single locus is of paramount importance in two such diverse species suggests that a single strong locus may be a general rule. 2. Patients who have survived for more than a year on a homografted kidney alone represent a chance compatibility at the S-locus. They were almost certainly incompatible with their donors in respect of weaker antigens (as can be inferred from the almost invariable breakdown in the course of weeks of skin homografts in man); but the immunosuppressive treatment given to all these patients was presumably effective enough to induce specific _
tolerance to the minor antigens. 3. The different alleles have equal gene frequenciesi.e., they have the same probability of occupying the Davies, D. A. L. Transplantation: Ciba Foundation Symposium. Boston, 1962. 6. Calne, R. Y. Renal Transplantation. London, 1963. 7. Simonsen, M. Unpublished. 8. Murray, J. E., Gleason, R., Bartholomay, A. Transplantation, 1964, 2,
5.
660. 9. 10.
Starzl, T. E., Marchino, T. L., Porter, K. A., Moore, C. A., Rif kind, D., Waddell, W R. Ann. intern. Med. 1964, 61, 470. Moorhead, J. F., Patel, A. R. Brit. med. J. 1964, ii, 1111.
erythrocytes of some cases of polycythxmia vera, chronic myeloid leukxmia, and hereditary spherocytosis after splenectomy. 22 By contrast, the higher activity in mongol erythrocytes seems to be a consistent finding, and the difference between the mean levels of activity for mongols and normals is approximately 50°o,as might be expected if it
due
the dose-effect of a gene on chromothe absence of overlap between the results for normal and mongol subjects may distinguish these results from results previously published 8 12 for leucocyte enzymes in mongolism, where overlaps with results for normal subjects were considerable. The finding, with minor exceptions, of normal levels of activity for all the enzymes of the Embden-Meyerhof pathway except phosphohexokinase, and of normal values for all the other constituents of the mongol erythrocytes suggests that the increased phosphohexokinase activity is not merely a reflection of a general and non-specific derangement of erythrocyte metabolism. By contrast, the results of Mellman et al.12 may be regarded as indicating a non-specific disturbance of enzyme activity in leucocytes in mongolism. The activities of two enzymes-galactose1-phosphate uridyl transferase and glucose-6-phosphate dehydrogenase-have now been studied in both the erythrocytes and leucocytes of mongols. The results indicate that the activities of these enzymes do not vary in parallel in the two cell series. It has been shown 11 that the increased activity of galactose-1-phosphate uridyl transferase in the blood of mongols resides in the leucocytes and not in the erythrocytes. Similarly, Mellman et al.12 have found increased activity of glucose-6phosphate dehydrogenase in mongol leucocytes, and we have found normal activity in the erythrocytes. It is at present impossible to see any relation between the increased level of phosphohexokinase activity reported here and the other abnormalities described in mongol some
were
21.
A. G. BAIKIE
M.B.
to
tion of genes on autosomes available to us have been concerned with much simpler organisms where the one-gene one-enzyme relation may seem more likely. Nevertheless, the varied cytogenetic findings in effective trisomy for chromosome 21 in man and in partial deletion of that chromosome provide several potentially informative situations for the study of the possible genic control of
erythrocyte-phosphohexokinase activity. SUMMARY
statistically significant increase in the activity of the enzyme phosphohexokinase was found in the erythrocytes of mongols. Other enzymes of the Embden-Meyerhof glycolytic pathway, as well as glucose-6-phosphate dehydrogenase and glutathione reductase, displayed no abnormal activity. Of 21 mongols, only 1 had a level of phosphohexokinase activity within the distribution of our results in healthy controls. Chromosome studies showed this patient to be a normal mongol mosaic in whom the bone-marrow was composed predominantly of cells of normal chromosome constitution. The level of phosphohexokinase activity in mongol erythrocytes may be directly related to the action of genes on chromosome 21, and not merely a concomitant of cell function altered by A
other mechanisms. This study was financed partly by
a
grant from the National Health
Medical Research Council and in part by the Anti-Cancer Council 22. Loder. P
B , de Gruchy, G C. Unpublished.
Glasg.,
F.R.C.P.E.,
M.C.PATH.
P. BRONWEN LODER B.SC. Sydney. G. C. DE GRUCHY M.D. Melb., F.R.A.C.P.,
Furthermore,
erythrocytes.16-18a The possibility exists of complex multigenic control of the activity of a single enzyme in differentiated cells in man. At present, the only model studies for the localisa-
and
of Victoria, Australia. We are grateful to Dr. 0. Margaret Garson and Mrs. J. Brasch, B.sc., for the chromosome studies; to Miss G. Babarczy and Miss K. Esmonde, B.sc., for assistance with the erythrocyte studies; and to Dr. G. Gregory and Miss Sandra Weste, B.Sc., for statistical advice and analysis
University of Melbourne Department of Medicine, St. Vincent’s Hospital, Melbourne, Australia
M.R.C.P., M.C.P.A.
Children’s Cottages, Kew, Victoria, Australia
M.D.
D. B. PITT Melb., M.R.A.C.P.
PROLONGED SURVIVAL OF SKIN-GRAFTS FROM CANCER PATIENTS ON NORMAL
RECIPIENTS MANY studies on the antigenicity of cells from tumour tissue in animals and in man have revealed quantitative as well as qualitative differences. 1 There have been considerably fewer studies on antigenic alterations in normal tissues from cancer patients or from tumourbearing animals. We are at present investigating a series of twelve patients with recurrent cancer. The reactivity of the patients to foreign antigens is being studied by . means of skin-grafts, their response to bacterial antigens, and the reactions of their blood-lymphocytes in tissue culture. In addition we are investigating the antigens of normal tissues from these patients. Skin-grafting from cancer patients, studies on the reactivity of normal lymphocytes in cancer patients and of lymphocytes from
patients in healthy persons, and in-vitro tests for cytotoxic damage by a battery of known potent antisera are also in progress and will be reported in detail elsewhere. Some of the findings in skin-grafting have been so unexpected and striking as to warrant a preliminary cancer
report. METHODS
specimens of full-thickness skin (11 mm. in were taken from the upper arm of five of the twelve diameter) with advanced cancer and were applied to similarly patients prepared sites on six healthy volunteers. The technique used was that of Rapaport and Converse.3 The grafts were examined at short intervals, and graft survival was determined by gross observations. Criteria for graft rejection included cyanosis and oedema of the graft and the appearance of erythema and induration around the graft. The subsequent escharification of the graft confirmed the diagnosis of rejection. The distinctive appearance of the white graft permits ready recognition of this type of response. Biopsy specimens of the graft sites were taken at intervals and were fixed in phosphate-buffered formalin, and Feulgen and hxmatoxylin-and-eosin stains were made of 5[1. sections. Circular
RESULTS
Skin from
white male
with rhabdomyosarcoma, was transplanted to two normal healthy persons, one male and one female. Both grafts took well, and there were no signs of rejection of either graft 60 days and 34 days after grafting when the recipients moved away and contact with them was lost. A Feulgen-stained biopsy specimen at 29 days on the female recipient showed the graft to be male. Skin from case 2, a white female with carcinoma of the breast, was rejected at 3 days with a typical white-graft reaction (fig. 1). The recipient was a 23-year-old healthy 1. 2. 3.
case
1,
a
patient
Old, L. T., Boyse, E. A. Ann. Rev. Med. 1964, 15, 167. Day, E. D. The Immunochemistry of Cancer. New York, 1964. Rapaport, F. T., Converse, J. M. Ann. Surg. 1956, 143, 306.
415
2: white-graft action at 3 days.
Fig. 1-Case
white male who had
no
re-
Fig.
2-Case 5:
at 32
history
graft-survival
days.
of transfusion
or
previous
skin-grafting. Skin from case 3, a white female with mammary carcinoma, was still in situ with no signs of rejection when contact was lost with the male recipient after 40 days. Feulgen staining of a biopsy specimen at 33 days showed the graft to be female. Case 4 was a patient with carcinoma of the breast. Skin was placed on a healthy male and was rejected indolently between the 21st and 24th day. No biopsy specimen was taken. Case 5, also a patient with carcinoma of the breast, donated skin to a healthy male volunteer. Biopsy specimens were taken on the 22nd and 35th days. This graft was also rejected indolently between the 35th and 39th day. On the 22nd day, Feulgen-positive cells were present in the graft. Fig. 2 shows the appearance of this
the 32nd day. all grafts, on microscopy there was extensive infiltration with lymphocytes, but no hxmorrhage and surprisingly little damage to the epidermal cells. Excluding the very
graft
on
In
atypical
response
to
skin from patient 2, Fig. 3-Skin-grafts from cancer patthe median survivalients on healthy recipients. time of the remaining five grafts was over 38 days (fig. 3). This is over three times the average duration of grafts between unrelated individuals and considerably longer than the rejection-time of normal skin placed upon cancer patients in our series. DISCUSSION
Chance
compatibility between donor and recipient was in five of the six cases. Typing of lymphocytes with a battery of isoimmune sera indicated numerous antigenic differences in the five pairs tested. Impaired reactivity of the recipients was also unlikely, since all were in good health and several were skin-tested with bacterial antigens and showed excellent delayed hypersensitivity reactions at 48 hours. Further, an exchange of skin-grafts between two of the recipients after they had rejected skin-grafts from the patients resulted in white grafts. Diminished antigenicity of the skin itself seems possible, and the lymphocytes from cancer or chronically debilitated patients often fail to react in tests of cytotoxicity or agglutination with known potent isoantisera.4 unlikely
4
Amos, D. B., Zmijewski, C. M., Metzgar, R S. Unpublished
It is generally assumed that transplantation antigens are structural components,5 but this is unproven, and decreased production of antigen resulting from a general disturbance of metabolism is possible. Of other possibilities, the presence of an enhancing type of antibody on the tissues, or a generally increased resistance to attack by homograft-rejection mechanisms seem worth exploring. Experiments to determine the cause should be sought in laboratory animals in which the antigenic patterns of the tissues are better understood. The findings suggest a major change in the normal tissues of patients with advanced cancer. D. B. A. was supported by United States Public Health Service grants Gm-Al 10356 and A1-K6 18 399; and B. G. H. was supported by United States Public Health Service grant 3T1GM-946.
D. B. AMOS M.D.
Lond.
B. G. HATTLER Department of Microbiology and Immunology and Department of Surgery, Duke University Medical Center, Durham, North Carolina, U.S.A.
M.D.
Cornell
W. W. SHINGLETON M.D.
Bowman
Gray
Hypothesis STRONG TRANSPLANTATION ANTIGENS IN MAN in man is still highly it is arguable if the continued use of live human donors (apart from identical twins) is really justifiable until more progress has been made in the identification of transplantation antigens in man. 6These antigens are all unidentified as yet. Nevertheless, given a few reasonable assumptions, the recent report from the Kidney Transplant Registry8 suggests that serological identification of some-perhaps no more than four to seven-of the strongest antigens could lead More recent to a radical improvement in the results. data 9 10 suggest that the identification of even two or three would suffice.
RENAL
homotransplantation experimental: so much so that
ASSUMPTIONS
1. The strongest transplantation antigens are all determined by different alleles of a single unidentified histocompatibility locus, S (strong). This is an extrapolation from the factual situation in mice (H-2) and chickens (B)-the only species sufficiently investigated to provide adequate information on this point. The evidence that a single locus is of paramount importance in two such diverse species suggests that a single strong locus may be a general rule. 2. Patients who have survived for more than a year on a homografted kidney alone represent a chance compatibility at the S-locus. They were almost certainly incompatible with their donors in respect of weaker antigens (as can be inferred from the almost invariable breakdown in the course of weeks of skin homografts in man); but the immunosuppressive treatment given to all these patients was presumably effective enough to induce specific _
tolerance to the minor antigens. 3. The different alleles have equal gene frequenciesi.e., they have the same probability of occupying the Davies, D. A. L. Transplantation: Ciba Foundation Symposium. Boston, 1962. 6. Calne, R. Y. Renal Transplantation. London, 1963. 7. Simonsen, M. Unpublished. 8. Murray, J. E., Gleason, R., Bartholomay, A. Transplantation, 1964, 2,
5.
660. 9. 10.
Starzl, T. E., Marchino, T. L., Porter, K. A., Moore, C. A., Rif kind, D., Waddell, W R. Ann. intern. Med. 1964, 61, 470. Moorhead, J. F., Patel, A. R. Brit. med. J. 1964, ii, 1111.