Brifish J w n d of Hcematology , I 979, 42, 3 9 I -3 98.
Red Blood Cell Enzyme Abnormalities in Patients Treated with Chemotherapy J. ETIEMBLE, J. F. BERNARD, CH. PICAT,D. BELPOMME A N D P. BOIVIN
Laboratoire d’Enzymologie des Cellules Sanguines ( I N S E R M U I ~ O - C N RESR A 573) et Service d’Hkmatologie Clinique, Hapita1 Beaujon, Clichy, France (Received 2 M a y 1978; accepted for publication 1 8 October 1978) SUMMARY. Sixteen red blood cell enzyme activities and fetal haemoglobin level have been assayed in 60 patients treated for haematologic or nonhaematologic malignant diseases with various combinations of cytostatic drugs. Acquired enzyme deficiency was found in 20 patients. The most frequently decreased activities were those of G6PD (12cases), PK (seven cases), PFK (six cases) and AK (three cases). In many patients decreased activity of some enzymes contrasted with increased activity of others such as ALD, TPI, G3PD, PGK, ENOL and 6PGD. The number ofabnormalities seems to be related to the duration of the treatment. Incidence of enzyme deficiencies was similar in patients previously treated or not with radiotherapy. Enzyme abnormalities were correlated neither with haemoglobin level nor with reticulocyte count. They were very similar to those observed in primary acquired dyserythropoietic and preleukaemic states. Their prognostic value and mechanism are discussed. Acquired erythroenzymopathies are much more frequent than the congenital ones. They are encountered in dyserythropoietic states such as primary refractory anaemia and acute or smouldering leukaemia (Boivin et al, 1970, 1975; Dreyfus et al, 1969; Valentine et al, 1973). In recent years many cases have been reported of refractory anaemia and/or leukaemia in patients undergoing chemotherapy for malignant diseases especially myeloma and Hodgkin’s disease (Allen, 1970; Cadman et a l , 1977; Catovsky et all 1971;Gonzales et all 1977;Khaleeli et all 1973; Krulik et al, 1977; Morlock et al, 1977; Penn, 1976; Rosner & Grunwald, 1974; Tchernia et al, 1976); leukaemia has also been described in patients treated with cytostatic drugs for nonmalignant diseases (Blanc et al, 1977; Cobau et al, 1973; Penn, 1976). O u r purpose in this study was to determine whether or not Chemotherapy was able to induce biochemical red cell abnormalities similar to those observed in primary dyserythropoietic states. The results bear out the high incidence of chemotherapy-induced erythroenzymopathies and show that the biochemical abnormalities may be observed before morphological features are apparent. Correspondence: Dr J. Etiemble, Laboratoire d’Enzymologie des Cellules Sanguines, H6pital Beaujou, 92 1 I 8 Clichy Cedex, France. 0007-1048/79/070043 9 I $02 .OO
0I 979 Black well Scientific Publications 391
3 92
1. Etiemble et a1 MATERIALS AND METHODS
Sixty hospitalized patients have been studied: 42 with malignant haemopathies and 18 with cancers other than those of the haematopoietic system. In the first group there were 23 patients with Hodgkin’s disease, 19 with non-Hodgkin lymphoma (lymphocytic, lymphoblastic and immunoblastic sarcoma); the second group included nine patients with breast cancer, three with Ewing sarcoma, three with carcinoma of the digestive tract, two with ovary cancer, one with cancer of the uterus. We have excluded from this study patients with primary bone marrow diseases such as leukaemia and refractory anaemia in which biochemical red cell abnormalities are frequently encountered. No blood transfusion had been given to any of the patients for a t least 3 months before the study. Twenty-six patients were submitted to chemotherapy for 3 months to I year, 3 I between 1 and 5 years, and three between 7 and I I years.
Two control groups have been studied. The first one was composed of 3 0 volunteer blood donors, considered as normal subjects. The second one included 20 patients (seven with Hodgkin’s disease, six with non-Hodgkin malignant lymphoma, seven with cancer) whose red cell enzymes were studied before any treatment was initiated. Most patients were treated with polychemotherapy: 24 with MOPP (Mustard-OncovinProcarbazide-Prednisone), 1 5 with COPP (Cyclophosphamide-Oncovin-ProcarbazidePrednisone), eight with a combination of Adriamycine, Vm26, Cyclophosphamide and Oncovin, four with the same combination excepted Vm 26, two with BACOP (BleomycineAdriamycine-Cyclophosphamide-Oncovin-Prednisone) the others by different but no codified drug combinations. Thirty-one patients had received radiotherapy before chemotherapy. The following red blood cell enzyme activities were studied: ( I ) glycolytic enzymes: hexokinase (Hx),phosphohexose isomerase (PHI), phosphofructokinase (PFK), aldolase (ALD), triose phosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (G3PD), 2-3-diphosphoglycerate mutase (2-3-DPGM), 3-phosphoglycerate kinase (PGK), 3-phosphoglycerate mutase (PGM), enolase (ENOL),pyru’vatekinase (PK); ( 2 ) hexose monophosphate shunt enzymes: glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6-PGD); (3) non-glycolytic enzymes: adenylate kinase (AK), glutathione reductase (GR), glutathione peroxidase (GP). All the enzyme activities were assayed by the methods of Beutler (1975). Fetal haemoglobin level was determined by alkali denaturation in 36 out of60 patients.
RESULTS
Frequency of Enzyme Abnormalities We considered activities below the lowest level in the controls (blood donors) as deficient. No enzymatic deficiency has been observed in patients before treatment (Fig I ) . In patients studied after treatment, we observed enzyme deficiencies in 20 cases (Fig 2), 13 with deficiency of only one activity, five with deficiency of two activities, two with a t least three deficiencies. The most frequently decreased activities were those of G6PD (12 cases), PK (seven cases), PFK (six cases), AK (three cases). Deficiency of glutathione reductase was observed in only one patient. Furthermore, some activities, especially those of GPI and z-j-DPGM, though not
Chemotherapy and Red Cell Enzymes
39 3
deficient, were found in the lowest part of the normality zone (Fig 2 ) . Deficiencies were of moderate grade: assayed activities were generally 50-66% of the normal mean. In contrast, activity of several enzymes was increased in many patients. As shown in Fig 2, this was obvious for ALD, TPI, G6PD, PGK, ENOL and 6PGD; in some casesactivity reached twice the normal mean. Injuence of the Treatment Duration Of 26 patients treated for less than I year, eight had an enzyme deficiency ( 3 1 % ) . O f 3 4 patients who had received chemotherapy for more than I year, enzyme deficiencies were observed in 1 2 (35%). Among 1 3 patients with deficiency ofonly one enzyme activity, six had been treated for less than I year; among the five having deficiency of two activities, three were treated for more than I year; the two patients having three deficiencies had both been treated for more than I year. Influence of Associated Radiotherapy O f 3 1 patients who had been treated previously with radiotherapy, 1 1 had enzyme deficiencies (3 5 %); whilst of 27 who did not receive radiation, nine had enzyme deficiencies (33%).
Influence ofthe Chemotherapeutic Protocols Enzyme deficiencieswere observed in nine out of 24 patients treated with MOPP: seven had G6PD deficiency. Two patients had three deficiencies: PK, PFK and G6PD. Two out of 1 5 patients treated with COPP had enzyme deficiencies: both had a PK deficiency, associated in one with AK deficiency. Four out of eight patients having received AVmCP combination had deficiencies: one of PFK, two of PK and one of G6PD activity associated with AK. Enzyme deficiencies were also observed in the four patients receiving Vm26, 5-Fluorouracil ( 5 FU), Ameticyne associated in one with cyclophosphamide and in the other with cyclophosphamide and Melphalan. Fetal Haemoglobin Level Nine out of 36 patients had an increased fetal haemoglobin, up to 6.2%. Five of them had an enzyme deficiency. Relationship of Enzyme Abnormalities with Haemoglobin Level and Red Cell Morphology About half of the patients had moderate anaemia and haemoglobin level 10-12.5 g/dl.. However, there was no correlation between red cell enzyme deficiencies and haemoglobin level. Reticulocyte count differed from one patient to another according to the time when blood samples were taken with respect to the time of chemotherapy. No correlation was found between the reticulocyte count and the enzyme activities. Macrocytosis with MCV > 9 5 microncubes was found in 27 patients: 1 3 of them had enzyme deficiency; MCV was normal in the other 3 3 patients; and enzyme deficien'cy was present in seven.
J . Etiemble
3 94
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FIG I. Activities of hexokinase (Hx), phosphohexose isomerase (PHI), phosphofructokinase (PFK), aldolase (ALD), triose phosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (G3PD). 2-3-diphosphoglycerate mutase (2-3-DPGM). phosphoglycerate kinase (PCK), phosphoglycerate mutase (PGM), enolase (ENOL), pyruvate kinase (PK), adenylate kinase (AK), glucosed-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD). glutathione reductase (GR) and glutathione peroxidase (GP) in 20 patients before treatment by chemotherapy. The results are expressed in international units /g Hb. Normality zones are defined by the mean I SD.
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FIG 2. Activities of the same enzymes as in Fig I in 60 patients after treatment by chemotherapy.
3 96
J . Etiemble et a1 DISCUSSION
Red blood cell enzyme abnormalities are frequently observed in acute and smouldering leukaemia and in the primary bone marrow insufficiency termed refractory anaemia with or without sideroblastosis. This later condition is often considered as a preleukaemic state. In contrast such abnormalities are very seldom observed in Hodgkin’s disease, non-Hodgkin lymphoma and visceral cancers, as demonstrated by the study of the red cells of such patients before treatment. In recent years many reports incriminate chemotherapy for the development of acute myeloid leukaemia and sideroblastic anaemia in patients treated for malignant or non-malignant disease. The present study shows that erythrocyte enzyme abnormalities are present in about 33 % of the patients treated by various chemotherapies. Such abnormalities are very similar to those observed in primary dyserythropoietic states (Boivin et a l , 1970, 1975; Valentine e f al, 1973) with the same contrast between decreased and increased activities, and involvement of the same enzymes, mainly pyruvate kinase, phosphofructokinase and adeny late kinase. However, we have also found in many patients a decreased G6PD activity which is not frequently observed in primary acquired dyserythropoiesis; differences in the technical assay procedures may be a possible explanation of this fact. As demonstrated by the control group of patients, enzyme abnormalities do not occur o r arc a t least quite exceptional in Hodgkin’s disease, non-Hodgkin lymphoma and cancer before treatment. From this study it is not clear at what time enzyme abnormalities appeared; however, in another group of patients with myeloma or cancer in whom a limited prospective study has been carried out, abnormalities sometimes appeared 3 months after theonset of chemotherapy, which is the time necessary to renew the whole erythrocyte population. Thus the drugs can induce biochemical abnormalities soon after their application (Renoux et al, 1978); furthermore red cell injuries could appear later in the course of long continued chemotherapy. Comparison between patients previously treated o r not treated by radiotherapy did not show any difference in the frequency of blood cell abnormalities. Thus it appears that chemotherapy alone is responsible for their production. One of the most important results of the present study is the discovery of erythrocyte enzyme abnormalities in patients in the absenceof any other haematological sign of dyserythropoiesis. Except for a moderate macrocytosis observed in some cases, erythrocyte morphology, corpuscular constants and reticulocyte counts were normal. Thus, enzyme abnormalities very similar to those observed in so-called ‘preleukaemic states’ could be an early sign of chemotherapy-induced dyserythropoiesis. Further studies and long-continued patient observation are needed to know whether the abnormalities are definitive or transitory and whether or not patients with such abnormalities are statistically more exposed to the risk of leukaemia or refractory anaemia than patients with normal red cell enzyme activities. The mechanism of the chemotherapy-induced enzyme abnormalities is unknown. Frischer & Ahmad (1977) recently noted that BCNU, a nitrosourea derivative, constantly produces an immediate and profound glutathione reductase deficiency by direct interaction with the enzyme protein molecule; BCNU was not used in the present series of patients, but nitrogen mustard could have a similar property (Roth et a l , 1975), although we have not observed
Chemotherapy and Red Cell Enzymes
3 97
significant glutathione reductase deficiency in the patients treated with MOPP. The mechanism of the abnormalities in our series is certainly different; enzyme activity disturbances seem to appear only some months after the treatment was initiated and in some cases to persist for a long time after the drug withdrawal; consequently they cannot be due to the direct action of drugs on the enzyme molecules. We cannot exclude the hypothesis of a metabolic abnormality induced by the drugs leading to a secondary post-synthetic modification of some enzyme proteins (Kahn e f a l , 1976).However, the most probableexplanation is an injury of the bone marrow stem cells inducing the production of abnormal clones and disturbance of protein synthesis: a similar mechanism has been observed in primary acquired dyserythropoiesis such as leukaemias and preleukaemic states (Valentine et a l , 1973). Increased fetal haemoglobin level is a supplementary argument for this hypothesis. For ethical reasons we cannot use drugs separately when drug combinations are proven to be more efficient for therapeutic purposes. As almost all the patients of the present study were treated by drug combinations it has not been possible to distinguish any specific actions of individual drugs. ACKNOWLEDGMENT
This work was supported by a grant of DGRST no. 77.7.1357.
REFERENCES ALLEN, W.S.A. (1970) Acute myeloid leukaemia after treatment with cytostatic agents. (Letter). Lancet, ii, 77s. BEUTLER, E. (1975) Red Cell Metabolism. A Manual of Biochemical Methods, 2nd edn. Grune & Stratton, New York. BLANC, A.P., GASTAUT, J.A., DALIVOUST, P. & CARCASSONNE, Y. (1977) HCmopathies malignes survenant au cours d’un traitement immunosuppressew. Quatre nouvelles observations. Nouvefle Presse Me‘dicale, 6 , 2503. M. (1970) BOIVIN,P., GALAND, C. & AUDOLLEN~, Enzymopathies acquises. I. Anomalies quantitatives observkes dans IOO cas d’hemopathies diverses. Pathologie et Biologie, 18, 175-187. BOIVIN, P., GALAND, C., HAKIN, J. & KAHN,A. (1975) Acquired erythroenzymopathies in blood disorders: study of zoo cases. Britishjournal of Haematolog)’, 31, 53 1-543. CADMAN, E.C., CAPIZZI, R.L. 81 BERTINO, J.R. (1977) Acute nonlymphocytic leukemia: a delayed complication of Hodgkin’s disease therapy: analysis of log cases. Cancer, 40, 1 2 8 ~ ~ 1 2 9 6 . CATOVSKY, D., SHAW,M.T., HOFFBRAND, A.V. 81 DACIE.J.V. (1971) Sideroblastic anaemia and its association with leukaemia and myelomastosis: a report of five cases. British Journal of Haematulogy, 20,385-393,
COBAU, C.D., SHEON,R.P. & KIRSNER, A.B. (1973) Immunosuppressive drugs and acute leukemia. (Letter). Annalr oflnternal Medicine, 79, 131-132. DREYFUS, B., ROCHANT,H. 81 SULTAN,C.P. (1969) AnCmies rkfractaires: enzymopathies acquises des cellules souches himatopoietiques. Nouvelle Revue Frangaise d’He‘matologie, 9, 65-86. FRISCHER, H. & AHMAD, T . (1977) Severe generalized glutathione reductase deficiency after antitumor chemotherapy with B C N U i1.3 bis(chlorethy1)-Initrosourea]._lournal of Laboratory and Clinical Medicine, @, 1080-1091. GONZALES, F., TRUJILLO,J.M. & ALEXANIAN, R. (1977) Acute leukemia in multiple myeloma. Annals of Internal Medicine, 86, 440-443. D. & KAHN,A., MARIE,]., BERNARD, J.F., COITREALI, BOIVIN,P. (1976) Mechanisms of the acquired erythrocyte enzyme deficiencies in blood disease. Clinica Chimica A d a , 71, 379-387. M., KEANE,W.M. 81 LEE, G.R. (1973) KHALEELI, Sideroblastic anemia in multiple myeloma: a preleukemic change. Blood, 41,17-25. KRULIK,M., GROLLEAU, J.Y., AUDEBEXT, A.A., MouJ.L.,H E R V O U ~ , GEOT-MAKTIN, M., HAROUSSEAU, D. & DEBRAY, J. (1977) Leuctmie aigue granuleuse au cows du myklome multiple. A propos de 5 observations. Semaine des Hdpitaux (Paris), 53, 2425.
3 98
1.Etiemble et a1
MORLOCK, G., BATAILLE, R., SANY,J. & SERRE,H.
cases and review of the literature. American joirmal
(1977) Ankmie rifractaire avec mykloblastose pattielle au cows d’un myklome multiple traiti par le Melphalan. Sentaine des Hdpiiaux (Paris), 53, 8 53-8 56. PENN, 1. (1976) Second malignant neoplasms asso-
of Medicine, 57, 927-940.
ciated with immunosuppressive medications. Comer, 37 (Supplement) , I 024- 103 2.
RENOUX.M . , BERNARD, J.F., TORRES, M., SCHLECEL, N., AMAR,M . , LOPEZ,M. & BOIVIN,P. (1978) Erythrocyte abnormalities induced by chemotherapy and radiotherapy: induction ofthe preleukemic states? Scandinavian]ournal ofHaematology, 21, 323-3 32. ROSNER, F. & GRUNWALD, H. (1974) Multiple myeloma terminating in acute leukemia. Report of 12
ROTH, E.F., JR, NAGEL, R.L., NEUMAN, G . , VAN DER HOFF,G., KAPLAN, B.H. & JAFFB, E.R. (197s)Metabolic effects of antisickling amounts of nitrogen and nor-nitrogen mustard on rabbit and human erythrocytes. Blood, 45, 779-788. TCHERNIA, G., MIELOT,F . , SUBTIL, E. & PARMENTIER, C. (1976) Acute myeloblastic leukemia after immunodepressive therapy of primary nonmalignant disease. Blood Cells, 2, 67-Xo. VALENTINE, W.N., KONRAD,P . N . & PAGLIA,D.E. (1973) Dyserythropoiesis, refractory anemia and ‘preleukemia’: metabolic features of the erythrocytes. Blood, 41, 857-875.
Red Blood Cell Enzyme Abnormalities in Patients Treated with Chemotherapy J. ETIEMBLE, J. F. BERNARD, CH. PICAT,D. BELPOMME A N D P. BOIVIN
Laboratoire d’Enzymologie des Cellules Sanguines ( I N S E R M U I ~ O - C N RESR A 573) et Service d’Hkmatologie Clinique, Hapita1 Beaujon, Clichy, France (Received 2 M a y 1978; accepted for publication 1 8 October 1978) SUMMARY. Sixteen red blood cell enzyme activities and fetal haemoglobin level have been assayed in 60 patients treated for haematologic or nonhaematologic malignant diseases with various combinations of cytostatic drugs. Acquired enzyme deficiency was found in 20 patients. The most frequently decreased activities were those of G6PD (12cases), PK (seven cases), PFK (six cases) and AK (three cases). In many patients decreased activity of some enzymes contrasted with increased activity of others such as ALD, TPI, G3PD, PGK, ENOL and 6PGD. The number ofabnormalities seems to be related to the duration of the treatment. Incidence of enzyme deficiencies was similar in patients previously treated or not with radiotherapy. Enzyme abnormalities were correlated neither with haemoglobin level nor with reticulocyte count. They were very similar to those observed in primary acquired dyserythropoietic and preleukaemic states. Their prognostic value and mechanism are discussed. Acquired erythroenzymopathies are much more frequent than the congenital ones. They are encountered in dyserythropoietic states such as primary refractory anaemia and acute or smouldering leukaemia (Boivin et al, 1970, 1975; Dreyfus et al, 1969; Valentine et al, 1973). In recent years many cases have been reported of refractory anaemia and/or leukaemia in patients undergoing chemotherapy for malignant diseases especially myeloma and Hodgkin’s disease (Allen, 1970; Cadman et a l , 1977; Catovsky et all 1971;Gonzales et all 1977;Khaleeli et all 1973; Krulik et al, 1977; Morlock et al, 1977; Penn, 1976; Rosner & Grunwald, 1974; Tchernia et al, 1976); leukaemia has also been described in patients treated with cytostatic drugs for nonmalignant diseases (Blanc et al, 1977; Cobau et al, 1973; Penn, 1976). O u r purpose in this study was to determine whether or not Chemotherapy was able to induce biochemical red cell abnormalities similar to those observed in primary dyserythropoietic states. The results bear out the high incidence of chemotherapy-induced erythroenzymopathies and show that the biochemical abnormalities may be observed before morphological features are apparent. Correspondence: Dr J. Etiemble, Laboratoire d’Enzymologie des Cellules Sanguines, H6pital Beaujou, 92 1 I 8 Clichy Cedex, France. 0007-1048/79/070043 9 I $02 .OO
0I 979 Black well Scientific Publications 391
3 92
1. Etiemble et a1 MATERIALS AND METHODS
Sixty hospitalized patients have been studied: 42 with malignant haemopathies and 18 with cancers other than those of the haematopoietic system. In the first group there were 23 patients with Hodgkin’s disease, 19 with non-Hodgkin lymphoma (lymphocytic, lymphoblastic and immunoblastic sarcoma); the second group included nine patients with breast cancer, three with Ewing sarcoma, three with carcinoma of the digestive tract, two with ovary cancer, one with cancer of the uterus. We have excluded from this study patients with primary bone marrow diseases such as leukaemia and refractory anaemia in which biochemical red cell abnormalities are frequently encountered. No blood transfusion had been given to any of the patients for a t least 3 months before the study. Twenty-six patients were submitted to chemotherapy for 3 months to I year, 3 I between 1 and 5 years, and three between 7 and I I years.
Two control groups have been studied. The first one was composed of 3 0 volunteer blood donors, considered as normal subjects. The second one included 20 patients (seven with Hodgkin’s disease, six with non-Hodgkin malignant lymphoma, seven with cancer) whose red cell enzymes were studied before any treatment was initiated. Most patients were treated with polychemotherapy: 24 with MOPP (Mustard-OncovinProcarbazide-Prednisone), 1 5 with COPP (Cyclophosphamide-Oncovin-ProcarbazidePrednisone), eight with a combination of Adriamycine, Vm26, Cyclophosphamide and Oncovin, four with the same combination excepted Vm 26, two with BACOP (BleomycineAdriamycine-Cyclophosphamide-Oncovin-Prednisone) the others by different but no codified drug combinations. Thirty-one patients had received radiotherapy before chemotherapy. The following red blood cell enzyme activities were studied: ( I ) glycolytic enzymes: hexokinase (Hx),phosphohexose isomerase (PHI), phosphofructokinase (PFK), aldolase (ALD), triose phosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (G3PD), 2-3-diphosphoglycerate mutase (2-3-DPGM), 3-phosphoglycerate kinase (PGK), 3-phosphoglycerate mutase (PGM), enolase (ENOL),pyru’vatekinase (PK); ( 2 ) hexose monophosphate shunt enzymes: glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6-PGD); (3) non-glycolytic enzymes: adenylate kinase (AK), glutathione reductase (GR), glutathione peroxidase (GP). All the enzyme activities were assayed by the methods of Beutler (1975). Fetal haemoglobin level was determined by alkali denaturation in 36 out of60 patients.
RESULTS
Frequency of Enzyme Abnormalities We considered activities below the lowest level in the controls (blood donors) as deficient. No enzymatic deficiency has been observed in patients before treatment (Fig I ) . In patients studied after treatment, we observed enzyme deficiencies in 20 cases (Fig 2), 13 with deficiency of only one activity, five with deficiency of two activities, two with a t least three deficiencies. The most frequently decreased activities were those of G6PD (12 cases), PK (seven cases), PFK (six cases), AK (three cases). Deficiency of glutathione reductase was observed in only one patient. Furthermore, some activities, especially those of GPI and z-j-DPGM, though not
Chemotherapy and Red Cell Enzymes
39 3
deficient, were found in the lowest part of the normality zone (Fig 2 ) . Deficiencies were of moderate grade: assayed activities were generally 50-66% of the normal mean. In contrast, activity of several enzymes was increased in many patients. As shown in Fig 2, this was obvious for ALD, TPI, G6PD, PGK, ENOL and 6PGD; in some casesactivity reached twice the normal mean. Injuence of the Treatment Duration Of 26 patients treated for less than I year, eight had an enzyme deficiency ( 3 1 % ) . O f 3 4 patients who had received chemotherapy for more than I year, enzyme deficiencies were observed in 1 2 (35%). Among 1 3 patients with deficiency ofonly one enzyme activity, six had been treated for less than I year; among the five having deficiency of two activities, three were treated for more than I year; the two patients having three deficiencies had both been treated for more than I year. Influence of Associated Radiotherapy O f 3 1 patients who had been treated previously with radiotherapy, 1 1 had enzyme deficiencies (3 5 %); whilst of 27 who did not receive radiation, nine had enzyme deficiencies (33%).
Influence ofthe Chemotherapeutic Protocols Enzyme deficiencieswere observed in nine out of 24 patients treated with MOPP: seven had G6PD deficiency. Two patients had three deficiencies: PK, PFK and G6PD. Two out of 1 5 patients treated with COPP had enzyme deficiencies: both had a PK deficiency, associated in one with AK deficiency. Four out of eight patients having received AVmCP combination had deficiencies: one of PFK, two of PK and one of G6PD activity associated with AK. Enzyme deficiencies were also observed in the four patients receiving Vm26, 5-Fluorouracil ( 5 FU), Ameticyne associated in one with cyclophosphamide and in the other with cyclophosphamide and Melphalan. Fetal Haemoglobin Level Nine out of 36 patients had an increased fetal haemoglobin, up to 6.2%. Five of them had an enzyme deficiency. Relationship of Enzyme Abnormalities with Haemoglobin Level and Red Cell Morphology About half of the patients had moderate anaemia and haemoglobin level 10-12.5 g/dl.. However, there was no correlation between red cell enzyme deficiencies and haemoglobin level. Reticulocyte count differed from one patient to another according to the time when blood samples were taken with respect to the time of chemotherapy. No correlation was found between the reticulocyte count and the enzyme activities. Macrocytosis with MCV > 9 5 microncubes was found in 27 patients: 1 3 of them had enzyme deficiency; MCV was normal in the other 3 3 patients; and enzyme deficien'cy was present in seven.
J . Etiemble
3 94
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FIG I. Activities of hexokinase (Hx), phosphohexose isomerase (PHI), phosphofructokinase (PFK), aldolase (ALD), triose phosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (G3PD). 2-3-diphosphoglycerate mutase (2-3-DPGM). phosphoglycerate kinase (PCK), phosphoglycerate mutase (PGM), enolase (ENOL), pyruvate kinase (PK), adenylate kinase (AK), glucosed-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD). glutathione reductase (GR) and glutathione peroxidase (GP) in 20 patients before treatment by chemotherapy. The results are expressed in international units /g Hb. Normality zones are defined by the mean I SD.
. .. .. .... .. ......
PHI
HX
PFK
. .' ...*-:. ... .... .......
ALD .*.V
0.
*-*-
50
8 -1 .
*.
*:
.a.
-0 4
'
... ...
.... .:+;:# ....... .....
-5 1
.=
GSPD
2.3DPGM
.
....
*:'
PGK
.. ..
..*.. ::a.
a '
..... ... . .
2
...*. ... ..... .'
:: :*.;* .
ENOL
0 .
21 2 !+&,-
0 .
......
-
-4 -1 .
....
..
.*: *.
110
PGM
.... ....
em
*. :*
150
.
0..
::*.y :.
.*
700
8 .
..
TPI
. ..... ...... . ..... : .-.. ..... . 0..
3
PK
. ..... :.'.
A
190 -
. AK
....
.... . ; . . .::..... : .....:~:-: ..... ... . .... . . **.
*--*
3.5
0..
*;
a .
3 2 -.
6,0
0 .
00 '
-..=**
.:*.
6PGD
. ......
60
.... ...... ....
.*
....... :.*.
...
.a:
b .
...
.='
.b
b:
*-,'
GP
..
7 b : -
4.0
GR
4.5
.
FIG 2. Activities of the same enzymes as in Fig I in 60 patients after treatment by chemotherapy.
3 96
J . Etiemble et a1 DISCUSSION
Red blood cell enzyme abnormalities are frequently observed in acute and smouldering leukaemia and in the primary bone marrow insufficiency termed refractory anaemia with or without sideroblastosis. This later condition is often considered as a preleukaemic state. In contrast such abnormalities are very seldom observed in Hodgkin’s disease, non-Hodgkin lymphoma and visceral cancers, as demonstrated by the study of the red cells of such patients before treatment. In recent years many reports incriminate chemotherapy for the development of acute myeloid leukaemia and sideroblastic anaemia in patients treated for malignant or non-malignant disease. The present study shows that erythrocyte enzyme abnormalities are present in about 33 % of the patients treated by various chemotherapies. Such abnormalities are very similar to those observed in primary dyserythropoietic states (Boivin et a l , 1970, 1975; Valentine e f al, 1973) with the same contrast between decreased and increased activities, and involvement of the same enzymes, mainly pyruvate kinase, phosphofructokinase and adeny late kinase. However, we have also found in many patients a decreased G6PD activity which is not frequently observed in primary acquired dyserythropoiesis; differences in the technical assay procedures may be a possible explanation of this fact. As demonstrated by the control group of patients, enzyme abnormalities do not occur o r arc a t least quite exceptional in Hodgkin’s disease, non-Hodgkin lymphoma and cancer before treatment. From this study it is not clear at what time enzyme abnormalities appeared; however, in another group of patients with myeloma or cancer in whom a limited prospective study has been carried out, abnormalities sometimes appeared 3 months after theonset of chemotherapy, which is the time necessary to renew the whole erythrocyte population. Thus the drugs can induce biochemical abnormalities soon after their application (Renoux et al, 1978); furthermore red cell injuries could appear later in the course of long continued chemotherapy. Comparison between patients previously treated o r not treated by radiotherapy did not show any difference in the frequency of blood cell abnormalities. Thus it appears that chemotherapy alone is responsible for their production. One of the most important results of the present study is the discovery of erythrocyte enzyme abnormalities in patients in the absenceof any other haematological sign of dyserythropoiesis. Except for a moderate macrocytosis observed in some cases, erythrocyte morphology, corpuscular constants and reticulocyte counts were normal. Thus, enzyme abnormalities very similar to those observed in so-called ‘preleukaemic states’ could be an early sign of chemotherapy-induced dyserythropoiesis. Further studies and long-continued patient observation are needed to know whether the abnormalities are definitive or transitory and whether or not patients with such abnormalities are statistically more exposed to the risk of leukaemia or refractory anaemia than patients with normal red cell enzyme activities. The mechanism of the chemotherapy-induced enzyme abnormalities is unknown. Frischer & Ahmad (1977) recently noted that BCNU, a nitrosourea derivative, constantly produces an immediate and profound glutathione reductase deficiency by direct interaction with the enzyme protein molecule; BCNU was not used in the present series of patients, but nitrogen mustard could have a similar property (Roth et a l , 1975), although we have not observed
Chemotherapy and Red Cell Enzymes
3 97
significant glutathione reductase deficiency in the patients treated with MOPP. The mechanism of the abnormalities in our series is certainly different; enzyme activity disturbances seem to appear only some months after the treatment was initiated and in some cases to persist for a long time after the drug withdrawal; consequently they cannot be due to the direct action of drugs on the enzyme molecules. We cannot exclude the hypothesis of a metabolic abnormality induced by the drugs leading to a secondary post-synthetic modification of some enzyme proteins (Kahn e f a l , 1976).However, the most probableexplanation is an injury of the bone marrow stem cells inducing the production of abnormal clones and disturbance of protein synthesis: a similar mechanism has been observed in primary acquired dyserythropoiesis such as leukaemias and preleukaemic states (Valentine et a l , 1973). Increased fetal haemoglobin level is a supplementary argument for this hypothesis. For ethical reasons we cannot use drugs separately when drug combinations are proven to be more efficient for therapeutic purposes. As almost all the patients of the present study were treated by drug combinations it has not been possible to distinguish any specific actions of individual drugs. ACKNOWLEDGMENT
This work was supported by a grant of DGRST no. 77.7.1357.
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