Europ..7: Cancer Vol. 14, pp. 183-187. Pergamon Press 1978. Printed in Great Britain
Enzymes, Lymph Node Involvement and Possible Prognosis in Human Breast Cancer N. DESHPANDE, IRENE MITCHELL and ROSEMARY MILLIS* Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, England and *Hedley Atkins Unit, New Cross Hospital, London SE14 5ER, England
Abstract--The activities of seven enzymes associatedwith carbohydrate metabolism were measured in tumoursfrom patients with primary carcinomas and the results were analysed in three different ways, i.e. according to tissue weight and enzyme/6PGDH and enzyme/PFK ratios. The tumours were then grouped together according to the number of nodesfound to contain metastases and thefollowing significant differences were observed. 1. Significantly lower activity of e-G P D H was observed in tumoursfrom patients in whomfour or more lymph nodes werefound to contain metastatic deposits than in those with no nodal involvement. 2. Enzyme/6PGD H and enzyme/PFK ratios were significantly lower in tumoursfrom patients withfour o÷more nodes involved than in those with no nodal involvement. Possibleprognostic significance of these enzymes is discussed.
INTRODUCTION
cinoma may be related to the rate of glycolysis and therefore by measuring the activities of certain enzymes it might be possible to predict the clinical course of the disease [5-7]. If so, they should correlate~ith the recurrence and survival rates. It is well known that in women with primary breast cancer, the disease-free interval and 5 yr survival is greatly reduced if lymph nodes are found to contain metastatic deposits at the time ofmastectomy [8, 9]. Therefore, analysis of the results obtained on enzyme activities in the primary carcinomas related to the presence or absence of nodal involvement at the time of mastectomy might give some insight into the usefulness of these measurements in prognosis. This paper reports the findings after such an analysis.
IT IS generally accepted than an early indication of the clinical course of any carcinoma will greatly assist clinicians in deciding on a suitable treatment. A considerable effort therefore is being made to devise biochemical parameters for this purpose. It is hoped that carcinomas will either secrete specific factor(s) which are not present in non-cancerous women or alter the body pool concentrations of parameter (s) normally present so that there are significant differences between the two categories. These hypotheses are then extended in clinical studies to test whether such parameter (s) can correlate with the prognosis. In no field is this more relevant than in carcinoma of the breast. Although the ideal factor(s) for this purpose has not been observed, several workers have attempted to investigate hormones, tumour antigens, proteins and enzymes as likely candidates and found some abnormalities in patients with breast carcinoma [1-4]. In recent studies activities of enzymes associated with carbohydrate metabolism in primary breast carcinomas have been measured to test the hypothesis that the energy requirements of a rapidly growing car-
MATERIAL AND METHODS All the chemicals used in the study were purchased from British Drug Houses Ltd. and were of analytical grade, a-GPDH, TPI, G6PDH, aldolase, NADH, NADP +, ATP, F-6-P, G-6-P, 6PG and pyruvate were purchased from Boehringer Mannheim. G-1-P was purchased from Koch-Light Ltd. DHAP was prepared from the dicyclohexylammonium salt of the dimethylketal according to the manufacturer's (Boehringer Mannheim)
Accepted 15 September 1977. 183
184
N. Deshpande, Irene Mitchell and Rosemary Millis
instructions.* Primary tumours were obtained from patients with carcinoma of the breast undergoing mastectomy. The malignancy of the tumours was confirmed by histological examination. The tissues were wrapped in aluminium foil, frozen on solid carbon dioxide and transferred to the laboratory where they remained frozen (-20°C) until further processing. All the tissues taken for histology were processed routinely and section from two levels of each lymph node block examined in order to increase the chances of finding small deposits of metastatic tumour. In this series an average of 23 axillary lymph nodes (range 8-58) were found in radical mastectomy specimens. The tissues were cleared of any surrounding fat, weighed and cut into small pieces. The semifrozen tissues were then homogenised in a Silverson homogeniser according to the procedure o f Shonk and Boxer [10]. The homogenates were centrifuged (800g for 15min) at 4°C, the cytosol fraction was decanted off and used as a source of enzymes. A 5% solution of tumours was used in these estimations. The activities of PFK, G6PDH, 6PGDH, PGM, LDH, ~-GPDH and PHI were measured. Enzyme assays were performed in a Beckman recording spectrophotometer according to the procedure of Shonk and Boxer [10], with the exception that one minute readings were recorded automatically for five minutes and a mean of the last four was taken as an average change in optical density (O.D.). The results were calculated on the basis of units (U) of enzyme activity per gram wet weight of tissue. A unit is defined as that amount of enzyme which will catalyse the transformation of 1/tmole ofsubstrate per minute. The results were analysed in three different ways: as units of enzyme activity/g and as enzyme/6PGDH and enzyme/PFK ratios. They are presented as mean _ S.D. The means were tested for significance by a Student's '/'-test.
RESULTS This study is based on a total of 95 patients, 47 of these were free of nodal involvement. O f the remainder there were 10 patients with only one involved lymph node, 8 with two, 3 with three and 27 in whom four or more nodes were involved. For the purpose of analysis, patients with two or three involved nodes were grouped together. The values (U/g) obtained in these four categories are shown in Table 1. There were no statistically significant differences in the activities ofG6PDH, 6PGDH, PGM, L D H and PHI between patients with no node involvement and those in the one, 2 + 3 or 4 + categories. 0~-GPDH activities were significantly lower in patients in the 4 + category than in those in whom no nodes were found to contain metastases. Further analyses of the results on the basis of enzyme/6PGDH and enzyme / P F K ratios are shown in Tables 2 and 3, respectively. In both cases significantly higher c~G P D H / 6 P G D H and 0~--GPDH/PFK ratios were observed in tumours from patients with no nodal involvement than those in whom four or more nodes were found to contain metastatic deposits. Furthermore, ~-GPDH/PFK ratios in tumours from patients who had two or three involved nodes were higher than those with four or more involved nodes.
DISCUSSION Recurrence and survival after mastectomv are usually considered to be dependent on certain clinical factors such as size of carcinoma, histology, anatomic type, grade of malignancy and lymph node involvement. The most closely related amongst these variables was the spread of the disease to the surrounding lymph nodes. Fisher and his colleagues [8] have shown tlaat after surgical removal of the breast the 5yr survival rate in patients with localised disease and O/ absence of nodal involvement is 76/o which drops to -rA5°/,owhen up to three nodes are involved and 31°L when four or more are found to contain metastatic deposits. The same also applies to recurrence rates. In the absence of axillary metastases the 5 yr recurrence rate is 21 °/o, which rises to 81 o.Ljif four or more nodes are involved. Similar findings have been reported by others [11, 12]. However, these are gross classifications ancl as has been pointed out t3y Gutler and Myers [11] the patients falling into the same prognostic class show marked variation in clinical characteristics. Therefore, further classification •
*The followingabbreviations are used in this paper. Enzymes--~-GPDH, ~-glycerolphosphate dehydrogenase;TPI, triosephosphateisomerase;G6PDH,glucose6-phosphate dehydrogenase;PFK, phosphofructokinase; 6PGDH, 6-phosphoglueonate dehydrogenase; PGM, phosphoglucomutase;LDH, lactatedehydrogenase;PHI, phosphohexose isomerase. Co-enzymes--NADH, reduced nicotinamide adenine dinucleotide; NADP÷, nicotinamide adenine dinucleotide phosphate; ATP, adenosine triphosphate. Substrates--F-6:P, fructose-6-phosphate;G-6-P, glucose6-phosphate; 6PG, 6-phosphogluconate; G-l-P, glucose1-phosphate; DHAP, dihydroxyacetonephosphate.
Enzymes, Lymph Node Involvement and Possible Prognosis
185
Table 1. Enzyme activities (#mole of pyridine nucleotide reduced or oxidised/min/g tissue) in human breast carcinomas. The tumours were separated according to the number of lymph nodesfound to contain metastatic deposits 0
1
2+3
0.057 + 0.044* 0.197+_0.149 0.132_+0.091 1.396 +_0.829 7.019 _ 4.781 0.310 _+0.249* 7.859 +_4.337
0.093 + 0.057 0.212+_0.110 0.134+_0.064 1.466 _+0.698 6.939 +- 3.072 0.214_+0.192 8.027 +- 3.655
0.060 _ 0.040 0.150_+0.090 0.130+_0.070 1.560 -+0.780 8.950 _+6.530 0.260 +-0.130 7.270 +-3.680
4+
Enzyme PFK G6PDH 6PGDH PGM LDH e-GPDH PHI
0.094 +-0.079 0.178_+0.11.9 0.137+_0.065 1.339 _+0.688 8.582 +- 3.982 0.182 +-0.168 8.868 +-4.406
O~;-No nodes involved; 1--One node involved; 2 + 3--Two or three nodes involved; 4 + - - F o u r or more nodes involved. *Significant differences between 0 and 4 + P
Enzymes, Lymph Node Involvement and Possible Prognosis in Human Breast Cancer N. DESHPANDE, IRENE MITCHELL and ROSEMARY MILLIS* Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, England and *Hedley Atkins Unit, New Cross Hospital, London SE14 5ER, England
Abstract--The activities of seven enzymes associatedwith carbohydrate metabolism were measured in tumoursfrom patients with primary carcinomas and the results were analysed in three different ways, i.e. according to tissue weight and enzyme/6PGDH and enzyme/PFK ratios. The tumours were then grouped together according to the number of nodesfound to contain metastases and thefollowing significant differences were observed. 1. Significantly lower activity of e-G P D H was observed in tumoursfrom patients in whomfour or more lymph nodes werefound to contain metastatic deposits than in those with no nodal involvement. 2. Enzyme/6PGD H and enzyme/PFK ratios were significantly lower in tumoursfrom patients withfour o÷more nodes involved than in those with no nodal involvement. Possibleprognostic significance of these enzymes is discussed.
INTRODUCTION
cinoma may be related to the rate of glycolysis and therefore by measuring the activities of certain enzymes it might be possible to predict the clinical course of the disease [5-7]. If so, they should correlate~ith the recurrence and survival rates. It is well known that in women with primary breast cancer, the disease-free interval and 5 yr survival is greatly reduced if lymph nodes are found to contain metastatic deposits at the time ofmastectomy [8, 9]. Therefore, analysis of the results obtained on enzyme activities in the primary carcinomas related to the presence or absence of nodal involvement at the time of mastectomy might give some insight into the usefulness of these measurements in prognosis. This paper reports the findings after such an analysis.
IT IS generally accepted than an early indication of the clinical course of any carcinoma will greatly assist clinicians in deciding on a suitable treatment. A considerable effort therefore is being made to devise biochemical parameters for this purpose. It is hoped that carcinomas will either secrete specific factor(s) which are not present in non-cancerous women or alter the body pool concentrations of parameter (s) normally present so that there are significant differences between the two categories. These hypotheses are then extended in clinical studies to test whether such parameter (s) can correlate with the prognosis. In no field is this more relevant than in carcinoma of the breast. Although the ideal factor(s) for this purpose has not been observed, several workers have attempted to investigate hormones, tumour antigens, proteins and enzymes as likely candidates and found some abnormalities in patients with breast carcinoma [1-4]. In recent studies activities of enzymes associated with carbohydrate metabolism in primary breast carcinomas have been measured to test the hypothesis that the energy requirements of a rapidly growing car-
MATERIAL AND METHODS All the chemicals used in the study were purchased from British Drug Houses Ltd. and were of analytical grade, a-GPDH, TPI, G6PDH, aldolase, NADH, NADP +, ATP, F-6-P, G-6-P, 6PG and pyruvate were purchased from Boehringer Mannheim. G-1-P was purchased from Koch-Light Ltd. DHAP was prepared from the dicyclohexylammonium salt of the dimethylketal according to the manufacturer's (Boehringer Mannheim)
Accepted 15 September 1977. 183
184
N. Deshpande, Irene Mitchell and Rosemary Millis
instructions.* Primary tumours were obtained from patients with carcinoma of the breast undergoing mastectomy. The malignancy of the tumours was confirmed by histological examination. The tissues were wrapped in aluminium foil, frozen on solid carbon dioxide and transferred to the laboratory where they remained frozen (-20°C) until further processing. All the tissues taken for histology were processed routinely and section from two levels of each lymph node block examined in order to increase the chances of finding small deposits of metastatic tumour. In this series an average of 23 axillary lymph nodes (range 8-58) were found in radical mastectomy specimens. The tissues were cleared of any surrounding fat, weighed and cut into small pieces. The semifrozen tissues were then homogenised in a Silverson homogeniser according to the procedure o f Shonk and Boxer [10]. The homogenates were centrifuged (800g for 15min) at 4°C, the cytosol fraction was decanted off and used as a source of enzymes. A 5% solution of tumours was used in these estimations. The activities of PFK, G6PDH, 6PGDH, PGM, LDH, ~-GPDH and PHI were measured. Enzyme assays were performed in a Beckman recording spectrophotometer according to the procedure of Shonk and Boxer [10], with the exception that one minute readings were recorded automatically for five minutes and a mean of the last four was taken as an average change in optical density (O.D.). The results were calculated on the basis of units (U) of enzyme activity per gram wet weight of tissue. A unit is defined as that amount of enzyme which will catalyse the transformation of 1/tmole ofsubstrate per minute. The results were analysed in three different ways: as units of enzyme activity/g and as enzyme/6PGDH and enzyme/PFK ratios. They are presented as mean _ S.D. The means were tested for significance by a Student's '/'-test.
RESULTS This study is based on a total of 95 patients, 47 of these were free of nodal involvement. O f the remainder there were 10 patients with only one involved lymph node, 8 with two, 3 with three and 27 in whom four or more nodes were involved. For the purpose of analysis, patients with two or three involved nodes were grouped together. The values (U/g) obtained in these four categories are shown in Table 1. There were no statistically significant differences in the activities ofG6PDH, 6PGDH, PGM, L D H and PHI between patients with no node involvement and those in the one, 2 + 3 or 4 + categories. 0~-GPDH activities were significantly lower in patients in the 4 + category than in those in whom no nodes were found to contain metastases. Further analyses of the results on the basis of enzyme/6PGDH and enzyme / P F K ratios are shown in Tables 2 and 3, respectively. In both cases significantly higher c~G P D H / 6 P G D H and 0~--GPDH/PFK ratios were observed in tumours from patients with no nodal involvement than those in whom four or more nodes were found to contain metastatic deposits. Furthermore, ~-GPDH/PFK ratios in tumours from patients who had two or three involved nodes were higher than those with four or more involved nodes.
DISCUSSION Recurrence and survival after mastectomv are usually considered to be dependent on certain clinical factors such as size of carcinoma, histology, anatomic type, grade of malignancy and lymph node involvement. The most closely related amongst these variables was the spread of the disease to the surrounding lymph nodes. Fisher and his colleagues [8] have shown tlaat after surgical removal of the breast the 5yr survival rate in patients with localised disease and O/ absence of nodal involvement is 76/o which drops to -rA5°/,owhen up to three nodes are involved and 31°L when four or more are found to contain metastatic deposits. The same also applies to recurrence rates. In the absence of axillary metastases the 5 yr recurrence rate is 21 °/o, which rises to 81 o.Ljif four or more nodes are involved. Similar findings have been reported by others [11, 12]. However, these are gross classifications ancl as has been pointed out t3y Gutler and Myers [11] the patients falling into the same prognostic class show marked variation in clinical characteristics. Therefore, further classification •
*The followingabbreviations are used in this paper. Enzymes--~-GPDH, ~-glycerolphosphate dehydrogenase;TPI, triosephosphateisomerase;G6PDH,glucose6-phosphate dehydrogenase;PFK, phosphofructokinase; 6PGDH, 6-phosphoglueonate dehydrogenase; PGM, phosphoglucomutase;LDH, lactatedehydrogenase;PHI, phosphohexose isomerase. Co-enzymes--NADH, reduced nicotinamide adenine dinucleotide; NADP÷, nicotinamide adenine dinucleotide phosphate; ATP, adenosine triphosphate. Substrates--F-6:P, fructose-6-phosphate;G-6-P, glucose6-phosphate; 6PG, 6-phosphogluconate; G-l-P, glucose1-phosphate; DHAP, dihydroxyacetonephosphate.
Enzymes, Lymph Node Involvement and Possible Prognosis
185
Table 1. Enzyme activities (#mole of pyridine nucleotide reduced or oxidised/min/g tissue) in human breast carcinomas. The tumours were separated according to the number of lymph nodesfound to contain metastatic deposits 0
1
2+3
0.057 + 0.044* 0.197+_0.149 0.132_+0.091 1.396 +_0.829 7.019 _ 4.781 0.310 _+0.249* 7.859 +_4.337
0.093 + 0.057 0.212+_0.110 0.134+_0.064 1.466 _+0.698 6.939 +- 3.072 0.214_+0.192 8.027 +- 3.655
0.060 _ 0.040 0.150_+0.090 0.130+_0.070 1.560 -+0.780 8.950 _+6.530 0.260 +-0.130 7.270 +-3.680
4+
Enzyme PFK G6PDH 6PGDH PGM LDH e-GPDH PHI
0.094 +-0.079 0.178_+0.11.9 0.137+_0.065 1.339 _+0.688 8.582 +- 3.982 0.182 +-0.168 8.868 +-4.406
O~;-No nodes involved; 1--One node involved; 2 + 3--Two or three nodes involved; 4 + - - F o u r or more nodes involved. *Significant differences between 0 and 4 + P
