Journal of Surgical Oncology 46:203-207 (1991)
Significance of Surgical Adjuvant Chemotherapy for Gastric Cancer NOBUHIKO TANIGAWA, MD, AND HlDEKl MORIMOTO, MD From the Second Department of Surgery, Fukui Medical School, Yoshida, Fukui, lapan
One hundred and twenty-four specimens (72 primaries and 52 metastases) in 107 patients with gastric cancer were tested using the human tumor clonogenic assay (HTCA). Assays for 64 specimens (52%, 33 primaries and 31 metastases) were considered evaluable. The clonogenicity of the primaries was less than that of the metastases (PP>0.05) ml of 10% formalin was poured over the top layer of one (Table 11). plate, and the preparation was stored at 4°C. This plate was counted on the day of colony counting to obtain the background colony count. Experiments were considered TABLE 1. Colony-Forming Ability of Primary vs. Metastatic evaluable for assessment of chemosensitivity if ( 1 ) the Specimens net number of colonies in the untreated controls was Colony count >30, (2) the mercuric chloride (100 pg/ml) treated Specimen n ~ n e a n k S D(range) ~ dishes showed >70% inhibition of colony formation 33 60 83 (30-355) when compared with the controls; and (3) the coefficient Primary KO.01 of variation for the newly formed colony count of the Metastasis 31 I06 f 200 (30- 1,000) control group was less than 50%.
*
In Vitro Drug Exposure
Overall
]
80 f 147 (30-1.000)
64
"SD, standard deviation.
Drug solutions were prepared from standard intravenous formulations at the concentrations (pgiml) indicated: adriamycin (0.4),bleomycin (2.0), 5-fluorouracil (5-FU) (10.0), mitomycin C (MMC) (3.0), melphalan TABLE 11. Colony-Forming Ability in Patients (n=15) in Whom Both the Primary and Imoregional Lymph Node Metastatic (1 .(I), cisplatinum (2.01, and 1.3-bis-2-chloroethyl-l- Specimens Were Evaluably Assayed nitrosourea (BCNU) (2.0).Each drug was tested at adose Colony count comparable with the highest concentration pharmacologmeanzkSDa (range) ically achievable in the patient's serum [ 131. Aliquots of Specimen solutions were prepared and stored at -70°C. Drugs were Primary 53 89 (30-355) 0.1>P>0.05 thawed immediately before use, and appropriate dilutions 118 rf- 204 (30-789) were made in PBS and added in 100 pl volume over the Lymph node metastasis upper layer. For untreated controls, 100 p1 of PBS alone was added. The drug effect was calculated as the aSD. standard deviation.
*
Surgical Adjuvant Chemotherapy
moderately
69
(n=7)
P60 pm in diameter after about 3 weeks of culture. Each colony was generally made of more than 30 clonogenic cells, although cells from different tumors were different in size. To form a cluster of >30 cells, a cell must divide five or more times (25 = 32>30 cells). Cells with the ability to achieve only a few divisions were not regarded as clonogenic in this study. Recognition of the correct target cells to be analyzed is important in the performance of chemosensitivity tests. For example, in a rapid assay using [3H]thymidine uptake [9, 14181, the cell to be assayed is one which goes through its cycle at least once. To form a cluster within 3 weeks, the cell division time must be S 4 days (4 days X 5 divisions = 20 days0.05), and decreased as the tumors advanced. Moreover, the clonogenicity of the metastatic specimens, all of which were from locoregional lymph nodes, was higher than that of the primaries (Table I, K 0 . 0 1 ) . A similar tendency was seen in 15 cases in which both the primaries and the locoregional lymph node metastases were defined as evaluable with this assay (Table 11, 0.1>P>0.05). These data suggest that some of the clonogenic cells of the primary tumor might enter Go phase or cycle more slowly after some cycles have passed, and the fractions defined in this assay would thus be lost as the primary tumor advanced. Alternatively, metastatic lesions in the locoregional lymph nodes might be formed by cells with a high clonogenic potential. The in vitro chemosensitivity of the gastric primary tumor specimens corresponded well to their clonogenicity when tumors were stratified by their degree of progression (Table IV; Fig. 3). Although the in vitro sensitivity to drugs varied with each specimen, tumor cells with a high clonogenicity were generally more chemosensitive. Thus, at the early phase of primary tumor and locoregional lymph node metastasis, the clonogenicity and chemosensitivity were high. Clinically, metastatic lesions remained after surgery affected the patient’s prognosis. These data suggest that aggressive chemotherapy after surgery may improve the clinical results for the patient with early phase of metastasis.
4.
5. 6.
7. 8.
9. 10.
11. 12. 13.
14. 15.
16.
17.
ACKNOWLEDGMENTS This work was supported in part by Grants-in-Aid for Cancer Research from the Japanese Ministry of Health and Welfare. We are indebted to Miss Yamakawa for her skillful technical assistance.
19.
REFERENCES
20.
Hamburger AW, Salmon SE: Primary bioassay of human tumor stem cells. Science 197:461463, 1977. Salmon SE, Hamburger AW, Soehnlen B, Durie BGM, Alberts DS, Moon TE: Quantitation of differential sensitivity of humantumor stem cells to anticancer drugs. N Engl J Med 298:13211327, 1978 Bertelsen CA, Sondak VK, Mann BD, Korn EL, Kern DH: Chemosensitivity testing of human solid tumors: A review of 1582
18.
21. 22.
207
assays with 258 clinical correlations. Cancer 53: 1240-1 245, 1984. Selby P, Buick R , Tannock I: A critical appraisal of the “human tumor stem cell assay.” N Engl J Med 308:129-134, 1983. Weisenthal LM, Lippman ME: Clonogenic and nonclonogenic in vitro chemosensitivity assays. Cancer Treatm Rep 69:615-63 I , 1985. Shoemaker RH, Wolpert-DeFilippes MK, Kern DH, Lieber MM, Makuch RW, Melnick NR, Miller WT, Salmon SE, Simon RM, Venditti JM, Von Hoff DD: Application of human tumor colonyforming assay to new drug screening. Cancer Res 45:2145-2153, 1985. Von Hoff DD: Human tumor cloning assays: Applications in clincial oncology and new antineoplastic agent development. Cancer Metast Rev 7:357-371, 1988. Ottestad L, Tveit KM, Hannisdal E, Skrede M, Nesland JM, Gundersen S , Pihl A: Colony forming ability of human breast carcinomas: Lack of prognostic significance. Br J Cancer 6 0 : 2 1 6 219, 1989. Tanigawa N, Kern DH, Hikasa Y, Morton DL: Rapid assay for evaluating the chemosensitivity of human tumors in soft agar culture. Cancer Res 42:2159-2164, 1982. Tanigawa N, Mizuno Y, Hashimura T, Honda K, Satomura K, Hikasa Y, Niwa 0, Sugahara T, Yoshida 0, Kern DH, Morton DL: Comparison of drug sensitivity among tumor cells within a tumor, between primary tumor and metastases, and between different metastases in the human tumor colony-forming assay. Cancer Res 44:2309-2312, 1984. Tanigawa N, Morimoto H: Colony-forming ability in vitro and clonology of colorectal cancer. Jpn J Cancer Res 81:687-691, 1990. Morimoto H, Tanigawa N: The significance of postoperative early chzmotherapy for locoregional lymph node metastases of gastric and colorectal cancer. Oncology in press. Alberts DS, Salmon SE, Chen H-SG, Moon TE, Young L, Surwit EA: Pharmacologic studies of anticancer drugs with the human tumor stem cell assay. Cancer Chemother Pharmacol 6:253-264, 1981, Hildebrand-Zanki SU, Kern DH: A rapid bioassay for in vitro drug stabilities. In Salmon SE, Trent JM (eds): “Human Tumor Cloning.” New York: Grune & Stratton, Inc, 1984, pp. 451458. Jones CA, Tsukamoto T, O’Brien PC, Uhl CB, Alley MC, Lieber MM: Soft agarose culture human tumor colony forming assay for drug sensitivity testing: 3H-Thymidine incorporation vs. colony counting. Br J Cancer 52:303-310, 1985. Kern DH, Drogemuller CR, Kennedy MC, Hildebrand-Zanki SU, Tanigawa N, Sondak VK: Development of a miniaturized, improved nucleic acid precursor incorporation assay for chemosensitivity testing of human solid tumors. Cancer Res 4554365441, 1985. Sondak VK, Bertelsen CA, Kern DH, Morton DL: Evolution and clinical application of a rapid chemosensitivity assay. Cancer 55:1367-1371, 1985. Kern DH, Morgan CR, Hildebrand-Zanki SU: In vitro pharmacodynamics of 1-b-o-arabinofuranosykytosine: Synergy of antitumor activity with cis-diamminedichloroplatinum. Cancer Res 48:117-121, 1988. Hamburger AW, Salmon SE, Kim MB, Trent JM, Soehnlen BJ, Alberts DS, Schmidt HJ: Direct cloning of human ovarian carcinoma cells in agar. Cancer Res 38:3438-3444, 1978. Sandback RN, Von Hoff DD, Clark G: Direct cloning of human breast cancer in soft agar culture. Cancer 50:1315-1321, 1982. Nomura Y, Tashiro H, Hisamatsu K: In vitro clonogenic growth and metastatic potential of human operable breast cancer. Cancer Res 495288-5293, 1989. Japanese Research Society for Gastric Cancer (eds): “The General Rules for the Gastric Cancer Study in Surgery and Pathology.” 10th Ed. Tokyo: Kanehara, Inc., 1979.
Significance of Surgical Adjuvant Chemotherapy for Gastric Cancer NOBUHIKO TANIGAWA, MD, AND HlDEKl MORIMOTO, MD From the Second Department of Surgery, Fukui Medical School, Yoshida, Fukui, lapan
One hundred and twenty-four specimens (72 primaries and 52 metastases) in 107 patients with gastric cancer were tested using the human tumor clonogenic assay (HTCA). Assays for 64 specimens (52%, 33 primaries and 31 metastases) were considered evaluable. The clonogenicity of the primaries was less than that of the metastases (PP>0.05) ml of 10% formalin was poured over the top layer of one (Table 11). plate, and the preparation was stored at 4°C. This plate was counted on the day of colony counting to obtain the background colony count. Experiments were considered TABLE 1. Colony-Forming Ability of Primary vs. Metastatic evaluable for assessment of chemosensitivity if ( 1 ) the Specimens net number of colonies in the untreated controls was Colony count >30, (2) the mercuric chloride (100 pg/ml) treated Specimen n ~ n e a n k S D(range) ~ dishes showed >70% inhibition of colony formation 33 60 83 (30-355) when compared with the controls; and (3) the coefficient Primary KO.01 of variation for the newly formed colony count of the Metastasis 31 I06 f 200 (30- 1,000) control group was less than 50%.
*
In Vitro Drug Exposure
Overall
]
80 f 147 (30-1.000)
64
"SD, standard deviation.
Drug solutions were prepared from standard intravenous formulations at the concentrations (pgiml) indicated: adriamycin (0.4),bleomycin (2.0), 5-fluorouracil (5-FU) (10.0), mitomycin C (MMC) (3.0), melphalan TABLE 11. Colony-Forming Ability in Patients (n=15) in Whom Both the Primary and Imoregional Lymph Node Metastatic (1 .(I), cisplatinum (2.01, and 1.3-bis-2-chloroethyl-l- Specimens Were Evaluably Assayed nitrosourea (BCNU) (2.0).Each drug was tested at adose Colony count comparable with the highest concentration pharmacologmeanzkSDa (range) ically achievable in the patient's serum [ 131. Aliquots of Specimen solutions were prepared and stored at -70°C. Drugs were Primary 53 89 (30-355) 0.1>P>0.05 thawed immediately before use, and appropriate dilutions 118 rf- 204 (30-789) were made in PBS and added in 100 pl volume over the Lymph node metastasis upper layer. For untreated controls, 100 p1 of PBS alone was added. The drug effect was calculated as the aSD. standard deviation.
*
Surgical Adjuvant Chemotherapy
moderately
69
(n=7)
P60 pm in diameter after about 3 weeks of culture. Each colony was generally made of more than 30 clonogenic cells, although cells from different tumors were different in size. To form a cluster of >30 cells, a cell must divide five or more times (25 = 32>30 cells). Cells with the ability to achieve only a few divisions were not regarded as clonogenic in this study. Recognition of the correct target cells to be analyzed is important in the performance of chemosensitivity tests. For example, in a rapid assay using [3H]thymidine uptake [9, 14181, the cell to be assayed is one which goes through its cycle at least once. To form a cluster within 3 weeks, the cell division time must be S 4 days (4 days X 5 divisions = 20 days0.05), and decreased as the tumors advanced. Moreover, the clonogenicity of the metastatic specimens, all of which were from locoregional lymph nodes, was higher than that of the primaries (Table I, K 0 . 0 1 ) . A similar tendency was seen in 15 cases in which both the primaries and the locoregional lymph node metastases were defined as evaluable with this assay (Table 11, 0.1>P>0.05). These data suggest that some of the clonogenic cells of the primary tumor might enter Go phase or cycle more slowly after some cycles have passed, and the fractions defined in this assay would thus be lost as the primary tumor advanced. Alternatively, metastatic lesions in the locoregional lymph nodes might be formed by cells with a high clonogenic potential. The in vitro chemosensitivity of the gastric primary tumor specimens corresponded well to their clonogenicity when tumors were stratified by their degree of progression (Table IV; Fig. 3). Although the in vitro sensitivity to drugs varied with each specimen, tumor cells with a high clonogenicity were generally more chemosensitive. Thus, at the early phase of primary tumor and locoregional lymph node metastasis, the clonogenicity and chemosensitivity were high. Clinically, metastatic lesions remained after surgery affected the patient’s prognosis. These data suggest that aggressive chemotherapy after surgery may improve the clinical results for the patient with early phase of metastasis.
4.
5. 6.
7. 8.
9. 10.
11. 12. 13.
14. 15.
16.
17.
ACKNOWLEDGMENTS This work was supported in part by Grants-in-Aid for Cancer Research from the Japanese Ministry of Health and Welfare. We are indebted to Miss Yamakawa for her skillful technical assistance.
19.
REFERENCES
20.
Hamburger AW, Salmon SE: Primary bioassay of human tumor stem cells. Science 197:461463, 1977. Salmon SE, Hamburger AW, Soehnlen B, Durie BGM, Alberts DS, Moon TE: Quantitation of differential sensitivity of humantumor stem cells to anticancer drugs. N Engl J Med 298:13211327, 1978 Bertelsen CA, Sondak VK, Mann BD, Korn EL, Kern DH: Chemosensitivity testing of human solid tumors: A review of 1582
18.
21. 22.
207
assays with 258 clinical correlations. Cancer 53: 1240-1 245, 1984. Selby P, Buick R , Tannock I: A critical appraisal of the “human tumor stem cell assay.” N Engl J Med 308:129-134, 1983. Weisenthal LM, Lippman ME: Clonogenic and nonclonogenic in vitro chemosensitivity assays. Cancer Treatm Rep 69:615-63 I , 1985. Shoemaker RH, Wolpert-DeFilippes MK, Kern DH, Lieber MM, Makuch RW, Melnick NR, Miller WT, Salmon SE, Simon RM, Venditti JM, Von Hoff DD: Application of human tumor colonyforming assay to new drug screening. Cancer Res 45:2145-2153, 1985. Von Hoff DD: Human tumor cloning assays: Applications in clincial oncology and new antineoplastic agent development. Cancer Metast Rev 7:357-371, 1988. Ottestad L, Tveit KM, Hannisdal E, Skrede M, Nesland JM, Gundersen S , Pihl A: Colony forming ability of human breast carcinomas: Lack of prognostic significance. Br J Cancer 6 0 : 2 1 6 219, 1989. Tanigawa N, Kern DH, Hikasa Y, Morton DL: Rapid assay for evaluating the chemosensitivity of human tumors in soft agar culture. Cancer Res 42:2159-2164, 1982. Tanigawa N, Mizuno Y, Hashimura T, Honda K, Satomura K, Hikasa Y, Niwa 0, Sugahara T, Yoshida 0, Kern DH, Morton DL: Comparison of drug sensitivity among tumor cells within a tumor, between primary tumor and metastases, and between different metastases in the human tumor colony-forming assay. Cancer Res 44:2309-2312, 1984. Tanigawa N, Morimoto H: Colony-forming ability in vitro and clonology of colorectal cancer. Jpn J Cancer Res 81:687-691, 1990. Morimoto H, Tanigawa N: The significance of postoperative early chzmotherapy for locoregional lymph node metastases of gastric and colorectal cancer. Oncology in press. Alberts DS, Salmon SE, Chen H-SG, Moon TE, Young L, Surwit EA: Pharmacologic studies of anticancer drugs with the human tumor stem cell assay. Cancer Chemother Pharmacol 6:253-264, 1981, Hildebrand-Zanki SU, Kern DH: A rapid bioassay for in vitro drug stabilities. In Salmon SE, Trent JM (eds): “Human Tumor Cloning.” New York: Grune & Stratton, Inc, 1984, pp. 451458. Jones CA, Tsukamoto T, O’Brien PC, Uhl CB, Alley MC, Lieber MM: Soft agarose culture human tumor colony forming assay for drug sensitivity testing: 3H-Thymidine incorporation vs. colony counting. Br J Cancer 52:303-310, 1985. Kern DH, Drogemuller CR, Kennedy MC, Hildebrand-Zanki SU, Tanigawa N, Sondak VK: Development of a miniaturized, improved nucleic acid precursor incorporation assay for chemosensitivity testing of human solid tumors. Cancer Res 4554365441, 1985. Sondak VK, Bertelsen CA, Kern DH, Morton DL: Evolution and clinical application of a rapid chemosensitivity assay. Cancer 55:1367-1371, 1985. Kern DH, Morgan CR, Hildebrand-Zanki SU: In vitro pharmacodynamics of 1-b-o-arabinofuranosykytosine: Synergy of antitumor activity with cis-diamminedichloroplatinum. Cancer Res 48:117-121, 1988. Hamburger AW, Salmon SE, Kim MB, Trent JM, Soehnlen BJ, Alberts DS, Schmidt HJ: Direct cloning of human ovarian carcinoma cells in agar. Cancer Res 38:3438-3444, 1978. Sandback RN, Von Hoff DD, Clark G: Direct cloning of human breast cancer in soft agar culture. Cancer 50:1315-1321, 1982. Nomura Y, Tashiro H, Hisamatsu K: In vitro clonogenic growth and metastatic potential of human operable breast cancer. Cancer Res 495288-5293, 1989. Japanese Research Society for Gastric Cancer (eds): “The General Rules for the Gastric Cancer Study in Surgery and Pathology.” 10th Ed. Tokyo: Kanehara, Inc., 1979.
