Pnxmghdins @ Longman
Leukotrienes and Essential Group UK Ltd 1990
Fatty Acids (1W)
0952.327&90/W-0199/$10,Cnl
40.199-202
The Effect of Gamma-Linolenic Acid, an in vitro Cytostatic Substance Contained in Evening Primrose Oil, on Primary Liver Cancer. A Double-blind Placebo Controlled Trial C. F. van der Merwe*, J. Booyens+, H. F. Jouberts and C. A. van der Merwe$. Medical University of Southern Africa, Departments of *Internal Medicine, Division Gastroenterology, +Physiology and *Chemical Pathology, Post Ofice Medunsa 0204, South Africa. “The Institute for Biostatistics of the S.A. Medical Research Council, H. F. Verwoerd Hospital, Private Bag X169, Pretoria 0001, South Africa ABSTRACT.
The cytostatic effects of essential fatty acid metabolic intermediates and of some prostaglandms and leukotrienes in vitro have been extensively documented. The essential fatty acids (EFAs) exhibit no side-effects when taken as a die&y supplement, even in large doses. Primary Liver Cancer (PLC) is a fatal disease in our area as it is always multifocal in nature. In vitro studies have shown a cytostatic effect of gamma-linolenic acid (GLA) on primary liver cancer cells. In a double-blind placebo controlled trial, using Evening Primrose Oil (as a source of GLA) as a dietary supplement in PLC patients, no statistically significant effect was observed on survival time or liver size. There was however a statistical significant beneficial effect on Gamma Glutamyl transferase values as a measure of liver function. No side-effects were observed. The large size of tumour and the low doses of GLA used in this trial probably explain the lack of significant effect on survival times.
INTRODUCTION
MATERIALS AND METHODS
Primary Liver Cancer is a rather common disease in Southern Africa with an incidence of approximately 11 per 100,000 per year in the black population (8). Because of the typical multifocal nature of the lesion, surgical resection is never possible. The response to chemotherapy is poor. When toxicity is borne in mind, no chemotherapy regime (5F.U. Methyl-CCNU, Adreomycin, Streptozotocin or combinations of these) under investigation, can be recommended as a standard treatment (6). The mean survival of untreated cases was found to be 42 days after diagnosis in our hospital (9). The cytostatic effects of essential fatty acids and their metabolic intermediates on various malignant cell lines in vitro have been described extensively (2, 3, 4, 7). One of the cell lines tested in our laboratory was the Alexander Hepatoma cell line (1, 5). Its growth was suppressed significantly by gamma-linolenic acid, a metabolite of linoleic acid. Because of the absence of side-effects of Efamol (Evening Primrose Oil/EPO) a naturally occurring substance, containing about 70% linoleic acid and 9% gamma-linolenic acid (GLA), we decided to supplement the diet of our patients with this substance. EPO is one of the few nutritional sources of GLA.
Criteria for inclusion into the trial were: a histological diagnosis of primary liver cancer in a mobile patient able to care for his or her daily activities (Eastern Co-Op. Oncology Group Performance Status 2 or better) with an expected survival of more then 14 days. Patients refusing further supplementation and patients deemed not able to take the supplementation any longer could be removed from the trial according to the protocol. No patient in either group refused further treatment or was excluded for other reasons. Hepatomgealy was utilized in all patients as an objective measure, as it was usually more than 4 ems below the xiphoid or right costal margin on quiet respiration. At the start of the supplementation, the following determinations were performed: patients’ weight, liver size in centimetres below the costal margin, full blood count and haemoglobin, urea and electrolytes, bilirubin, total protein, albumin, alkaline phosphatase, gamma-glutamyl transferase (G.G.T.) and alpha fetoprotein. These estimations were repeated 2-weekly. Subjective assessment of patients’ well being was measured on a scale of 10 ems from best possible to worst possible. The patient was asked to mark his 199
PROST:
B
200
Prostaglandins Leukotrienes and Essential Fatty Acids
condition on this scale every 2 weeks, when coming to the clinic for assessment. A total of 62 patients, participating voluntarily, were randomly assigned to treatment in a doubleblind fashion using published random-number tables with the constraint that within each list of 6 letters, 3 would refer to the active substance and 3 to the placebo. Active treatment was Efamol (36 capsules per day), a variety of evening primrose seed oil derived from strains of the plant specially selected to yield oil of constant composition. It was supplied in the form of soft gelatine capsules containing 500 mg oil. Identical appearing capsules containing olive oil, 500 mg, were used as placebo (36 capsules per day). The first patient entered the trial on the 30th of September 1984 and the trial was completed on the 9th of September 1986. The statistical methods used were those of Siegel (8, 9) for non-parametric statistics and the Wilcoxon sign ranked Test. The statistical differences in survival between active and placebo treatment were determined using the Generalised Wilcoxon (Breslow) and Generalised Savage (Mantel-Cox) tests in the BMDP Statistical Software Incorporated Life tables and Survival Functions Program.
tical analysis of the results revealed no significant difference between the groups. Alkaline phosphatase (AlkPhos) and GammaGlutamyl-Transaminase (GGT) are the most sensitive liver function tests denoting hepatic space occupying lesions such as hepatoma. GGT values decreased during supplementation in 7 cases on EPO and 2 cases on placebo. Statistical analysis of these results revealed a significant difference between the two groups (p = 0.0192). Survival Survival is a key measure of efficacy of treatment in primary liver cancer. The mean survival time for the EPO supplemented group was 58 days and for the placebo group 42 days. The Figure depicts the cumulative proportion survival in both groups. The differences between the 2 groups were not significant (p = 0,45 Wilcoxon and 0,26 Savage).
_
E.P.O. PLACEBO p = 0,26
RESULTS 62 patients were entered into the trial, 31 into the placebo and 31 into the EPO group. Nine cases were lost to follow-up, 4 in the placebo and 5 in the EPO group. The groups were similar with respect to age, sex and tumour size. The high number of cases lost to follow-up is due to the rural habitation of many cases, poor transport facilities and defeatist attitudes of many of our cases. The Table gives the data of all patients on subjective response, liver size and GGT values.
10 10
M
lm TIME (DAYS)
180
200 220
Side-effects No side-effects were reported in any of the patients.
DISCUSSION Response 1. Subjective responses There were 4 cases showing subjective improvement in the treatment group and three in the placebo group. However, statistical analysis of the results revealed no significant difference between the two groups. 2. Objective responses Objective responses to supplementation were seen in liver function and liver size although these were transient and only observed in the first few weeks of the trial. Liver size in centimetres decreased in 6 cases on EPO (19%) and 3 (cases on placebo (10%). Statis-
Gamma-linolenic acid (GLA) has been shown to have cytostatic activity in vitro. In this clinical study, the effect of GLA (as Efamol) on survival in primary liver cancer was not significantly better than placebo. However, the survival curve showed that EPO was always better than placebo and as far as objective and subjective measurements were concerned, the Efamol group of patients showed better results than the placebo group. A factor of major importance is the freedom from side-effects of Efamol .
Efamol supplementation had a significant effect on G.G.T. values, showing a decrease towards normal which must be interpreted as showing an effect on the tumour, compared to placebo.
Effect of Gamma-Linolenic Table E.P.O./Placebo Patient
trial patient data. Group epo/placebo
ew pbo Pbo Pbo epo 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62
Acid on Primary Liver Cancer
epo epo epo
epo pbo Pbo pbo pbo epo pbo pbo epo epo epo cpo pbo pbo epo pbo pbo pbo pbo cpo cpo epo epo epo pbo epo pbo pbo pbo pbo pbo epo epo cpo pbo cpo cpo pbo pbo epo epo epo epo pbo pbo pbo cpo pbo pbo pbo cpo epo pbo cpo
Wellbeing in ems before after
Liver Size beffr; ems)
8.0 1.0 5.0 0.9 7.2 7.0 0.8 2.5 4.8 3.5 5.0 5.5 3.5 7.0 4.8 3.5 9.5 9.5 6.8 6.0 4.8 3.0 2.5 4.5 1.5 0.8 9.8 9.7 9.8 0.6 5.4 3.5 4.5 9.3 8.0 6.4 9.7 6.2 9.8 9.3 9.4 9.5 9.8 3.8 5.0 8.5 6.0 7.0
19.5 10.0 6.0 4.0 8.0 8.0 8.0 9.0 5.6 11.0 0.0 6.0 2.0 8.0 7.0 7.5 6.0 7.5 7.0 8.0 10.0 0.0 6.0 8.0 0.0 6.0 7.0
8.0
8.5 5.0 S.8 7.2 7.2 7.5 7.0 6.2 6.5 6.5 8.0 6.2 5.0
8.2 2.5 5.0 1.4 7.2 8.5 6.8 2.5 3.5 5.5 4.4 7.5 6.4
6.8 8.0 4.8 2.5 4.5 1.5 0.8 9.8 9.7 9.8 0.6 4.3 3.5 4.5 9.5 8.6 6.6 9.9 8.0 9.8 9.4 9.5 6.3 7.4 5.5 9.4 6.0 5.0 8.0 8.9 5.0
4.2 5.5 8.0 7.2 6.2 6.5 5.4 3.5 5.0
after
0.0
14.0 6.0 13.0 4.5 8.0 20.0 10.5 7.0 7.0 6.0 7.0 22.0 7.0 7.0 2.0 14.0 2.0 10.0 10.0 7.0 7.0 17.5 4.5 15.0 15.0 5.0 6.4 6.0 10.0 7.7 10.0 4.0 9.0 5.0
16.0 8.0 6.0 5.0 8.0 7.0 13.0 11.0
G.G.T. I.U./L before after 2045 51
1370
389
400
161 56
236 122 109
36s96 11.0 6.0 9.0 8.0 7.0
279 174 300 1145
279 229 135 1281
2.0 8.0 10.0
84 202 84
10s 23 80
6.0 8.0 0.0 4.0 14.0 0.0 14.0 6.0 12.0 9.0 10.0 19.0 10.5 7.0 7.0 6.0 7.0
532 347 492 116 171
548 361 499 289 216
501 575 271 110 778 614 18 513 68 40 209 149 504 272 153 318
515 600 299
7.0 7.0 3.0 16.5 2.0 10.0 7.0 8.5 7.0 17.5 4.5 15.0 15.0 5.0 6.0 10.0 7.7 10.0 0.0 5.0
1532 589 26 546 68 271 486 298 54 387
415 157 138 398
415 165 160 486
347 185 617 348 404 157 49
359 185
265 374 109 151
446 234 7s 259 200
The before score represents the value immediately prior to starting the trial, whereas the after score represents the last available value for that patient.
201
202
+staglandins
Leukotrienes and Essential Fatty Acids
It must be remembered that Efamol fatty acids contain only 9% GLA. When allowance is made for the glycerol a daily intake of 36 capsules of Efamol provides only 1,44 g GLA. If one takes into account that some patients had up to 3 kg of tumour in their liver, the lack of an overall response to Efamol is understandable. It is possible that higher doses of GLA will have a more profound effect on this tumour. Efforts are being made to produce a pure GLA for this purpose. The average survival of 42 days found in the placebo group, equals the 42 days average found for a group of 46 untreated cases in our hospital in an earlier study (9). References 1. Booyens J, Dippenaar N, Fabbri D et. al. Some effects of linoleic acid and gamma-linolenic acid on the proliferation of human hepatoma cells in culture. S Afr Med J 65: 607-612, 1984. 2. Botha J H. Robinson K M. Resoonse of human mammary cell lines to gamma-liiolenic acid, dihomo-gamma-linolenic acid and ethanol. S Afr J Science 82: 43, 1986.
3 Bregman M D, Meyskens F L. Inhibition of human malignant melanoma colony-forming cells in vitro by prostaglandin A,. Cancer Research 43: 1642-1645, 1983. 4 Dippenaar N. Booyens J. Fabbri D et. al. The reversibility of cancer. S Afr Med J 62: 505-509, 1982. 5. Dippenaar N, Booyens J, Fabbri D et. al. The reversibility of cancer: Evidence that malignancy in human hepatoma cells is gamma-linolenic acid deficiency-dependent. S Afr Med J 62: 683-685. 1982. 6. Falkson G. Macintyre J M, Moertel C H et. al. Primary liver cancer. An eastern co-operative oncology group trial. Cancer 54: 970-977, 1984. 7. Leary W P, Robinson K M, Booyens J, Dippenaar N. Some effects of gamma-linolenic acid on cultured human oesophageal carcinoma cells. S Afr Med J 62: 681-683, 1982. 8. Siegel S. non parametric statistics for the behavioural Sciences. MC Craw-Hill, New York p. 68-75. 9. Idem. p. 75-83. 10. Van dir Merwe CF. Primary liver cancer in the Transvaal. Henato-Gastroenteroloeica 28: 233-235. 1981. ’ 11. Van der Merwe C F. The reversibility of cancer. S Afr Med J 65: 712. 1984.
Leukotrienes and Essential Group UK Ltd 1990
Fatty Acids (1W)
0952.327&90/W-0199/$10,Cnl
40.199-202
The Effect of Gamma-Linolenic Acid, an in vitro Cytostatic Substance Contained in Evening Primrose Oil, on Primary Liver Cancer. A Double-blind Placebo Controlled Trial C. F. van der Merwe*, J. Booyens+, H. F. Jouberts and C. A. van der Merwe$. Medical University of Southern Africa, Departments of *Internal Medicine, Division Gastroenterology, +Physiology and *Chemical Pathology, Post Ofice Medunsa 0204, South Africa. “The Institute for Biostatistics of the S.A. Medical Research Council, H. F. Verwoerd Hospital, Private Bag X169, Pretoria 0001, South Africa ABSTRACT.
The cytostatic effects of essential fatty acid metabolic intermediates and of some prostaglandms and leukotrienes in vitro have been extensively documented. The essential fatty acids (EFAs) exhibit no side-effects when taken as a die&y supplement, even in large doses. Primary Liver Cancer (PLC) is a fatal disease in our area as it is always multifocal in nature. In vitro studies have shown a cytostatic effect of gamma-linolenic acid (GLA) on primary liver cancer cells. In a double-blind placebo controlled trial, using Evening Primrose Oil (as a source of GLA) as a dietary supplement in PLC patients, no statistically significant effect was observed on survival time or liver size. There was however a statistical significant beneficial effect on Gamma Glutamyl transferase values as a measure of liver function. No side-effects were observed. The large size of tumour and the low doses of GLA used in this trial probably explain the lack of significant effect on survival times.
INTRODUCTION
MATERIALS AND METHODS
Primary Liver Cancer is a rather common disease in Southern Africa with an incidence of approximately 11 per 100,000 per year in the black population (8). Because of the typical multifocal nature of the lesion, surgical resection is never possible. The response to chemotherapy is poor. When toxicity is borne in mind, no chemotherapy regime (5F.U. Methyl-CCNU, Adreomycin, Streptozotocin or combinations of these) under investigation, can be recommended as a standard treatment (6). The mean survival of untreated cases was found to be 42 days after diagnosis in our hospital (9). The cytostatic effects of essential fatty acids and their metabolic intermediates on various malignant cell lines in vitro have been described extensively (2, 3, 4, 7). One of the cell lines tested in our laboratory was the Alexander Hepatoma cell line (1, 5). Its growth was suppressed significantly by gamma-linolenic acid, a metabolite of linoleic acid. Because of the absence of side-effects of Efamol (Evening Primrose Oil/EPO) a naturally occurring substance, containing about 70% linoleic acid and 9% gamma-linolenic acid (GLA), we decided to supplement the diet of our patients with this substance. EPO is one of the few nutritional sources of GLA.
Criteria for inclusion into the trial were: a histological diagnosis of primary liver cancer in a mobile patient able to care for his or her daily activities (Eastern Co-Op. Oncology Group Performance Status 2 or better) with an expected survival of more then 14 days. Patients refusing further supplementation and patients deemed not able to take the supplementation any longer could be removed from the trial according to the protocol. No patient in either group refused further treatment or was excluded for other reasons. Hepatomgealy was utilized in all patients as an objective measure, as it was usually more than 4 ems below the xiphoid or right costal margin on quiet respiration. At the start of the supplementation, the following determinations were performed: patients’ weight, liver size in centimetres below the costal margin, full blood count and haemoglobin, urea and electrolytes, bilirubin, total protein, albumin, alkaline phosphatase, gamma-glutamyl transferase (G.G.T.) and alpha fetoprotein. These estimations were repeated 2-weekly. Subjective assessment of patients’ well being was measured on a scale of 10 ems from best possible to worst possible. The patient was asked to mark his 199
PROST:
B
200
Prostaglandins Leukotrienes and Essential Fatty Acids
condition on this scale every 2 weeks, when coming to the clinic for assessment. A total of 62 patients, participating voluntarily, were randomly assigned to treatment in a doubleblind fashion using published random-number tables with the constraint that within each list of 6 letters, 3 would refer to the active substance and 3 to the placebo. Active treatment was Efamol (36 capsules per day), a variety of evening primrose seed oil derived from strains of the plant specially selected to yield oil of constant composition. It was supplied in the form of soft gelatine capsules containing 500 mg oil. Identical appearing capsules containing olive oil, 500 mg, were used as placebo (36 capsules per day). The first patient entered the trial on the 30th of September 1984 and the trial was completed on the 9th of September 1986. The statistical methods used were those of Siegel (8, 9) for non-parametric statistics and the Wilcoxon sign ranked Test. The statistical differences in survival between active and placebo treatment were determined using the Generalised Wilcoxon (Breslow) and Generalised Savage (Mantel-Cox) tests in the BMDP Statistical Software Incorporated Life tables and Survival Functions Program.
tical analysis of the results revealed no significant difference between the groups. Alkaline phosphatase (AlkPhos) and GammaGlutamyl-Transaminase (GGT) are the most sensitive liver function tests denoting hepatic space occupying lesions such as hepatoma. GGT values decreased during supplementation in 7 cases on EPO and 2 cases on placebo. Statistical analysis of these results revealed a significant difference between the two groups (p = 0.0192). Survival Survival is a key measure of efficacy of treatment in primary liver cancer. The mean survival time for the EPO supplemented group was 58 days and for the placebo group 42 days. The Figure depicts the cumulative proportion survival in both groups. The differences between the 2 groups were not significant (p = 0,45 Wilcoxon and 0,26 Savage).
_
E.P.O. PLACEBO p = 0,26
RESULTS 62 patients were entered into the trial, 31 into the placebo and 31 into the EPO group. Nine cases were lost to follow-up, 4 in the placebo and 5 in the EPO group. The groups were similar with respect to age, sex and tumour size. The high number of cases lost to follow-up is due to the rural habitation of many cases, poor transport facilities and defeatist attitudes of many of our cases. The Table gives the data of all patients on subjective response, liver size and GGT values.
10 10
M
lm TIME (DAYS)
180
200 220
Side-effects No side-effects were reported in any of the patients.
DISCUSSION Response 1. Subjective responses There were 4 cases showing subjective improvement in the treatment group and three in the placebo group. However, statistical analysis of the results revealed no significant difference between the two groups. 2. Objective responses Objective responses to supplementation were seen in liver function and liver size although these were transient and only observed in the first few weeks of the trial. Liver size in centimetres decreased in 6 cases on EPO (19%) and 3 (cases on placebo (10%). Statis-
Gamma-linolenic acid (GLA) has been shown to have cytostatic activity in vitro. In this clinical study, the effect of GLA (as Efamol) on survival in primary liver cancer was not significantly better than placebo. However, the survival curve showed that EPO was always better than placebo and as far as objective and subjective measurements were concerned, the Efamol group of patients showed better results than the placebo group. A factor of major importance is the freedom from side-effects of Efamol .
Efamol supplementation had a significant effect on G.G.T. values, showing a decrease towards normal which must be interpreted as showing an effect on the tumour, compared to placebo.
Effect of Gamma-Linolenic Table E.P.O./Placebo Patient
trial patient data. Group epo/placebo
ew pbo Pbo Pbo epo 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62
Acid on Primary Liver Cancer
epo epo epo
epo pbo Pbo pbo pbo epo pbo pbo epo epo epo cpo pbo pbo epo pbo pbo pbo pbo cpo cpo epo epo epo pbo epo pbo pbo pbo pbo pbo epo epo cpo pbo cpo cpo pbo pbo epo epo epo epo pbo pbo pbo cpo pbo pbo pbo cpo epo pbo cpo
Wellbeing in ems before after
Liver Size beffr; ems)
8.0 1.0 5.0 0.9 7.2 7.0 0.8 2.5 4.8 3.5 5.0 5.5 3.5 7.0 4.8 3.5 9.5 9.5 6.8 6.0 4.8 3.0 2.5 4.5 1.5 0.8 9.8 9.7 9.8 0.6 5.4 3.5 4.5 9.3 8.0 6.4 9.7 6.2 9.8 9.3 9.4 9.5 9.8 3.8 5.0 8.5 6.0 7.0
19.5 10.0 6.0 4.0 8.0 8.0 8.0 9.0 5.6 11.0 0.0 6.0 2.0 8.0 7.0 7.5 6.0 7.5 7.0 8.0 10.0 0.0 6.0 8.0 0.0 6.0 7.0
8.0
8.5 5.0 S.8 7.2 7.2 7.5 7.0 6.2 6.5 6.5 8.0 6.2 5.0
8.2 2.5 5.0 1.4 7.2 8.5 6.8 2.5 3.5 5.5 4.4 7.5 6.4
6.8 8.0 4.8 2.5 4.5 1.5 0.8 9.8 9.7 9.8 0.6 4.3 3.5 4.5 9.5 8.6 6.6 9.9 8.0 9.8 9.4 9.5 6.3 7.4 5.5 9.4 6.0 5.0 8.0 8.9 5.0
4.2 5.5 8.0 7.2 6.2 6.5 5.4 3.5 5.0
after
0.0
14.0 6.0 13.0 4.5 8.0 20.0 10.5 7.0 7.0 6.0 7.0 22.0 7.0 7.0 2.0 14.0 2.0 10.0 10.0 7.0 7.0 17.5 4.5 15.0 15.0 5.0 6.4 6.0 10.0 7.7 10.0 4.0 9.0 5.0
16.0 8.0 6.0 5.0 8.0 7.0 13.0 11.0
G.G.T. I.U./L before after 2045 51
1370
389
400
161 56
236 122 109
36s96 11.0 6.0 9.0 8.0 7.0
279 174 300 1145
279 229 135 1281
2.0 8.0 10.0
84 202 84
10s 23 80
6.0 8.0 0.0 4.0 14.0 0.0 14.0 6.0 12.0 9.0 10.0 19.0 10.5 7.0 7.0 6.0 7.0
532 347 492 116 171
548 361 499 289 216
501 575 271 110 778 614 18 513 68 40 209 149 504 272 153 318
515 600 299
7.0 7.0 3.0 16.5 2.0 10.0 7.0 8.5 7.0 17.5 4.5 15.0 15.0 5.0 6.0 10.0 7.7 10.0 0.0 5.0
1532 589 26 546 68 271 486 298 54 387
415 157 138 398
415 165 160 486
347 185 617 348 404 157 49
359 185
265 374 109 151
446 234 7s 259 200
The before score represents the value immediately prior to starting the trial, whereas the after score represents the last available value for that patient.
201
202
+staglandins
Leukotrienes and Essential Fatty Acids
It must be remembered that Efamol fatty acids contain only 9% GLA. When allowance is made for the glycerol a daily intake of 36 capsules of Efamol provides only 1,44 g GLA. If one takes into account that some patients had up to 3 kg of tumour in their liver, the lack of an overall response to Efamol is understandable. It is possible that higher doses of GLA will have a more profound effect on this tumour. Efforts are being made to produce a pure GLA for this purpose. The average survival of 42 days found in the placebo group, equals the 42 days average found for a group of 46 untreated cases in our hospital in an earlier study (9). References 1. Booyens J, Dippenaar N, Fabbri D et. al. Some effects of linoleic acid and gamma-linolenic acid on the proliferation of human hepatoma cells in culture. S Afr Med J 65: 607-612, 1984. 2. Botha J H. Robinson K M. Resoonse of human mammary cell lines to gamma-liiolenic acid, dihomo-gamma-linolenic acid and ethanol. S Afr J Science 82: 43, 1986.
3 Bregman M D, Meyskens F L. Inhibition of human malignant melanoma colony-forming cells in vitro by prostaglandin A,. Cancer Research 43: 1642-1645, 1983. 4 Dippenaar N. Booyens J. Fabbri D et. al. The reversibility of cancer. S Afr Med J 62: 505-509, 1982. 5. Dippenaar N, Booyens J, Fabbri D et. al. The reversibility of cancer: Evidence that malignancy in human hepatoma cells is gamma-linolenic acid deficiency-dependent. S Afr Med J 62: 683-685. 1982. 6. Falkson G. Macintyre J M, Moertel C H et. al. Primary liver cancer. An eastern co-operative oncology group trial. Cancer 54: 970-977, 1984. 7. Leary W P, Robinson K M, Booyens J, Dippenaar N. Some effects of gamma-linolenic acid on cultured human oesophageal carcinoma cells. S Afr Med J 62: 681-683, 1982. 8. Siegel S. non parametric statistics for the behavioural Sciences. MC Craw-Hill, New York p. 68-75. 9. Idem. p. 75-83. 10. Van dir Merwe CF. Primary liver cancer in the Transvaal. Henato-Gastroenteroloeica 28: 233-235. 1981. ’ 11. Van der Merwe C F. The reversibility of cancer. S Afr Med J 65: 712. 1984.
