Growth patterns and cell kinetics of human osteosarcoma xenografts in serial passages in nude mice analysed byin vivo labelling with iododeoxyuridine

A human osteoblastic osteosarcoma was transplanted in nude mice and followed in seven serial passages. Tumour growth, structure and cell proliferation were studied in order to test the validity of the experimental tumour model. Tumour cell kinetics was analysed by in vivo labelling with the thymidine analogue iododeoxyuridine (IdUrd). Immunohistochemistry was used to measure the IdUrd labelling index. Duration of S phase (t _S) was estimated by flow cytometry. From these two parameters potential doubling time (t _pot) was calculated. Cell kinetic parameters showed low variations between passages and also between xenografts in the same passage. Smaller variations oft _S compared to labelling index andt _pot were found.t _pot was generally short with an interpassageal mean of 1.3 days and CV=14.8%. All xenografts showed DNA aneuploidy (mean DNA index=1.6). Homogeneous tumour growth was indicated by low variations of volume doubling time and lag time. There was no correlation between tumour growth and cell proliferation. Histopathological characteristics of the donor patient's tumour were retained during serial transplantation.Abbreviations IdUrd iododeoxyuridine - BrdUrd bromodeoxyuridine - t _pot potential doubling time - t _S duration of S phase - t _VD volume doubling time     

Effect of steroid hormones on human colorectal adenocarcinoma xenografts,of known steroid-receptor status,in nude mice

Summary The effect of hormone therapy on the growth of human colonic adenocarcinoma xenografts in nude mice was evaluated. Primary xeno-transplantation for ten different human colorectal adenocarcinomas into nude mice yielded a tumour take of 50%. One of these host tumours was found to contain androgen receptors (8 fmol/mg cytosol protein; K _d 0.73×10^-9 M), which were maintained in the xenograft at the third and ninth passages, but not expressed at the tenth and twelfth passages. The host tumour and its xenograft did not express either oestrogen or progesterone receptors. Administration of dihydrotestosterone led to inhibition of xenograft growth at the ninth passage compared with untreated controls (P<0.05), but had no effect on xenograft growth at the tenth and twelfth passages when androgen receptors were absent. Stilboestrol and progesterone failed to influence xenograft growth. In conclusion, dihydrotestosterone administration led to inhibition of xenograft growth only in the presence of androgen receptor, suggesting that some colorectal cancers might be considered steroid-hormone-sensitive tumours.Abbreviations AR androgen receptor M. J. G. Farthing is a Wellcome Trust Senior Lecturer and gratefully acknowledges financial support by the Wellcome Trust     

The heterogeneity of solid human xenotransplant tumours

Summary Fifteen xenograft tumour lines (4 osteosarcomas and 11 squamous carcinomas) were established on nude mice in numerous passages. Over a period of 4 years, samples were taken and studied systematically in more than 4000 histological slides. Of the tumour lines, 12 (4 osteosarcomas and 8 squamous carcinomas) showed remarkable dedifferentiation, but also redifferentiation towards a higher grade. There was a correlation between the rate of tumour takes in mice and poor prognosis in patients, but none between tumour doubling time and the phases of the cell cycle. Altogether, the results showed a great heterogeneity of human xenotransplants in terms of biological behaviour and morphological changes.     

Establishment and characteristics of two new human mammary carcinoma lines serially transplantable in nude mice

Two human mammary carcinomas of postmenopausal women were successfully transplanted into nude mice. Both tumours were classified as epidermal-growth-factor-, oestradiol- and progesterone-receptor-negative and c-erbB2-protein-positive. Histological studies of the primary tumours (4000 and 4151) revealed ductal invasive mammary carcinomas. In the first passages the precondition for the growth of breast carcinoma 4000 were pretreatments of the nude mice with oestradiol and peanut oil before transplantation. The mammary carcinomas 4000 and 4151 described here are suitable for in vivo testing of antineoplastic substances and for biological studies.This work was supported by Schering AG Berlin     

Cytokines and pancreatic cancer. Sensitivity of xenotransplants of predominantly pancreatic carcinomas to rIFN-gamma and rTFN-alpha in nude mice

Ten xenotransplants of human gastrointestinal carcinomas (eight human pancreatic carcinomas) were assayed for their sensitivity to human rIFN-gamma and human rTNF-alpha in nude mice. Both substances demonstrated a dose and route dependent antitumoral activity in principle. However, the extent of response varied distinctly between the tested xenografts. rTNF-alpha was clearly superior to rIFN-gamma at systemic application of comparable doses (0.8 mg/kg/d). Intramural administration of both cytokines could cause cytotoxic effects and was significantly more effective than systemic administration that predominantly resulted in antiproliferation. Growth inhibition of rIFN-gamma or rTNF-alpha alone could be clearly enhanced by combining both cytokines. In addition, the results suggest: the possibility to enhance the effects of rIFN-gamma alone also by combination with nIL 2, as well as a decrease of the effects of rTNF-alpha with time of therapy.     

DNA cell-cycle analysis of cervical cancer by flow cytometry using simultaneous cytokeratin labelling for identification of tumour cells

DNA ploidy and cell-cycle distribution were determined by flow cytometry in fresh tumour tissue of 53 cervical carcinomas. Epithelial cells were labelled by a fluorescein-isothiocyanate-conjugated cytokeratin antibody (CK6, CK18) to study the influence of contaminating stromal and inflammatory cells on results of cell-cycle analysis of tumour cells. Without identification of cytokeratin-positive cells 30/53 (57%) tumours were found to be DNA-aneuploid compared to 43/53 (81%) after gating for cytokeratin. Only 7 of 15 DNA-multiploid tumours could be detected without cytokeratin staining. In addition, cytokeratin-negative cells, which are found in all tumours, can be used as an internal standard for the calculation of ploidy and for quality control (coefficient of variation, linearity) of each individual sample. Cell-cycle analysis revealed significantly higher S-phase and G₂M-phase fractions in cytokeratin-gated compared to ungated samples (13.1% versus 10.0% and 8.0% versus 5.4%;P<0.001). This difference was more pronounced in DNA-diploid than DNA-aneuploid tumours. In conclusion, about 30% of DNA-aneuploid tumours could only be detected after cytokeratin labelling of epithelial cells. Owing to the identification of cytokeratin-positive cells the influence of non-tumoural cell elements on cell-cycle analysis was reduced markedly. Therefore, in cervical cancer, cytokeratin labelling can optimize both the determination of DNA ploidy and cell-cycle analysis.     

Phenotypic characteristics of Epstein-Barr-virus-associated gastric carcinomas

We identified Epstein-Barr-virus(EBV)-associated gastric carcinomas by in situ hybridization that targets EBV-encoded small RNA, and investigated their phenotypic characteristics by mucin histochemistry. Out of 132 gastric carcinomas, 15 were EBV-positive, and they were exclusively located at the proximal part of the stomach; 8 were early and 7 were advanced carcinomas. Out of the 15 EBV-positive carcinomas, 10 were so-called gastric carcinoma with lymphoid stroma (GCLS); 4 of the other 5 tumours were moderately differentiated adenocarcinoma (tub 2) showing a lace pattern. This pattern was also seen in the intramucosal parts of 6 (3 early and 3 advanced) GCLS. As for the mucin phenotype of the cancer cells, the gastric type was predominant in the EBV-positive tumours, while the EBV-negative tumours, even with the lace pattern or GCLS, showed phenotypes other than the gastric type (intestinal or mixture of intestinal and gastric). It was inferred that EBV-positive gastric carcinomas show tub 2 with the lace pattern and gastric phenotype in earlier stages, and become GCLS during the tumour progression, but the gastric phenotype tends to remain throughout the history.Abbreviations EBV Epstein-Barr virus - GCLS gastric carcinoma with lymphoid stroma - EBER EBV-encoded small RNA - GOS galactose oxidase Schiff - CPS concanavalin A paradoxical staining     

Effect of radiation therapy on the potential doubling time of tumours in colorectal cancers.

OBJECTIVE: The aim of the present study was to investigate the effects of standard fractionated radiation therapy on the kinetic parameters of colorectal adenocarcinomas. METHODS: The study of tumour kinetics involved in vivo injection of bromodeoxyuridine. Endoscopic biopsies were obtained from the tumour and analysed with flow cytometry. This procedure provides a rapid calculation of qualitative parameters such as ploidy and quantitative parameters such as the in vivo S-phase fraction labelling index which indicates the percentage of cells that have entered into the cycle, the duration of S-phase (Ts) and the potential tumour doubling time (Tpot). RESULTS: Thirty-eight colorectal carcinomas were studied without prior chemotherapy or radiation therapy (group 1) and ten rectal carcinomas were studied following radiation therapy (group 2). In diploid tumours, the labelling index was significantly lower in the post-radiotherapy group than in the pre-radiotherapy group (2.7 +/- 1.1% versus 6.4 +/- 4.2%, respectively; P= 0.01), and the Tpot was significantly longer after radiotherapy (group 2) (22.0 +/- 7.0 days versus 8.6 +/- 6.0 days, P = 0.002). Standard fractionated radiation therapy also appears to result in a longer Tpot in diploid adenocarcinomas of the colon and rectum. This effect was not observed in aneuploid tumours. CONCLUSIONS: The effectiveness of hyperfractionated schedules of radiation therapy for aneuploid rectal tumours with short Tpot warrants further investigation in a larger patient population.     

Proliferation of Human Lymphoid Cells Lymphocytopoiesis and Cell Cycle Parameters in Isolated Perfused Human Spleens

Parameters of the cell cycle of lymphoid cells and the rate of production of small lymphocytes and mature plasma cells were estimated in human spleens. 12 normal and 1 enlarged spleen with lymphatic hyperplasia were labelled either by a pulse or continuously with ³H-TdR-thymidine during normothermic perfusion of the isolated spleens. Biopsies of splenic tissue were taken at different intervals and evaluated autoradiographically. The mean initial labelling index of immunoblasts was 73% and for plasmoblasts 52%. The labelling index for small lymphocytes and mature plasma cells increased initially from 0 % to 1 %, and up to 25 % respectively after 14 h. The duration of the S-phase, as determined by double labelling, was 8.26 h for lymphoid blasts. Data from the percentage labelled mitosis curve resulted in a minimal t_G2 of about 1 h and t_G2 + t_M of about 4 h. The estimated generation time was about 11.5 h. The results found on the hyperplastic spleen were comparable to those on the normal spleen.     

Production and pre-clinical significance of haematopoietic peptide growth factors (HPGF) in human non-seminomatous germ cell tumour cell lines

Peptide growth factors involved in the regulation of haematopoiesis (HPGF), for example granulocyte-colony-stimulating factor (G-CSF) and granulocyte/macrophage-colony-stimulating factor (GM-CSF), are of clinical importance in the treatment of testicular germ cell tumour (GCT) patients with modern chemotherapy regimens since they ameliorate chemotherapy-induced neutropenia. Aberrant expression of and/or response to HPGF has been reported in several solid tumour types although no data are available on GCT with the exception of those on stem cell factor (SCF). The aims of this pre-clinical study were twofold: (1) to screen a panel of human non-seminomatous (NS)GCT for the production of HPGF and (2) to test the effects of G-CSF or SCF on the growth of NSGCT cell lines in vitro, and on the growth kinetics of two human NSGCT xenograft models. HPGF concentrations in cell culture supernatant from 11 NSGCT cell lines growing under routine culture conditions were measured by enzyme-linked immunosorbent assay. The growth kinetics of cell lines was quantified in vitro using the sulphorhodamine B assay. The growth kinetics of nude mouse NSGCT xenografts was followed by measuring tumour volumes every 2–3 days over days 1–30, following daily subcutaneous injection of nude mice (days 1–14). The cell lines produced G-CSF (1/11 cell lines), GM-CSF (2/11), SCF (2/11), M-CSF (6/11), and interleukin-6 (9/11). Growth stimulation of cell line H12.1 by SCF was observed in vitro, but no statistically significant differences in NSGCT xenograft tumour volume (V _T) or relative V _T (rV _T) in treated groups were observed on days 14 or 29 compared to the control. The change in rV _T of H12.1 xenografts treated with G-CSF alone compared to control (rV _T/rV _T,c) was 0.96 on day 29. The values for rV _T/rV _T,c for H12.1 xenografts treated with G-CSF in combination with low- or high-dose SCF were, respectively, 1.67 or 1.7 compared to 1.19 for SCF-treated mice. The results are in agreement with clinical data to date where no observations have been reported of stimulation or inhibition of tumours in patients receiving treatment with G-CSF. Before any clinical trials are initiated in GCT patients treated with G-CSF in combination with SCF, further pre-clinical experiments with this tumour type are recommended to investigate this phenomenon further in a greater number of NSGCT cell lines in vitro and in vivo and with a wider range of SCF/G-CSF schedules. The potential relevance of secretion of HPGF in NSGCT cell lines in vitro to the pathobiology of GCT in patients is also a subject of interest for future research. Received: 12 May 1997 / Accepted: 19 April 1998     

Gastric and intestinal phenotypic cell marker expressions in gastric differentiated-type carcinomas: association with E-cadherin expression and chromosomal changes

Gastric and intestinal phenotypic cell markers are widely expressed in gastric carcinomas, irrespective of their histological type. In the present study, the relations between the phenotypic marker expression of the tumour, histological findings, expression of cell adhesion molecules, and the chromosomal changes in gastric differentiated-type carcinomas were examined. The phenotypic marker expression of the tumour was determined by the combination of the expression of the human gastric mucin (HGM), MUC6, MUC2 and CD10, and was evaluated in comparison with the expression of cell adhesion molecules, such as E-cadherin and β-catenin, and chromosomal changes by comparative genomic hybridization (CGH) in 34 gastric differentiated-type carcinomas. Tumours were classified into the gastric- (G-), gastric and intestinal mixed- (GI-), intestinal- (I-), or unclassified- (UC-) phenotype according to the immunopositivity of staining for HGM, MUC6, MUC2, and CD10. G-phenotype tumours were significantly associated with a higher incidence of differentiated-type tumours mixed with undifferentiated-type component, compared with GI- and I-phenotype tumours (88.9 vs 33.3%, P=0.0498 and 88.9 vs 42.9%, P=0.0397; respectively). HGM-positive tumours were significantly associated with a higher incidence of tumours with abnormal expression of E-cadherin, compared with HGM-negative tumours (66.7 vs 21.1%, P=0.0135). GI-phenotype tumours were significantly associated with a higher incidence of tumours with abnormal expression of E-cadherin, compared with I-phenotype tumours (77.8 vs 21.4%, P=0.0131). HGM-negative tumours were significantly associated with higher frequencies of the gains of 19q13.2 and 19q13.3, compared with HGM-positive tumours (57.9 vs 20.0%, P=0.0382 and 63.2 vs 13.3%, P=0.0051; respectively). MUC6-positive tumours were significantly associated with higher frequencies of the gains of 20q13.2, compared with MUC6-negative tumours (71.4 vs 30.0%, P=0.0349). MUC2-positive tumours were significantly associated with the gain of 19p13.3, compared with MUC2-negative tumours (41.2 vs 5.9%, P=0.0391). I-phenotype tumours were significantly associated with higher frequencies of gains of 5p15.2 and 13q33-34, compared with G-phenotype tumours (66.7 vs 0%, P=0.0481, each) and also associated with higher frequencies of gain of 7p21, compared with GI-phenotype tumours (66.7 vs 0%, P=0.0481). Our present results show that gastric differentiated-type carcinomas have different characteristics according to the phenotypic marker expression of the tumour in terms of histological findings, E-cadherin expression and pattern of chromosomal changes.     

Morphology and modes of cell proliferation in earliest signet-ring-cell carcinomas induced in canine stomachs byN-ethyl-N′-nitro-N- nitrosoguanidine

Summary Signet-ring-cell carcinomas were induced in the stomach of 12 beagle dogs by p.o. administration ofN-ethyl-N-nitro-N-nitrosoguanidine (ENNG), and the morphology and modes of cell proliferation in an incipient stage of cancer growth were studied with bromodeoxyuridine (BrdUrd) incorporation. From 5 to 27 months after the completion of 8 months' carcinogen treatment, minute carcinomas were found in the stomachs of 9 dogs. Before sacrifice, the dogs were given a single or repeated i.v. injections of BrdUrd for 1–3 days. Minute signet-ring-cell carcinomas were found to form a layered structure, in which the cancer cells proliferated in the lamina propria at the gland-neck level and differentiated to postmitotic signet-ring cells at the upper and lower levels of the mucosa. From repeated injections of BrdUrd, the time required for all the proliferative cells to be labelled with BrdUrd (reflecting the maximum cellcycle time) was estimated to be 1.7 days for the normal glands, and 2.7 days for minute signet-ring-cell carcinomas. From the labelling index with BrdUrd as well as from the morphology, earliest carcinomas were identified in the single gland. There remained atrophic normal epithelium commonly in the single-gland lesions. Proliferative atypical cells appeared to be shed into the stroma passively through the atrophy and subsequent collapse of the gland rather than through active invasion. This may be a reason why cancer cells in minute signet-ring cell carcinomas preserved the normal pattern of cell renewal movement to form the layered structure.Abbreviations used ENNG N-ethyl-N-nitro-N-nitrosoguanidine - BrdUrd bromodeoxyuridine - LI labelling index - t _c cell cycle time - t _s duration of DNA synthetic phase This work was supported by Grants-in Aid for Cancer Research from the Ministry of Education. Science and Culture, Japan.     

Proliferative heterogeneity of human renal cell carcinomas and prevalence ofras gene point mutations

The variable prevalence and a possible stage-dependent increase ofras gene point mutations in human tumors might correspond to clonal growth advantages ofras-activated cells. Tumor areas with activatedras genes might thus differ in proliferative activity from those lackingras gene activation. This hypothesis is studied in a series of human renal cell carcinomas that had been used previously for an analysis of proliferative compartments after post-operative vascular [³H]/[¹⁴C]thymidine perfusion [Rabes et al. (1979) Cancer 44: 799–813]. The growth fraction of different subcompartments of these tumors was studied by immunohistochemistry with mib1 antibody, recognizing a fixation- and embedding-resistant epitope of Ki-67 protein. Thirty subpopulations of 14 human renal cell carcinomas that exhibited a broad spectrum of proliferative activity were chosen for an analysis of the prevalence of K-ras point mutations in exon 1 by a mutation-enriching primermediated restriction-fragment-length-polymorphism analysis and/or direct sequencing of polymerase-chain-reaction-amplified material. The combined autoradiographic and immunohistochemical analysis confirmed the intra- and intertumoral proliferative heterogeneity. Compared to [³H]/[¹⁴C]thymidine labeling indices, mib1 labeling indices are higher. The ratio of mib1 to [³H]/[¹⁴C]thymidine labeling indices varies from 1.9 to 4.1 for the individual tumor subcompartments. However, neither in K-ras codons 12/13 nor in adjacent codons did we detect any mutations in the various tumor compartments. The results suggest that neither mode of proliferation nor type of differentiation is related to K-ras exon 1 point mutations in human renal cell carcinomas.Abbreviations PCR polymerase chain reaction - RCC renal cell carcinoma - BrdUrd 5-bromo-2-deoxyuridine - [³H]dThd [³H]thymidine Supported by a grant from Dr. Mildred Scheel-Stiftung für Krebsforschung, Bonn, F. R. Germany     

In vivo induction of neoplastic growth in nude mouse connective tissue adjacent to xenografted human tumors

Summary Induction of neoplastic growth of murine stroma cells within the human tumor xenograft was observed after serial passage of CEA and 2-microglobulin producing human colonic SLu tumor xenografts in nu/nu BALB/c mice. Mouse tumors within the human tumor xenografts wre identified using specific immunohistologic staining techniques for mouse histocompatibility marker or human CEA. These mixed tumors could be distinguished from normal human tumor xenografts by a different relationship between development of the tumor marker in the serum and tumor size. We were able to establish transformed murine cells from human xenografts, either induced by SC injection of 1×10⁶ tumor cells of the SLu cell line or by human SLu or mammary carcinoma tissue serially passaged in athymic animals. The established human and murine cell lines were characterized by cytogenetic methods. Transformed murine cells were then continuously passaged in tissue culture. The transformed mouse fibroblasts proved to possess tumorigenicity in nude mice. In the case of SLu-derived mouse tumor cells, tumors also developed in the immunocompetent BALB/c mice using 1×10⁶ to 5×10⁶ tumor cells for SC transplantation.     

Calcitonin gene-related peptide in small cell lung carcinomas

OBJECTIVE Calcitonin gene-related peptide (CGRP) is a regulatory peptide encoded by the calcitonin gene. CGRP is expressed in increased amounts by the cells of medullary thyroid carcinomas and has been demonstrated by immunohistochemistry to occur in neuroendocrine cells and nerve fibres of lung tissue. MEASUREMENTS Serum CGRP levels were measured in patients with small cell lung carcinomas before treatment (n= 74) and immediately before the second course of chemotherapy (n= 30). In-situ hybridization and immunohistochemistry were performed on tumour tissue and CGRP was extracted from two tumours and characterized by gel chromatography and high pressure liquid chromatography. RESULTS Serum CGRP levels were elevated in small cell lung carcinomas when compared with healthy controls of similar age and sex (median values 55.0 vs 36.6 pmol/l, P<0.001), and 27% had levels above the upper normal range. Serum CGRP levels decreased following the initial course of chemotherapy (P < 0.05) but remained elevated when compared to the controls (P < 0.001). In-situ hybridization for CGRP mRNA was positive in three of 17 tumours and immunohistochemistry was positive in seven of 31 tumours investigated. CGRP immunoreactivity extracted from two tumours was characterized by gel chromatography and high pressure liquid chromatography. A major part of the immunoreactivity was demonstrated to represent the intact molecule. CONCLUSIONS We found that patients with small cell lung carcinomas had elevated concentration of serum calcitonin gene-related peptide but only 27% had values above the upper normal range. Serum CGRP is therefore of limited value as a tumour marker. Intact CGRP can be extracted from tumour tissue, but in-situ hybridization and immunohistochemistry showed positive reactions in only a few of the tumours investigated. The elevated serum CGRP levels are therefore likely to be largely of extratumoral origin.     

Two distinct cell lines derived from a human osteosarcoma

Summary Two cell lines were established from a human osteosarcoma transplanted into athymic nude mice after the second (O9N2) and fifth passages (HuO9). Both cell lines expressed 1,25(OH)₂D₃-responsive alkaline phosphatase activity and produced tumors in the dorsum of nude mice that were histologically similar to the original tumor. However, the morphological and growth characteristics of the two cell lines differed. O9N2 cells were large and polygonal, whereas HuO9 cells showed spindle shapes. HuO9 cells had a higher growth rate and saturation density than O9N2 cells. The c-myc oncogene was amplified 4-to 8-fold in HuO9 cells but not in O9N2 cells. Both cell lines had a homozygous internal deletion, lacking the 7.4-kb HindIII fragment in the Rb gene. The results suggest the importance of the c-myc oncogene in the growth and morphological control of human osteosarcoma cells and of the Rb gene in the pathogenesis of the tumor.Abbreviations used ALP alkaline phosphatase - PBS phosphatebuffered saline - 1,25(OH)₂D₃ 1,25-dihydroxyvitamin D₃ - Rb gene retinoblastoma gene     

Human monoclonal antibody SK1-mediated cytotoxicity against colon cancer cells

PURPOSE: Human monoclonal antibody (HuMAb) SK1, a human monoclonal IgM, has previously been shown to react selectively with a wide range of human carcinomas. In this study, the complement-dependent cytotoxicity (CDC) mediated by the HuMAb SK1 was investigated. METHODS: The presence of AgSK1 on the two studied cell lines, HT29 and PANC-1, was evaluated by the immunocytochemical staining. The intracellular and surface locations of the targeting antigen of HuMAb SK1 were further characterized by the study of flow cytometry. The specific lysis of target cells by the HuMAb SK1 in the CDC assay was studied. RESULTS: In the presence of human complement, the HuMAb SK1 was shown to be effective in the lysis of cultured human gastrointestinal cancer cells as well as the fresh colon cancer cells derived from the patient's specimens. In addition, our data suggested that HuMAb SK1 activated the mouse complement in a similar magnitude. CONCLUSIONS: We concluded that HuMAb SK1 showed some promise for future clinical trials. The in vitro CDC effect of HuMAb SK1 with mouse complement suggested that the antitumor effect of HuMAb SK1 might be successfully studied in the nude mouse model bearing xenografts of human colon cancer as a part of the preclinical evaluation.This study was supported in part by Grant R29 CA56299-01A1 from the National Cancer Institute.Poster presentation at the meeting of The American Society of Colon Rectal Surgeons, May 1991.     

The carcinogenic effect of 2,2-dioxopropylnitrosamine on the renal pelvic epithelium of Sprague-Dawley rats,after chronic subcutaneous injections

Summary The carcinogenicity of 2,2-dioxopropylnitrosamine on the urinary tract was investigated in three experimental groups of Sprague-Dawley rats (15 males, 15 females/group) by weekly subcutaneous administration for the life of the carcinogen at dose levels 1/5, 1/10 and 1/20 of the LD₅₀, and compared with that in a similar group of untreated controls. It resulted in the induction of urinary tract tumours in 42 out of 79 effective animals (53%). Of these animals, 38 developed tumours within the renal pelvis. In the high-dose group, females had a 100% incidence of renal pelvic tumours, and males 73%. In all experimental groups, renal pelvic tumours were more frequent than ureteral and vesical ones. Histologically, the tumours were transitional cell papillomas and carcinomas, except for one squamous carcinoma. Out of 66 tumours, 42 (64%) were low-grade. High-grade tumours arose mainly in the renal pelvis of animals belonging to the highest-dose group. This experiment offers a useful model for the study of mechanisms involved in renal pelvic carcinogenesis.Abbreviation DOPN 2,2-dioxopropylnitrosamine - DHPN dihydroxy-di-n-propylnitrosamine     

Role of KIT expression in the prognosis of clear cell renal cell carcinomas in Chinese patients

Objectives  To investigate the expression of KIT in clear cell renal cell carcinomas, and to reveal the relationships between KIT status and clinicopathological features and survival of clear cell renal cell carcinomas. Methods  The expression of KIT was tested immunohistochemically in 119 specimens of clear cell renal cell carcinoma. Their correlations to clinicopathological parameters were compared and discussed. Kaplan–Meier method was used to determine the survival between KIT-positive and KIT-negative patients. Multivariate analysis was performed using the Cox-regression model for overall survival. Results  A total of 13 out of 119 cases of clear cell renal cell carcinomas (10.9%) were demonstrated consistent overexpression of KIT. There was statistical significance in the correlation between KIT expression and tumor size (P < 0.01), pathological stage (P < 0.01), tumor grade (P < 0.01) and P53 (0.01 < P < 0.05). On multivariate analysis, positive KIT expression presented an independent predictive factor for decreased overall survival (hazard ratio 17.26, P = 0.005). The estimated mean survival time was 25.6 months for KIT-positive patients and 56.9 months for KIT negative patients, P < 0.001. Conclusions  The expression of KIT was significantly associated with tumor size, pathological stage, tumor grade and P53 in clear cell renal cell carcinomas. The expression of KIT is an important survival predicting factor for patients with clear cell renal cell carcinoma.     

Tirapazamine plus cisplatin and irradiation in a mouse model: improved tumor control at the cost of increased toxicity

Purpose Tirapazamine (TPZ) reportedly enhances the tumor cell killing effect of cisplatin up to fivefold and it is an attractive drug for combination with radiotherapy. We evaluated the toxicity of a fractionated combined treatment. Methods Murine RIF-1 fibrosarcomas growing on the right hind foot of C3-H mice were used. Within 2 weeks, animals were treated with six i.p. injections of TPZ (43.2–172.8 mg/kg total), and/or cisplatin (24 mg/kg total) and ten fractions of 2 Gy to the tumor. All treatments were carried out under anesthesia. Maximum follow-up was 35 days. The local tumor control was determined by calculating the tumor doubling time t _2vo. In addition to standard toxicity assessment, the major inner organs were examined histologically. Results The administration of low TPZ doses to the cisplatin/radiotherapy treatment caused only little changes in tumor doubling time (t _2vo) and led to a lethality rate of 15–30%. Higher TPZ doses caused an increase in t _2vo, but also a further increase in lethality and toxicity in particular to the heart, liver, kidney and stomach. Cisplatin/radiotherapy treatment without TPZ produced no severe toxicity. Conclusions This is a detailed study of both the acute and delayed toxicities of combined TPZ treatment in a mouse model. In our study the addition of TPZ to the cisplatin/radiotherapy treatment caused a significant increase in toxicity with only moderate effect on the tumor. This work was supported by: Deutsche Forschungsgemeinschaft (DFG grant Ad132/2-1).