不同个体膀胱癌组织培养化疗药物敏感性的研究

目的：探讨不同个体膀胱癌对化疗药物的敏感性。方法：采用组织培养药敏测试法，对37例膀胱癌的化疗药物敏感性进行检测。结果：不同化疗药物对不同个体膀胱癌的抑制率差异明显。膀胱癌THP加MMC的敏感率(81．8％)和平均抑制率(67．6％)均高于任何一种单一药物；复发膀胱癌的药物敏感率和平均抑制率均低于初发者；膀胱癌的药物敏感性与分级无关。结论：不同个体膀胱癌对不同化疗药物的敏感性差异明显，复发膀胱癌对化疗药物存在一定的耐受，联合用药可以提高化疗效果。组织培养药敏测试法可有效地为不同个体膀胱癌选择最佳的化疗方案。     

组织培养药敏法在表浅膀胱癌个体化化疗的应用

目的：研究组织培养药敏法在表浅膀胱癌个体化化疗中的意义。方法：用组织培养药敏法检测药物敏感性，选择一种最敏感药物对39例表浅膀胱癌患者术后膀胱灌注化疗，与按经验给药的42例表浅膀胱癌患者对比研究，所有患者随访6～24个月。结果：不同化疗药物对不同个体膀胱癌的抑制率差异明显。39例按药敏结果选择最佳化疗药物的表浅膀胱癌患者，8例（20．5％）复发，而按经验给药42例患者，20例（47．6％）复发，复发率前者显著低于后者（P〈0．05，X^2检验）。结论：用Hoffman式组织培养药敏法选择最合适的药物对术后膀胱癌进行化疗，可以明显提高表浅膀胱癌术后的治疗效果，实现表浅膀胱癌的个体化膀胱灌注化疗。     

The effect of the histoculture drug response assay (HDRA) based perioperative chemotherapy for non-small cell lung cancer

In this study we analyze the usefulness of the histoculture drug response assay (HDRA) based perioperative chemotherapy for non-small cell lung cancer. From 2001 to 2006, we examined the chemosensitivity of 70 lung cancer tissues to cisplatin (CDDP), carboplatin (CBDCA), paclitaxel, docetaxel, gemcitabine and irinotecan. In 16 patients with stage III lung cancer who treated induction therapy, the response rate was 100% of 5 patients treated chemotherapy using 2 HDRA-positive drugs, 50% of 8 patient treeated using 1 positive drugs and 0% of 3 patients treated using negative drugs, respectively. The 3-year survival rate of the 5 patients treated using 2 positive drugs was better than that of 11 patient treated using 1 or non positive drugs (p = 0.07). In 39 patients with stage III lung cancer who treated adjuvant chemotherapy, the survival rate of the 14 patients treated chemotherapy using 2 positive drugs was significantly better than that of 25 patients treated using 1 or non positive drugs (p = 0.03). Therefore, HDRA may useful to the improvement of the response to chemotherapy and survival.     

In vitro chemosensitivity test for human genito-urinary tumors using collagen gel matrix

BACKGROUND: Although the aim of chemosensitivity tests is to predict the efficacy of anticancer agents for individual patients, no generally accepted assay has been established. METHODS: A chemosensitivity test was conducted for solid tumors with an organ culture system using collagen gel matrix (CGM). Seventy-five samples of transitional cell carcinoma (TCC), 20 of germ cell tumor (GCT) and 13 of renal cell carcinoma (RCC) were used for the chemosensitivity test, and 20 patients were treated with anticancer drugs on the basis of the test results. RESULTS: Positive rates of anticancer drugs for the 75 TCC samples were 64.9% for carboplatin, 63.4% for cisplatin, 32.1% for etoposide, 19.7% for THP-adriamycin, 16.7% for vinblastine, and 12.3% for methotrexate, indicating that positive rates of the latter three agents consisting of an MVAC regimen were unexpectedly low. The GCT had higher positive rates than the other cancers while RCC had the lowest. In 20 eligible patients (seven patients with bladder tumors and 13 with GCT), the true positive and true negative rates were 42% (5/12) and 75% (6/8), respectively, and the sensitivity and specificity were 71% (5/7) and 46% (6/13), resulting in a 55% (11/20) accurate predictive value. CONCLUSION: Although predictive accuracy was moderate when combination chemotherapy was used, information about chemosensitivity may have some beneficial effect on the treatment of patients with invasive bladder cancer or advanced GCT, because insensitive drugs detected by the test could be deleted or replaced with more sensitive ones.     

ATP—TCA技术指导浅表性膀胱癌术后灌注化疗的临床应用

目的：探讨生物荧光体外肿瘤药敏检测技术（ATP—TCA）在浅表性膀胱癌肿瘤细胞药敏试验中的应用,探讨该技术在指导膀胱癌个体化治疗上的应用价值。方法：对40例浅表性膀胱癌标本进行肿瘤细胞分离,原代培养,应用ATP—TCA技术检测肿瘤标本对五种常用化疗药物的敏感率和耐药率。试验组术后选用最敏感的化疗药物对患者常规行膀胱灌注化疗,对照组选择丝裂霉素进行术后常规化疗。术后随访2年,评价两组膀胱癌复发情况。结果：40例标本中,吡柔比星（THP）、羟基喜树碱（HCPT）、丝裂霉素（MMC）、表柔比星（EPI）、吉西他滨（GEM）的敏感率分别为75．0％、10．0％、5．0％、37．5％、10．0％,肿瘤细胞对五种化疗药物的敏感率和耐药率差异有统计学意义（P〈0．01）。膀胱肿瘤对化疗药物的敏感性存在个体差异。术后随访2年,药敏组膀胱癌复发率为17．5％（7／40）,对照组膀胱癌复发率为37．5％（15／40）。两组肿瘤复发率差异有统计学意义（P〈0．05）。结论：应用ATP—TCA技术检测出的药敏结果能够反映个体对化疗药物的敏感性,可以作为选择灌注化疗用药的理论基础,指导临床用药进行个体化治疗。同时,应用ATP—TCA技术指导临床膀胱癌术后灌注化疗,可显著降低浅表性膀胱癌患者术后复发率,提高临床疗效。     

A new chemosensitivity test for cancer cells by measuring intracellular ATP content

Several in vitro chemosensitivity tests have been developed to select effective anticancer agents for individual cases. However, none of them is used routinely because of the low evaluability or the time consuming nature. We developed a new practical method which is simple, rapid, and applicable to fresh human tumors. The principle of the method is to measure the ATP content of cancer cells by bioluminescence after drug exposure. A linear relationship was observed between either the number of cells or their viability and light intensity. Four established human stomach cancer cell lines and five colon cancer cell lines were examined for their chemosensitivity with a test plate having 96 wells. A clear dose-dependent response was seen with almost all drugs tested in this study, and each cell line showed an identical response to drugs. For the clinical application, cancer cells taken from three human solid tumors were tested. In all cases, the chemosensitivity was clearly evaluable. This simple, rapid and sensitive method can be a good indicator for the determination of anticancer agents in cancer chemotherapy.     

Evaluation of the Histoculture Drug Response Assay as a Sensitivity Test for Anticancer Agents

Abstract. Purpose: To investigate whether the effects of anticancer agents are able to be predicted, the results of sensitivity tests on anticancer agents were compared with the results of preoperative chemotherapy. Methods: Biopsies were taken from 25 patients with esophageal cancer and 10 with gastric cancer, before chemotherapy, and the drug sensitivity was determined after culturing for 1 week. The chemotherapy consisted of low-dose cis-diamino-dichloroplatinum + 5-fluorouracil, and its clinical effect was determined after 3 or 5 weeks. Results: Sensitivity was determined in 20 of the esophageal cancer patients and 8 of the gastric cancer patients, accounting for 80% of all the patients. Of the 11 patients judged to have sensitivity by the histoculture drug response assay (HDRA), 7 had a partial response, and of the 17 judged to have no sensitivity, 16 had a minor response or no change (NC). It was thus demonstrated that predictions of the effect of anticancer agents could be made with considerable accuracy using HDRA. Conclusion: The prognosis of the NC patients was poor, and distant metastasis, thought to be an adverse effect, soon appeared. From the viewpoint of both medical costs and patient quality of life, treatments other than preoperative chemotherapy should be selected for patients assessed to have NC. We believe that these sensitivity tests should be introduced clinically. Received: October 23, 2000 / Accepted: July 17, 2001     

Histopathological study of metallothionein in bladder cancer and renal cell carcinoma]

Metallothionein (MT) is a low molecular-metal binding protein with multiple biological functions. Recently, MT has been implicated as a factor involved in resistance to anticancer drugs, which presumably inactivates anticancer drugs, including cisplatin, and doxorubicin. In this report, we investigated the relationship of MT expression with the clinical features in bladder cancer and renal cell carcinoma. In 35 cases of bladder cancer, 10 cases of renal cell carcinoma and 3 cases of normal mucosa of bladder, the expression of MT was immunohistologically examined by avidinebiotin-peroxidase (ABC) staining of paraffin-embedded tissue specimens with anti-MT antibody. Intense MT expression was noted in all cases of normal mucosa of bladder. MT was detected in 10 of 35 cases of bladder cancer, with the incidence of MT expression being significantly higher increases with lower pathological tumor grade. MT was detected in 8 of 10 cases of renal cell carcinoma, and all of the their normal renal tubules showed more intense staining. A number of hypotheses can be proposed from these observations. First, our observation of decreased MT expression in poorly differentiated carcinomas, which are the more proliferating tumors, this suggests correlation of MT expression with proliferative status of cancer. Second, the higher incidence of MT expression in renal cell carcinoma than in bladder cancer may suggest that it is a factor responsible for the lower efficacy of chemo-therapy in renal cell carcinoma than in bladder cancer.     

In vitro growth and chemosensitivity studies of childhood cancers using clonogenic assay

We evaluated the usefulness of the recently developed Human Tumor Clonogenic Assay (HTCA) for chemosensitivity studies in childhood cancers. A total of 20 childhood cancer specimens containing 8 neuroblastomas, 4 malignant lymphomas, three hepatoblastomas, two rhabdomyosarcomas, two germinomas and one adrenal carcinoma were assayed. Overall, 65 per cent (13/20) of tumor cell specimens showed adequate colony forming ability to yield the sensitivity of anticancer drugs. The pattern ofin vitro sensitivity of a particular tumor to most anticancer drugs tested was not dissimilar to the clinical experiences of these agents. The overallin vitro response rate of childhood cancers to anticancer agents was 51 per cent. A retrospective analysis of this assay revealed a true positive rate of 57 per cent and a true negative rate of 100 per cent.     

Presence of serum p53 antibodies is associated with decreased in vitro chemosensitivity in patients with esophageal cancer

Resistance to chemotherapy remains a serious problem inhibiting the successful treatment of advanced esophageal cancer. A number of studies have revealed that p53 genetic alteration and protein overexpression can predict chemosensitivity. Furthermore, p53 protein overexpression in cancer tissues has been found to induce serum p53 antibodies (p53-Abs). This study was conducted to examine whether analysis of serum p53 Abs could predict the chemosensitivity of esophageal cancer. Serum analysis of p53 antibodies was performed by enzyme-linked immunosorbent assay in 19 patients with esophageal squamous cell carcinoma preoperatively, then surgically resected specimens were stained immunohistochemically for p53 protein expression. Tumor tissues were also analyzed for chemosensitivity by the histoculture drug response assay (HDRA) using cis-dichlorodiammineplatinum(II) (CDDP), 5-fluorouracil (5-FU), and adriamycin (ADM). Serum p53-Abs were present in 47% (9/19) of the patients and immunohistochemical analysis revealed overexpression of p53 protein in 42% (8/19) of the tumors. The presence of serum p53 antibodies was significantly correlated with p53 immunoreactivity (P = 0.005). The inhibition index of patients positive for p53-Abs was significantly lower than that of patients negative for p53-Abs (P < 0.001). This tendency was also observed in the inhibition index to 5-FU. The presence of serum p53-Abs was associated with decreased in vitro chemosensitivity to CDDP and 5-FU. Thus, the detection of serum p53-Abs is suggested to be useful for predicting chemosensitivity in patients with esophageal cancer. Received: August 2, 2000 / Accepted: March 6, 2001     

A study of fibroblasts in the chemosensitivity testing on human lung cancer cell lines]

The purpose of this study is to assess effects of fibroblasts in the vitro chemosensitivity testing on human lung cancer cells and to remove them. Fourteen lung cancer cell lines and 14 fibroblasts derived from resected specimens of lung cancers were used, whose S.D (succinate dehydrogenase) activities were measured with MTT colorimetric assay. The chemosensitivity of a lung cancer cell alone was compared with that of mixed cancer cell and fibroblast. As results, S.D activities of fibroblasts were less 2-4 fold than those of lung cancer cells. Fibroblasts were as sensitive to CDDP, MMC and 5-FU as lung cancer cells, but more sensitive to ADM and VP-16 than them. When sensitivity testings were performed on mixed cancer cells and fibroblasts, or mixed cancer cells and conditioned media of fibroblasts to CDDP with 3 day's incubation times, the sensitivity was affected in 61%, or 10% of all the pairs, respectively. However, when these tests were done without any incubation times, the sensitivity was not affected. Therefore, it was suggested that anticancer drugs had to be simultaneously added when single cell suspensions were plated if resected specimens were used in a anticancer drug sensitivity test.     

组织培养加药物敏感性测定在表浅膀胱癌术后丝裂霉素灌注化疗中的指导作用

目的 探讨组织培养加药物敏感性测定在指导表浅膀胱癌术后丝裂霉素(MMC)膀胱灌注化疗中的应用价值.方法 原发表浅膀胱移行细胞癌患者41例.男30例,女11例.平均年龄55岁.TNM分期:Ta 10例、T1 31例.病理分级:G1 9例、G2 22例、G3 10例.采用经尿道电切或膀胱部分切除术保留膀胱.肿瘤标本依次行三维组织培养和改良噻唑盐(MTT)法药物敏感性测定.生长抑制率>70%为高度敏感,50%～70%为中度敏感,<50%为不敏感.41例患者术后均行MMC标准膀胱灌注化疗.无复发随访时间5年,中途复发者则随访结束.结果 41例患者癌组织经1 g/L浓度MMC作用2 h后不敏感13例,其中Ta 1例、T1 12例(P=0.009),G1 1例、G2 7例、G35例(P=0.101).灌注化疗后随访3～5年,复发16例(39%),其中Ta 1例、T1 15例(P=0.059),G210例、G3 6例(P=0.016).对MMC不敏感组的复发率为77%(10/13),敏感组仅为21%(6/28,P=0.004).单因素Kaplan-Meier生存分析和Log-rank检验结果显示,平均无肿瘤复发时间不敏感组为16.5个月,敏感组为49.2个月(P<0.001).多因素Cox回归分析显示对MMC敏感性是独立预后因子(P=0.008).此法预测MMC膀胱灌注化疗效果的准确率为78%.结论 利用组织培养药物敏感性测定方法指导表浅膀胱癌术后MMC灌注化疗,可提高疗效,降低肿瘤复发率.     

大肠癌细胞和外周血淋巴细胞对化疗药物的体外敏感性

目的:探讨大肠癌细胞及外周血淋巴细胞对化疗药物体外敏感性的及二者的相关性。方法:用MTT法检测40份大肠癌标本及外周血淋巴细胞对氟尿嘧啶(5-FU)、奥沙利铂(L-OHP)、伊立替康(CPT)单药、两药及三药(全量或半量)应用的敏感性。结果:单药最有效的药物依次为伊立替康、奥沙利铂和氟尿嘧啶,敏感率分别为35.0%、27.5%和20.0%;三药联合应用的抑制效果显著优于两药联合(P<0.05),三药全量及半量联合应用效果差异无显著性(P>0.05);癌细胞及外周血淋巴细胞对3种化疗药物的敏感性有很好的相关性(r=0.969)。结论:MTT可用于为大肠癌患者选择合适的化疗药物,由氟尿嘧啶、奥沙利铂及伊立替康的联合应用具有高效协同抑制大肠癌细胞的作用。     

Study on chemosensitivity test of urogenital tumors by ATP assay]

In order to improve the efficacy of chemotherapy against urogenital cancers, we developed a chemosensitivity test by measuring intracellular ATP in cancer cells. In the fundamental experiments using HeLa cells, a significant correlation was observed between intracellular ATP levels and numbers of viable cells. We employed 8 drugs, ADM, VCR, VLB, MTX, 5-FU, PEP, MMC, and CDDP for the assay Thirty-four renal tumors, 68 urothelial tumors and 19 testicular tumors were tested, and evaluable results were obtained in 25 specimens of renal tumors (76%) 55 specimens of urothelial tumors (80%) and 17 specimens of testicular tumors (98%). According to the ATP assay, renal tumors were sensitive to ADM and MMC, urothelial tumors to ADM, MMC and CDDP and testicular tumors to ADM and MMC. The ATP assay to determine the chemosensitivity to clinical specimens was sensitive and efficient. Thus the ATP assay could be applied for selection of anticancer drugs in the chemotherapy of urogenital cancers.     

Histoculture drug response assay for gefitinib in non-small-cell lung cancer

Objective  There are many predictive factors for gefitinib sensitivity, including epidermal growth factor receptor (EGFR) gene mutation, EGFR copy number, and k-ras mutation. To investigate all of them is too expensive. We evaluated the chemosensitivity for gefitinib in non-small-cell lung cancer (NSCLC) using a histoculture drug response assay (HDRA). Methods  Surgically resected fresh tumor specimens from 22 patients with NSCLC were used. There were 13 male and 9 female patients, ranging in age from 49 to 84 (average 70) years old. Sixteen patients (73%) were smokers. Sixteen adenocarcinomas, four squamous cell carcinomas, and two other histological types were included. Small pieces of viable cancer tissue were placed on the collagen gel and then cultured for 7 days in the presence of gefitinib. Results  The HDRA was successful in all specimens. A dose-response relation was observed between inhibition rates and gefitinib concentration (p = 0.016). The inhibition rate at 20 μg/ml (IR₂₀) in adenocarcinoma without smoking (39.2% ± 35.1%, n = 6) was higher than that with smoking (2.2% ± 5.0%, n = 10, P = 0.001) and that of nonadenocarcinoma (16.9% ± 23.6%, n = 6, P = 0.09). Gene mutation analysis was performed in two of three adenocarcinomas without smoking, which showed especially high IR₂₀ values, and sensitizing mutations were observed in these specimens. A cutoff inhibition rate of approximately 40%–50% appeared to be suitable for a concentration of 20 μg/ml. Conclusion  HDRA appears to be applicable for evaluating sensitivity to gefitinib in NSCLC. It provides a convenient method for predicting the response to gefitinib in patients with NSCLC whose fresh tumor specimens are available.     

Prediction of survival in patients with head and neck cancer using the histoculture drug response assay

BACKGROUND: Chemoresponse is a significant outcome predictor in patients with head and neck cancer, regardless of the treatment modality used. The histoculture drug response assay (HDRA) has been shown to be a reliable method for in vitro chemoresponse assessment. In this study, we have correlated the HDRA assessment with survival in patients with head and neck squamous cell carcinoma (HNSCC). METHOD: Tumor specimens from 41 of 42 patients undergoing treatment for HNSCC were successfully evaluated by the HDRA. Tumor tissue was histocultured on Gelfoam sponges gel in 24-well plates, followed by treatment with cisplatin (15 microg/mL) or 5-fluorouracil (40 microg/mL) in triplicate. A control group received no drug treatment. After completion of drug treatment, the relative cell survival in the tumors was determined using the MTT assay. The inhibition rate (IR) for each drug was calculated relative to the control for each case, and sensitivity was defined as a tumor IR of greater than 30%. Treatment was based on established protocols for the location and stage of the tumor and included surgery, radiation, and/or chemotherapy. Survival comparisons were performed using the generalized Wilcoxon test for the comparison of Kaplan-Meier survival curves. RESULTS: Resistance to 5-fluorouracil was present in 13 cases (32%), to cisplatinum in 13 cases (32%), and to both agents in 11 cases (27%). The 2-year cause-specific survival was significantly greater for patients sensitive to 5-fluorouracil (85% vs 64%; p =.04), cisplatinum (86% vs 64%; p =.05), or both agents (85% vs 63%; p =.01). The association between HDRA assessment of chemoresponse and clinical outcome remained significant even after controlling for the effects of TNM stage and the presence of recurrent cancer at presentation by multivariate analysis. CONCLUSIONS: Chemosensitivity determined by the HDRA seems to be a strong predictor of survival in patients with advanced HNSCC and should be considered further.     

Comparison of the cytotoxic activities of chemotherapeutic drugs using a human bladder cancer cell line

Summary Many chemotherapeutic drugs have been used to treat patients with advanced bladder cancer, but few of these have been evaluated adequately in phase II clinical trials. Continuous cell lines provide one means for comparing the in vitro cytotoxicities of anticancer agents. In this study, a continuous cell line derived from a transitional cell cancer of the human bladder, which still produces tumours histologically similar to the tumour of origin on xenotransplantation, was used to measure the in vitro cytotoxicities of twelve chemotherapeutic drugs by clonogenic assay. The most cytotoxic agents tested were methotrexate, mitoxantrone, adriamycin, mitomycin C and cisplatin. These in vitro findings are compatible with the activity of these drugs given systemically as single agents in phase II clinical trials in patients with advanced bladder cancer.     

In vitro growth and chemosensitivity studies of childhood cancers using clonogenic assay

We evaluated the usefulness of the recently developed Human Tumor Clonogenic Assay (HTCA) for chemosensitivity studies in childhood cancers. A total of 20 childhood cancer specimens containing 8 neuroblastomas, 4 malignant lymphomas, three hepatoblastomas, two rhabdomyosarcomas, two germinomas and one adrenal carcinoma were assayed. Overall, 65 per cent (13/20) of tumor cell specimens showed adequate colony forming ability to yield the sensitivity of anticancer drugs. The pattern of in vitro sensitivity of a particular tumor to most anticancer drugs tested was not dissimilar to the clinical experiences of these agents. The overall in vitro response rate of childhood cancers to anticancer agents was 51 per cent. A retrospective analysis of this assay revealed a true positive rate of 57 per cent and a true negative rate of 100 per cent.     

浅表性膀胱肿瘤对化疗药物的敏感性研究

目的:探讨敏感化疗药物预防浅表性膀胱癌术后复发的作用。方法:对30例表浅性膀胱癌体外原代细胞培养,用表阿霉素、羟基喜树碱、吡柔比星、丝裂霉素和盐酸米托蒽醌进行MTT法药物敏感实验(敏感实验组),用最敏感药物行膀胱灌注;同期选择30例患者用吡柔比星灌注作为对照(对照组)。结果:敏感实验组17例对吡柔比星最敏感,6例对盐酸米托蒽醌最敏感,3例对丝裂霉素最敏感,1例对羟基喜树碱最敏感,1例对表阿霉素最敏感,2例对5种化疗药物都不敏感。28例患者分别选择最敏感药物灌注后随访2年,2例复发(7.1%)。对照组吡柔比星灌注后随访2年,有8例复发(26.7%)。两组比较差异有统计学意义(P<0.05)。结论:5种化疗药物对膀胱肿瘤的体外细胞毒作用不同,其中吡柔比星的抑制作用最强。根据药物敏感实验对浅表性膀胱肿瘤行灌注治疗,对预防肿瘤复发有较好效果。     

A trial of the in vitro sensitivity test of anti-cancer drugs using primary cultured urogenital cancer cells

Summary An in vitro chemosensitivity test was applied to clinical specimens of urogenital cancer tissues obtained at operation. Incorporation of ³H-leucine into primary cultured cells 24 h after treatment with cytotoxic drugs was used as an index for cell viability. Primary cell culture was performed using specimens obtained from 37 patients including 20 with transitional cell carcinoma, 15 with renal cell carcinoma and 2 with testicular cancer. Primary cell growth was achieved in 27 (73%) out of 37 specimens and 10 were tested for chemosensitivity. Each specimen of the tumor revealed different sensitivity to drugs, and results of quadruplicate tests for each specimen were identical. It was concluded that the present method of measuring incorporation of radioactivity using urogenital cancer cells primarily cultured in microtiter plate is practically applicable to an in vitro chemosensitivity test.