Clinical meaning of ATP-tumor chemosensitivity assay in chemotherapy of primary liver cancer
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《中华医药杂志》英文版
Jiangxi Province Liver Tumor Treatment Center,Nanchang ,Jiangxi Province 330029,China
[Abstract] Objective To investigate the curative effect of 104 cases by hepatic arterial catheter reserved to infusion chemotherapy with ATP-tumor chemosensitivity assay(ATP-TCA)directed chemotherapy regimens.Methods Tumor tissue was cut in laparotomy to cultivate in vitro and assay its chemosensitivity.Compare the effect of single drug 5-FU,MMC and ADM with the combination drug.In the treatment,the two-combination chemotherapeutic schemes was adopted according to the result of the ATP-TCA.In the meantime,in control group 30 cases proven PLC equally was infused with the conventional three-combination drug.Two groups were evaluated after three courses of chemotherapy.Results The overall response rate of sensitivity test was 36%~44% in the single drug group and 81% in the combination drug group.The clinical overall response rate was 75% in the treatment group and in control group 56%.The treatment group was better than the control group.In the treatment group survival period over 6 months was 80% and over one year was 44%.In the control group survival period over 6 months was 60% and over one year was 30%.Conclusions The ATP-TCA has preferable directive value for terminal stages of PLC to decrease the dose of drugs,reduce the side-effect and improve the clinic curative effect.
[Key words] hepatocellular carcinoma;cell culture;sensitivity test;chemotherapy
The regular chemotherapeutic scheme is generally adopted in tumor chemotherapy,but each patient has different respond for the same scheme,and this has more or less influence upon curative effect.So for,the ATP-tumor chemosensitivity assay (ATP-TCA) have been applied to improve curative effect and decrease side-effects.The ATP-TCA has been studied for many years,it has been consistently taken for a sensitive and stable method for tumor chemosensitivity testing[1~4].Recent years,this assay has been gradually taken in domestic,and some clinical reports have appeared.However,application of the ATP-TCA in primary liver cancer has not been reported.We have treated 104 patients confirmed primary liver cancer with ATP-TCA directed chemotherapy regimens,the reports as follows.
MATERIALS AND METHODS
Data and Patients
From April 2002 to June 2003,104 patients confirmed primary liver cancer have been performed hepatic arterial catheter reserved to infusion chemotherapy through hypodermic implantation of drug pump in laparotomy.In the meantime,we excise tumor specimens and determined.Of these patients,there were 86 male and 18 female,the oldest was 73 and the youngest was 30.Pathologic histology classification：hepatocellular carcinoma were 89 cases,cholangiocarcinoma were 9 cases and mixed were 6 cases.Of these patients,81 cases AFP were positive and 21 cases CEA were positive.In addition,we stochastic selected 30 patients who had been performed a same operation before the April 2002,including 27 cases were hepatocellular carcinoma,2 cases were cholangiocarcinoma and 1 case was mixed,of these patients 27 cases AFP were positive and 3 cases CEA were positive.They all only been done the pathology checks.
Materials
The reagents and materials provided in this research included Complete Assay Medium(CAM),Tumor Dissociation Enzyme Reagent,Maximum ATP Inhibitor,Tumor Cell Extraction Reagent,Luciferin-Luciferase,Sterile Microwell Culture Plates,syringe,filter etc.DCS corporation，Luciferin-Luciferase Counting Reagent was the product of Germany Berthold Corporation).All chemotherapeutic drugs are made in China,including MMC,ADM and 5-FU.
Experiment Method and Clinical Observation
Measurement of ATP Standard
Each ATP standard was determined at 5 concentrations corresponding to 1×10-2,1×10-3,1×10-4,1×10-5,1×10-6mol/L.All were injected into the 96-microwell culture plates and mensurated luminescence in each concentration in triplicate wells
Cultivation of the Liver Tumor Cells and Measurement
The method in ATP-TCA followed manufacturer instructions.We minced the tumor specimen into 0.3~0.5 cm pieces using the sterile scalpels and washed them with 0.9% sodium chloride liquor,immerse in the 1640 Liquor,then excised and discarded excess fat and minced them,added tumor dissociation solution in them.After 4 days incubation(5% CO2,37°C and over 75% humidity),we centrifuged the tumor cell and got the suspension,inoculated the unicellular suspension on the 24-well culture plates,adjusted them to made the cell concentrations corresponding to 1.5~2.5×104 per well,then each chemotherapeutic drug or drug combination was tested at concentrations corresponding to 1×PPC or 2×PPC.The control groups added the culture liquor equally.Every drug was tested in three well respectively.After 5 days cultivation we distilled the ATP and counted the percentage of tumor growth inhibition by this equation:Percent Tumor Growth Inhibition(TGI) = (1-the ATP content in treatment groups/the ATP content in control groups×100%)
Evaluation and Interpretation of Results
In this subject,we divided the chemotherapeutic drug or drug combination into four ranks,they are strong sensitivity,partial sensitivity,weak sensitivity and resistance(Table 1).
Table 1 The Sensitivity Rank of Chemotherapeutic Drug or Drug Combination
Above that the IC90 and IC50 were drug concentration that achieve 90% and 50% growth inhibition calculated by interpolation respectively,100%TDC,25%TDC were the chemotherapeutic drug concentration tested in the research.
Clinical Observation
In this study,we observed the clinical meaning on the basis of ATP-TCA,we divided all patients into two groups,the treatment groups was treated with the two-drug combinations as indicated by the ATP-TCA assay.The control group was treated with the regular three-drug combinations(ADM,MMC,5-FU).Two group were equal in dose,every patients underwent 3 courses of ATP-TCA directed chemotherapy consecutively.To research the alleviation of the symptom，the tumor sizes,the hepatic function,AFP and survival period.
Clinical Effect Evaluation
Two groups have underwent three courses,the effect was evaluated as follows.
A.Complete responses(CR):the clinic symptom all disappeared,the volume of tumor reduced 50% or above,hepatic function was natural,AFP reduced 50% or and above.
B.Partial responses (PR):after 3 months,the symptom meliorated,volume of tumor reduced less than 50%,hepatic function improved,APF decreased in a certain extent.
C.Stable disease (SD):the symptom,volume of tumor,hepatic function and AFP have not meliorated even worse.RESULTS
Result for Individual Chemotherapeutic Drug and Drug Combination See Table 2.
Table 2 Chemotherapeutic Sensitivity Comparison between Individual Chemotherapeutic Drug and Drug Combination in 104 Cases of Primary Liver Cancer
Relation of the Clinical Curative Effect and the Tumor Chemosensitivity Assay
All the treatment groups were treated with two-drug combinations as indicated by the ATP-TCA assay and the control groups were treated with three-drug combination(MMC+ADM+5-FU).Two groups all underwent three courses of treatment and the data recorded as follows.
The data of two groups in symptom,sign,hepatic function and AFP after three months research,it could be seen that the groups with ATP-TCA werebetter in hepatic function,AFP and alleviation in symptom.
Table 3 Relation between Clinical Effect and Results of ATP-TCAC
Table 4 Comparison Between Two Groups in Hepatic Function,Symptom and AFPGroups〖〗Alleviation in symptom〖〗Volume of tumor〖〗HF〖〗Decrease in AFPTreatment group(N=104)〖〗79(76%)〖〗71(68%)〖〗81(78%)〖〗84(81%)Control group(N=30)〖〗19(63%)〖〗16(53%)〖〗19(63%)〖〗21(70%)
Survival
There were 83 cases occupied 80% who live for over six months,46 cases for over one year occupied 44% in treatment group; and there were 18 cases occupied 60% live for over six months,9 cases for over one year occupied 30%.
DISCUSSION
The ex vitro adenosine triphosphate tumor chemosensitivity assay(ATP-TCA) has not yet much developed in domestic,Multilabel Counter is a multifunctional detector that can distinguish fluorescence,make spectrophotometric analysis and detect biological and chemical luminescence.In this study we adopted the ATP-TCA only to utilize its function of detecting biological luminescence.The Multilabel Counter is better than the general analytic instruments in function.In this assay the 96-microwell culture plate be used and make it possible to make microanalysis. And it is economical due to the dosage of fluorescein and luciferase is consumedly saved,Moreover,the specimen can be mensurated at the same time,which can reduce the operative procedure and make an error,make the result more veracious,and the method would more convenient.According to the reference,when the ATP standard curve be perform at concentrations range from 1×10-5 to 1×10-9 mol/L.It can be seen the fluorescence-luminescence have a linear correlation with the ATP concentration,It was showed that the Multilabel Counter,can be used in assay the fluorescence and it is advantageous to be used widely.
There is obvious difference of survival probability between the treatment group and the control group during the fifth day to the seventh day[2,5,6],especially on the fifth day it is most obvious. The ATP-TCA using agar culture can suppress the group of no-tumor cell,and this can reduce interfering factor,so this assay is better to reflect the effect of the chemotherapeutic drug on the tumor cell.
The chemotherapy applied in carcinoma has been greatly advanced since the achievement of curing lymphoma with HNL in 1945.However,the encouraging advance in the chemotherapy of PLC has not be achieved,recent years,with more and more new drugs were published and improving of the method of using drugs,especially the inspiring effect of TACE.Lately the new method ATP-TCA provide a much more exact,effective scientific derive for chemotherapy of PLC.
The formal chemotherapy of PLC adopted individual 5-FU,but the effect of which was not so good[7].In this study the sensitive ratio about 5-FU is 26%,ADM 45%,MMC 44%,which are all higher than 8.33% in the conference data.It probably has correlated with biological characteristic of PLC and the high drug concentration of hepatic arterial catheter reserved to infusion,in addition,in order to compare with the effect of individual drug and combination drug,we compared the effects of the individual drug with the combination drugs.The outcome indicates that the response with the combination drug was 81%,the response of individual drug chemotherapy was between 36% and 44%,that is,the curative effect with combined drug was evidently better,it was showed that the combination chemotherapeutics therapy was much better than individual drug chemotherapy.
Based on the outcome of the test,the cases who were strong sensitivity and partial sensitivity for MMC+ADM+5-FU were treated with two-combination drug,and to compare effect with the control groups which used the traditional three-combination drug therapy at the same dosage.The result showed the clinical effect of the treatment groups directed by ATP-TCA was more better than the control groups.The clinical effect was improved greatly.CR+PR was 75%.The symptoms of the consequent reviewed patient were improved,the hepatic function was more normal,together with the decrease of AFP,as well as the survival quality was improved,one-year survival period reached 80%,one-year survival period 44%,whereas the toxic effect of the contrast groups with traditional three-combination drugs was higher.The hepatic function that is seriously damaged,the clinical effect is not as good as the treatment groups. The one-year survival period was 60% and the one-year survival period was 30%,all were lowed than the treatment groups.
In conclusion,the ex vitro ATP-TCA lead a constructive role in the treatment of PLC,it can cut down the dosage of chemotherapy drug,lessen the poisonous side-effect and improve the curative effect as well.
REFERENCES
1. Petru E，Sevin JP，et al.Comparative chemosensitivity profiles in four human Ovarian Carcinoma cell lines measuring ATP bioluminescence. J. Gynecol Oncology, 1990,38(2):155-160.
2. Sevin BU ,Peng ZL, Perras JP, et al. Application of an ATP-bioluminescence assay in human tumor chemosensitivity testing J. Gynecol Oncology, 1988,31:191-204.
3. Ng TY, Ngan HY, Cheng DK, et al. Clinical applicability of the ATP cell viability assay as a predictor of chemoresponse in platinum-resistant epithelial ovarian cancer using nonsurgical tumor cell samples. J. Gynecol Oncology, 2000, 76:405-408.
4. Cree IA et al . Correlation of the clinical response to chemotherapy in breast cancer with ex vivo chemosemsitivity. Anti Cancer Drugs , 1966, 7:630.
5. Liu Shanling .The exploratory development of ATP-biology fluorometric method in tumor vitro drug sensitivity test.Acta Academiae Medicinae Militaris, 2000,31(3):330-333.
6. Liu Shanling. ATP-biology fluorometric intra liquid assay detect viable count. Acta Academiae Medicinae Militaris, 2000,31(2):260-261,268.
7. Chu Zhonghua The application of the ex vitro adenosine triphosphate tumor chemosensitivity assay in the chemotherapy of primary live cancer. Chinese Journal Of Experimental Surgery , 2002 ,19(3):235.
(Editor Jaque)(RAO Rong-sheng,ZHOU Xin-w)
[Abstract] Objective To investigate the curative effect of 104 cases by hepatic arterial catheter reserved to infusion chemotherapy with ATP-tumor chemosensitivity assay(ATP-TCA)directed chemotherapy regimens.Methods Tumor tissue was cut in laparotomy to cultivate in vitro and assay its chemosensitivity.Compare the effect of single drug 5-FU,MMC and ADM with the combination drug.In the treatment,the two-combination chemotherapeutic schemes was adopted according to the result of the ATP-TCA.In the meantime,in control group 30 cases proven PLC equally was infused with the conventional three-combination drug.Two groups were evaluated after three courses of chemotherapy.Results The overall response rate of sensitivity test was 36%~44% in the single drug group and 81% in the combination drug group.The clinical overall response rate was 75% in the treatment group and in control group 56%.The treatment group was better than the control group.In the treatment group survival period over 6 months was 80% and over one year was 44%.In the control group survival period over 6 months was 60% and over one year was 30%.Conclusions The ATP-TCA has preferable directive value for terminal stages of PLC to decrease the dose of drugs,reduce the side-effect and improve the clinic curative effect.
[Key words] hepatocellular carcinoma;cell culture;sensitivity test;chemotherapy
The regular chemotherapeutic scheme is generally adopted in tumor chemotherapy,but each patient has different respond for the same scheme,and this has more or less influence upon curative effect.So for,the ATP-tumor chemosensitivity assay (ATP-TCA) have been applied to improve curative effect and decrease side-effects.The ATP-TCA has been studied for many years,it has been consistently taken for a sensitive and stable method for tumor chemosensitivity testing[1~4].Recent years,this assay has been gradually taken in domestic,and some clinical reports have appeared.However,application of the ATP-TCA in primary liver cancer has not been reported.We have treated 104 patients confirmed primary liver cancer with ATP-TCA directed chemotherapy regimens,the reports as follows.
MATERIALS AND METHODS
Data and Patients
From April 2002 to June 2003,104 patients confirmed primary liver cancer have been performed hepatic arterial catheter reserved to infusion chemotherapy through hypodermic implantation of drug pump in laparotomy.In the meantime,we excise tumor specimens and determined.Of these patients,there were 86 male and 18 female,the oldest was 73 and the youngest was 30.Pathologic histology classification：hepatocellular carcinoma were 89 cases,cholangiocarcinoma were 9 cases and mixed were 6 cases.Of these patients,81 cases AFP were positive and 21 cases CEA were positive.In addition,we stochastic selected 30 patients who had been performed a same operation before the April 2002,including 27 cases were hepatocellular carcinoma,2 cases were cholangiocarcinoma and 1 case was mixed,of these patients 27 cases AFP were positive and 3 cases CEA were positive.They all only been done the pathology checks.
Materials
The reagents and materials provided in this research included Complete Assay Medium(CAM),Tumor Dissociation Enzyme Reagent,Maximum ATP Inhibitor,Tumor Cell Extraction Reagent,Luciferin-Luciferase,Sterile Microwell Culture Plates,syringe,filter etc.DCS corporation，Luciferin-Luciferase Counting Reagent was the product of Germany Berthold Corporation).All chemotherapeutic drugs are made in China,including MMC,ADM and 5-FU.
Experiment Method and Clinical Observation
Measurement of ATP Standard
Each ATP standard was determined at 5 concentrations corresponding to 1×10-2,1×10-3,1×10-4,1×10-5,1×10-6mol/L.All were injected into the 96-microwell culture plates and mensurated luminescence in each concentration in triplicate wells
Cultivation of the Liver Tumor Cells and Measurement
The method in ATP-TCA followed manufacturer instructions.We minced the tumor specimen into 0.3~0.5 cm pieces using the sterile scalpels and washed them with 0.9% sodium chloride liquor,immerse in the 1640 Liquor,then excised and discarded excess fat and minced them,added tumor dissociation solution in them.After 4 days incubation(5% CO2,37°C and over 75% humidity),we centrifuged the tumor cell and got the suspension,inoculated the unicellular suspension on the 24-well culture plates,adjusted them to made the cell concentrations corresponding to 1.5~2.5×104 per well,then each chemotherapeutic drug or drug combination was tested at concentrations corresponding to 1×PPC or 2×PPC.The control groups added the culture liquor equally.Every drug was tested in three well respectively.After 5 days cultivation we distilled the ATP and counted the percentage of tumor growth inhibition by this equation:Percent Tumor Growth Inhibition(TGI) = (1-the ATP content in treatment groups/the ATP content in control groups×100%)
Evaluation and Interpretation of Results
In this subject,we divided the chemotherapeutic drug or drug combination into four ranks,they are strong sensitivity,partial sensitivity,weak sensitivity and resistance(Table 1).
Table 1 The Sensitivity Rank of Chemotherapeutic Drug or Drug Combination
Above that the IC90 and IC50 were drug concentration that achieve 90% and 50% growth inhibition calculated by interpolation respectively,100%TDC,25%TDC were the chemotherapeutic drug concentration tested in the research.
Clinical Observation
In this study,we observed the clinical meaning on the basis of ATP-TCA,we divided all patients into two groups,the treatment groups was treated with the two-drug combinations as indicated by the ATP-TCA assay.The control group was treated with the regular three-drug combinations(ADM,MMC,5-FU).Two group were equal in dose,every patients underwent 3 courses of ATP-TCA directed chemotherapy consecutively.To research the alleviation of the symptom，the tumor sizes,the hepatic function,AFP and survival period.
Clinical Effect Evaluation
Two groups have underwent three courses,the effect was evaluated as follows.
A.Complete responses(CR):the clinic symptom all disappeared,the volume of tumor reduced 50% or above,hepatic function was natural,AFP reduced 50% or and above.
B.Partial responses (PR):after 3 months,the symptom meliorated,volume of tumor reduced less than 50%,hepatic function improved,APF decreased in a certain extent.
C.Stable disease (SD):the symptom,volume of tumor,hepatic function and AFP have not meliorated even worse.RESULTS
Result for Individual Chemotherapeutic Drug and Drug Combination See Table 2.
Table 2 Chemotherapeutic Sensitivity Comparison between Individual Chemotherapeutic Drug and Drug Combination in 104 Cases of Primary Liver Cancer
Relation of the Clinical Curative Effect and the Tumor Chemosensitivity Assay
All the treatment groups were treated with two-drug combinations as indicated by the ATP-TCA assay and the control groups were treated with three-drug combination(MMC+ADM+5-FU).Two groups all underwent three courses of treatment and the data recorded as follows.
The data of two groups in symptom,sign,hepatic function and AFP after three months research,it could be seen that the groups with ATP-TCA werebetter in hepatic function,AFP and alleviation in symptom.
Table 3 Relation between Clinical Effect and Results of ATP-TCAC
Table 4 Comparison Between Two Groups in Hepatic Function,Symptom and AFPGroups〖〗Alleviation in symptom〖〗Volume of tumor〖〗HF〖〗Decrease in AFPTreatment group(N=104)〖〗79(76%)〖〗71(68%)〖〗81(78%)〖〗84(81%)Control group(N=30)〖〗19(63%)〖〗16(53%)〖〗19(63%)〖〗21(70%)
Survival
There were 83 cases occupied 80% who live for over six months,46 cases for over one year occupied 44% in treatment group; and there were 18 cases occupied 60% live for over six months,9 cases for over one year occupied 30%.
DISCUSSION
The ex vitro adenosine triphosphate tumor chemosensitivity assay(ATP-TCA) has not yet much developed in domestic,Multilabel Counter is a multifunctional detector that can distinguish fluorescence,make spectrophotometric analysis and detect biological and chemical luminescence.In this study we adopted the ATP-TCA only to utilize its function of detecting biological luminescence.The Multilabel Counter is better than the general analytic instruments in function.In this assay the 96-microwell culture plate be used and make it possible to make microanalysis. And it is economical due to the dosage of fluorescein and luciferase is consumedly saved,Moreover,the specimen can be mensurated at the same time,which can reduce the operative procedure and make an error,make the result more veracious,and the method would more convenient.According to the reference,when the ATP standard curve be perform at concentrations range from 1×10-5 to 1×10-9 mol/L.It can be seen the fluorescence-luminescence have a linear correlation with the ATP concentration,It was showed that the Multilabel Counter,can be used in assay the fluorescence and it is advantageous to be used widely.
There is obvious difference of survival probability between the treatment group and the control group during the fifth day to the seventh day[2,5,6],especially on the fifth day it is most obvious. The ATP-TCA using agar culture can suppress the group of no-tumor cell,and this can reduce interfering factor,so this assay is better to reflect the effect of the chemotherapeutic drug on the tumor cell.
The chemotherapy applied in carcinoma has been greatly advanced since the achievement of curing lymphoma with HNL in 1945.However,the encouraging advance in the chemotherapy of PLC has not be achieved,recent years,with more and more new drugs were published and improving of the method of using drugs,especially the inspiring effect of TACE.Lately the new method ATP-TCA provide a much more exact,effective scientific derive for chemotherapy of PLC.
The formal chemotherapy of PLC adopted individual 5-FU,but the effect of which was not so good[7].In this study the sensitive ratio about 5-FU is 26%,ADM 45%,MMC 44%,which are all higher than 8.33% in the conference data.It probably has correlated with biological characteristic of PLC and the high drug concentration of hepatic arterial catheter reserved to infusion,in addition,in order to compare with the effect of individual drug and combination drug,we compared the effects of the individual drug with the combination drugs.The outcome indicates that the response with the combination drug was 81%,the response of individual drug chemotherapy was between 36% and 44%,that is,the curative effect with combined drug was evidently better,it was showed that the combination chemotherapeutics therapy was much better than individual drug chemotherapy.
Based on the outcome of the test,the cases who were strong sensitivity and partial sensitivity for MMC+ADM+5-FU were treated with two-combination drug,and to compare effect with the control groups which used the traditional three-combination drug therapy at the same dosage.The result showed the clinical effect of the treatment groups directed by ATP-TCA was more better than the control groups.The clinical effect was improved greatly.CR+PR was 75%.The symptoms of the consequent reviewed patient were improved,the hepatic function was more normal,together with the decrease of AFP,as well as the survival quality was improved,one-year survival period reached 80%,one-year survival period 44%,whereas the toxic effect of the contrast groups with traditional three-combination drugs was higher.The hepatic function that is seriously damaged,the clinical effect is not as good as the treatment groups. The one-year survival period was 60% and the one-year survival period was 30%,all were lowed than the treatment groups.
In conclusion,the ex vitro ATP-TCA lead a constructive role in the treatment of PLC,it can cut down the dosage of chemotherapy drug,lessen the poisonous side-effect and improve the curative effect as well.
REFERENCES
1. Petru E，Sevin JP，et al.Comparative chemosensitivity profiles in four human Ovarian Carcinoma cell lines measuring ATP bioluminescence. J. Gynecol Oncology, 1990,38(2):155-160.
2. Sevin BU ,Peng ZL, Perras JP, et al. Application of an ATP-bioluminescence assay in human tumor chemosensitivity testing J. Gynecol Oncology, 1988,31:191-204.
3. Ng TY, Ngan HY, Cheng DK, et al. Clinical applicability of the ATP cell viability assay as a predictor of chemoresponse in platinum-resistant epithelial ovarian cancer using nonsurgical tumor cell samples. J. Gynecol Oncology, 2000, 76:405-408.
4. Cree IA et al . Correlation of the clinical response to chemotherapy in breast cancer with ex vivo chemosemsitivity. Anti Cancer Drugs , 1966, 7:630.
5. Liu Shanling .The exploratory development of ATP-biology fluorometric method in tumor vitro drug sensitivity test.Acta Academiae Medicinae Militaris, 2000,31(3):330-333.
6. Liu Shanling. ATP-biology fluorometric intra liquid assay detect viable count. Acta Academiae Medicinae Militaris, 2000,31(2):260-261,268.
7. Chu Zhonghua The application of the ex vitro adenosine triphosphate tumor chemosensitivity assay in the chemotherapy of primary live cancer. Chinese Journal Of Experimental Surgery , 2002 ,19(3):235.
(Editor Jaque)(RAO Rong-sheng,ZHOU Xin-w)