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Genomic imprinting of LIT1 gene in colorectal cancer
AGAA1413
April 2000
and the intensity of caspase-3 expression was clearly weaker in the lymphoma cells compared with the normal lymphoid cells.Conclusions : The present data suggested that anti-apoptotic property of MALT lymphoma shown by the lower expression of caspase- 3 rather than acceleration of cell proliferation, may have pathogenic significance. This might contribute to the indolent characteristics of the MALT type lymphoma.
6417 CENTRAL NEUROTOXICITY INDUCED BY OXALIPLATIN REPORT ON 4 CASES. Sarah Taieb, Gilles Freyer, Loic Rambaud, Louis Descos, Veronique Trillet-Lenoir, Ctr Hospitalier Lyon Sud, Pierre Benite, France. Background: Oxaliplatin in association to fluoro-uracil and folinic acid is a new drug widely used as a first line treatment in patients with metastatic colorectal cancer. Oxaliplatin is responsible for a dose-dependent neurotoxicity which affects peripheral nerves resulting in quadridistal and peribuccal dysesthesia which occur at low temperature. These symptoms usually decrease and disappear a few days after completion of chemotherapy. At elevated cumulative doses of oxaliplatin, paresthesia may become persistent and unrelated to cold inducing patients' functional disability. Paresthesia usually resolve a few weeks or months after treatment discontinuation. To our knowledge, oxaliplatin-induced neurotoxicity has been described as mainly peripheral. We report here the first 4 cases of central neurotoxicity induced by oxaliplatin. Case reports: From 1998 to 1999, 100 patients with metastatic colorectal cancer were treated by oxaliplatin (85mg/sqm), 5-fluoro-uracil (bolus: 800mg/sqm + continuous infusion: 1200mg/sqm) and folinic acid (200mg/sqm) as a 48-hour bimonthly regimen. Among these patients, 4 patients experienced central neurotoxicity. A Lherrnitte's sign (paresthesia or dysesthesia induced by cervical spine flexion) was observed in 3 cases, in association with genito-sphincteral deficiency in I case and in association with proprioception deficit in I case ; I patient experienced genito-sphincteral deficiency with saddle type of hypoesthesia and pyramidal syndrome. In the 4 cases, the cumulative dose of oxaliplatin was always higher than JOOOmg. Central neurologic symptoms resolved a few weeks after discontinuation of the treatment. Somatosensory evoked potentials were recorded in 2 patients and showed central toxicity signs with disappearance of cervico-medullary response. In one patient somatosensory evoked potentials recording contributed to the decision of the investigator to stop oxaliplatin administration. Conclusions: Oxaliplatin might be responsible for central nervous system toxicity in a limited number of patients. Since this drug is very promising in colorectal cancer treatment, careful clinical follow-up is required. Somatosensory evoked potentials recording could help to display infraclinical signs of central neurotoxicity before the occurrence of severe functional disability.
6418 DETECTION OF TELOMERASE ACTIVITY IN CANCER CELLS OBTAINED FROM BILE. Itoi Takao, Shinohara Yasushi, Takeda Kazuya, Nakamura Kazuto, Shimizu Masafumi, Takei Kazuo, Ohno Hiroyuki, Tokyo Med Univ, Tokyo, Japan. Telomerase is detected by the telomeric repeat protocol (TRAP) assay in more than 85% of primary cancers. In the present study, we determined telomerase activity using exfoliated bile cells obtained from biliary tract cancers. The aim of this study was to provide additional information regarding minimally invasive approachs to the detection of biliary tract cancer in combination with routine cytologic examination. We analyzed for telomerase activity in the bile from 3 patients with gallbladder carcinoma, 7 patients with cholangiocarcinoma, 4 patients with cholecystitis and 3 patients with cholangitis. Semiquantitative determination of telomerase activity was performed using both a fluorescence-based TRAP assay on cell extracts and at celluar level by an in situ TRAP assay. The fluorescence-based TRAP assay detected bile telomerase activity in samples from 4 of 10 patients with biliary tract cancer. In contrast, the in situ TRAP assay detected telomerase positive cells in samples from 6 of 10 patients with biliary tract cancers. However only one of these samples showed class V cytology. No non-malignant disorder detected telomerase activity by both an in situ TRAP and fluorescence-based TRAP assay. In conclusion, a combination of semiquantitative analysis and an in situ TRAP assay to detect telomerase positive cells may improve the diagnosis of biliary tract cancers with the combination of routine cytologic examination.
6419 THYMIDINE PHOSPHORYLASE (TP) IS ASSOCIATED WITH TUMOR INVASION AND METASTASIS: EFFECT OF ITS INHIBITOR ON EXPERIMENTAL LIVER METASTASIS. Sonshin Takao, Shin-ichi Akiyama, Tetsuhiro Nakajou, Hiroyoshi You, Masaki Kitazono, Futoshi Miyazono, Youichirou Matsuo, Hidetoshi Noma, Chikara Kusano, Hiroyuki Shinchi, Shouji Natsugoe, Takashi Aikou, Kagoshima Univ Sch of Medicine, Kagoshima, Japan. Backgrounds: Thymidine phosphorylase (TP) catalyzes the reversible phosphorolysis of thymidine, deoxyuridine, and their analogs. In mammals, TP is a homodimer of 55 kDa subunits, and we found that TP is identical to platelet-derived endothelial cell growth factor, and has angiogenic activity in vivo. In the mouse dorsal air sac assay model, TP inhibitor (TPI) completely suppressed the angiogenesis induced by KBrrp cells. We determined whether TPI can suppress blood-borne metastasis such as
hepatic metastasis. Cells: KBrrp cells (KB cells transfected with RSVrrp) and KB/CV cells (KB cells transfected with RSV). Methods: Immunoblotting analysis, Immunocytochemical staining, Invasion-MTT Assay (We defined the in vitro invasive ability of the tumor cells as percent invasion), chemotaxis assay, and quantitative PCR. Experimental liver metastasis: KBrrP cells (I x 105 ) were injected into the spleens of nude mice, and TPI (100 mg/Kg/day) was administered orally to the mice for 4 weeks. To detect micrometastasis in the liver, whole livers were removed. Expression of human f3-globin in the livers of nude mice was detected by PCR 3, 7, 14, 19 and 24 days after injection of the KBrrP cells into the spleens. Results: I) The growth rate of cultured KBrrp was similar to that of KB/CV cells, and TPI (500 JLM) did not affect the growth rate of either cell line in vitro. 2) KBrrp tumors in nude mice grew significantly faster than KB/CV tumors, and TPI decreased the growth rate of KBrrp tumors in vivo. 3) The chemotactic motility of KBrrp cells was much higher than that of KB/CV cells, but TPI inhibited the level of KBrrP cells to that of KB/CV cells. 4) The percent invasion level of KBrrP cells was higher than that of KB/CV cells, but TPI inhibited the percent invasion level of KBrrp cells to that of KBfCV cells. 5) In experimental liver metastasis, the expression level of human l3-globin mRNA in the livers of the control group was 6-fold higher than that of the TPI-treated group at day 24. 6) The number of macroscopic metastatic nodules (115 ± 58) in the livers of the control group (n = 7) was significantly higher than in the livers (18 ± 14) of the TPI treated group (n = 7) 4 weeks after injection of KBrrp cells. Discussion: Our findings indicate that the inhibition of TP acting by TPI causes suppression of motility and invasion in addition to inhibition of angiogenesis. TPI might be valuable for treating patients with metastatic TP-expressing tumors. 6420 GENOMIC IMPRINTING OF LITI GENE IN COLORECTAL CANCER. Kiwamu Tanaka, Goshi Shiota, Hironaka Kawasaki, Tottori Univ, Yonago, Japan; Tottori Univ, Yonaga, Japan. Aims. Genomic imprinting is epigenetic modification leading to the functional inequality of paternal and maternal alleles in somatic cells and loss of imprinting (LOl) has been shown to play an important role in carcinogenesis. Recently, the transcript, called LIT! (long QT intoronic transcript I), has been identified within the KvLQT! locus and expressed preferentially from the paternal allele and produced in most human tissues. LOI of LIT! was found in Beckwith-Wiedemann syndrome (BWS) patients and reported to be associated with the etiology of BWS. However, there is no information about genomic imprinting of LIT! in adult cancers. To clarify genomic imprinting of LIT! in colorectal cancer, we examined LOl in surgically resected tissues. Methods. 44 patients who underwent surgical resection of colorectal cancer were enrolled in this study. We first searched for informative cases using DNA samples judged by PCR-restriction fragment length polymorphism. Next, we studied the status of LOl using RNA samples of those informative cases in the tumor tissues and the normal tissues. Results. Of 44 patients with colorectal cancer, 10 patients were informative case. LOl of LIT I was observed in 4 of 10 (40%) patients, although it was not seen in noncancerous tissues. Conclusion. Our results suggest that LOI of LIT! is one of the causes which promote colorectal carcinogenesis. 6421 MUTATION ANALYSIS IN BELGIAN FAMILIAL COLORECTAL CANCER KINDREDS: HIGH PROPORTION OF NOVEL MUTA· TIONS IN THE MISMATCH REPAIR GENES. Sabine Tejpar, Marijke Spaepen, Bruno Vankeirseblick, Eric Van Cutsem, Karel Geboes. Gert Matthijs, Eric Legius, Jean-Pierre Fryns, Ctr for Human Genetics KULeuven, Leuven, Belgium; Dept of Gastroenterology, KU Leuven, Leuven, Belgium; Dept of Pathology KU Leuven, Leuven, Belgium. Background: Colorectal Cancer(CRC) families complying to the modified Amsterdam Criteria 1 are predicted to have mutations in the mismatch repair genes, in upto 45 % The modified Amsterdam criteria are: At least three relatives with CRC or extra-intestinal cancers, one of which should be a first degree relative of the other two, two succesive generations affected. at least one CRC diagnosed before the age of 50. Methods: Forty Amsterdam criteria positive families and 32 families not complying to the criteria but with familial clustering of CRC, were selected for mutation analysis of hMSH2 and hMLHI, using the DGGE technique. Results: - In the Amsterdam criteria positive group II mutations were identified, 7 mutations in MSH2 and 4 in MLHI. - Nine of these mutations are novel, previously undescribed mutations. - In some of the mutation positive families unusual phenotypes were observed. - No mutations were identified in the Amsterdam criteria negative group. Discussion: The clinical phenotype of Amsterdam criteria positivity, does indeed select well for patients with mutations in Mismatch Repair genes, whereas Amsterdam criteria negative families rarely have mutations. Altough a high proportion of novel mutations were identified in our study, these mutations still behaved according to the predicted model. Some unusual phenotypes were found in our mutation positive families, highlighting the difficulties in clinical selection and counselling of these families. 'Vasen et al. Gastroenterology, 1999, 116;1453-1456 2Wijnen et al. The new England Journal of Medicine, 1998, 339(8); 511-518
April 2000
and the intensity of caspase-3 expression was clearly weaker in the lymphoma cells compared with the normal lymphoid cells.Conclusions : The present data suggested that anti-apoptotic property of MALT lymphoma shown by the lower expression of caspase- 3 rather than acceleration of cell proliferation, may have pathogenic significance. This might contribute to the indolent characteristics of the MALT type lymphoma.
6417 CENTRAL NEUROTOXICITY INDUCED BY OXALIPLATIN REPORT ON 4 CASES. Sarah Taieb, Gilles Freyer, Loic Rambaud, Louis Descos, Veronique Trillet-Lenoir, Ctr Hospitalier Lyon Sud, Pierre Benite, France. Background: Oxaliplatin in association to fluoro-uracil and folinic acid is a new drug widely used as a first line treatment in patients with metastatic colorectal cancer. Oxaliplatin is responsible for a dose-dependent neurotoxicity which affects peripheral nerves resulting in quadridistal and peribuccal dysesthesia which occur at low temperature. These symptoms usually decrease and disappear a few days after completion of chemotherapy. At elevated cumulative doses of oxaliplatin, paresthesia may become persistent and unrelated to cold inducing patients' functional disability. Paresthesia usually resolve a few weeks or months after treatment discontinuation. To our knowledge, oxaliplatin-induced neurotoxicity has been described as mainly peripheral. We report here the first 4 cases of central neurotoxicity induced by oxaliplatin. Case reports: From 1998 to 1999, 100 patients with metastatic colorectal cancer were treated by oxaliplatin (85mg/sqm), 5-fluoro-uracil (bolus: 800mg/sqm + continuous infusion: 1200mg/sqm) and folinic acid (200mg/sqm) as a 48-hour bimonthly regimen. Among these patients, 4 patients experienced central neurotoxicity. A Lherrnitte's sign (paresthesia or dysesthesia induced by cervical spine flexion) was observed in 3 cases, in association with genito-sphincteral deficiency in I case and in association with proprioception deficit in I case ; I patient experienced genito-sphincteral deficiency with saddle type of hypoesthesia and pyramidal syndrome. In the 4 cases, the cumulative dose of oxaliplatin was always higher than JOOOmg. Central neurologic symptoms resolved a few weeks after discontinuation of the treatment. Somatosensory evoked potentials were recorded in 2 patients and showed central toxicity signs with disappearance of cervico-medullary response. In one patient somatosensory evoked potentials recording contributed to the decision of the investigator to stop oxaliplatin administration. Conclusions: Oxaliplatin might be responsible for central nervous system toxicity in a limited number of patients. Since this drug is very promising in colorectal cancer treatment, careful clinical follow-up is required. Somatosensory evoked potentials recording could help to display infraclinical signs of central neurotoxicity before the occurrence of severe functional disability.
6418 DETECTION OF TELOMERASE ACTIVITY IN CANCER CELLS OBTAINED FROM BILE. Itoi Takao, Shinohara Yasushi, Takeda Kazuya, Nakamura Kazuto, Shimizu Masafumi, Takei Kazuo, Ohno Hiroyuki, Tokyo Med Univ, Tokyo, Japan. Telomerase is detected by the telomeric repeat protocol (TRAP) assay in more than 85% of primary cancers. In the present study, we determined telomerase activity using exfoliated bile cells obtained from biliary tract cancers. The aim of this study was to provide additional information regarding minimally invasive approachs to the detection of biliary tract cancer in combination with routine cytologic examination. We analyzed for telomerase activity in the bile from 3 patients with gallbladder carcinoma, 7 patients with cholangiocarcinoma, 4 patients with cholecystitis and 3 patients with cholangitis. Semiquantitative determination of telomerase activity was performed using both a fluorescence-based TRAP assay on cell extracts and at celluar level by an in situ TRAP assay. The fluorescence-based TRAP assay detected bile telomerase activity in samples from 4 of 10 patients with biliary tract cancer. In contrast, the in situ TRAP assay detected telomerase positive cells in samples from 6 of 10 patients with biliary tract cancers. However only one of these samples showed class V cytology. No non-malignant disorder detected telomerase activity by both an in situ TRAP and fluorescence-based TRAP assay. In conclusion, a combination of semiquantitative analysis and an in situ TRAP assay to detect telomerase positive cells may improve the diagnosis of biliary tract cancers with the combination of routine cytologic examination.
6419 THYMIDINE PHOSPHORYLASE (TP) IS ASSOCIATED WITH TUMOR INVASION AND METASTASIS: EFFECT OF ITS INHIBITOR ON EXPERIMENTAL LIVER METASTASIS. Sonshin Takao, Shin-ichi Akiyama, Tetsuhiro Nakajou, Hiroyoshi You, Masaki Kitazono, Futoshi Miyazono, Youichirou Matsuo, Hidetoshi Noma, Chikara Kusano, Hiroyuki Shinchi, Shouji Natsugoe, Takashi Aikou, Kagoshima Univ Sch of Medicine, Kagoshima, Japan. Backgrounds: Thymidine phosphorylase (TP) catalyzes the reversible phosphorolysis of thymidine, deoxyuridine, and their analogs. In mammals, TP is a homodimer of 55 kDa subunits, and we found that TP is identical to platelet-derived endothelial cell growth factor, and has angiogenic activity in vivo. In the mouse dorsal air sac assay model, TP inhibitor (TPI) completely suppressed the angiogenesis induced by KBrrp cells. We determined whether TPI can suppress blood-borne metastasis such as
hepatic metastasis. Cells: KBrrp cells (KB cells transfected with RSVrrp) and KB/CV cells (KB cells transfected with RSV). Methods: Immunoblotting analysis, Immunocytochemical staining, Invasion-MTT Assay (We defined the in vitro invasive ability of the tumor cells as percent invasion), chemotaxis assay, and quantitative PCR. Experimental liver metastasis: KBrrP cells (I x 105 ) were injected into the spleens of nude mice, and TPI (100 mg/Kg/day) was administered orally to the mice for 4 weeks. To detect micrometastasis in the liver, whole livers were removed. Expression of human f3-globin in the livers of nude mice was detected by PCR 3, 7, 14, 19 and 24 days after injection of the KBrrP cells into the spleens. Results: I) The growth rate of cultured KBrrp was similar to that of KB/CV cells, and TPI (500 JLM) did not affect the growth rate of either cell line in vitro. 2) KBrrp tumors in nude mice grew significantly faster than KB/CV tumors, and TPI decreased the growth rate of KBrrp tumors in vivo. 3) The chemotactic motility of KBrrp cells was much higher than that of KB/CV cells, but TPI inhibited the level of KBrrP cells to that of KB/CV cells. 4) The percent invasion level of KBrrP cells was higher than that of KB/CV cells, but TPI inhibited the percent invasion level of KBrrp cells to that of KBfCV cells. 5) In experimental liver metastasis, the expression level of human l3-globin mRNA in the livers of the control group was 6-fold higher than that of the TPI-treated group at day 24. 6) The number of macroscopic metastatic nodules (115 ± 58) in the livers of the control group (n = 7) was significantly higher than in the livers (18 ± 14) of the TPI treated group (n = 7) 4 weeks after injection of KBrrp cells. Discussion: Our findings indicate that the inhibition of TP acting by TPI causes suppression of motility and invasion in addition to inhibition of angiogenesis. TPI might be valuable for treating patients with metastatic TP-expressing tumors. 6420 GENOMIC IMPRINTING OF LITI GENE IN COLORECTAL CANCER. Kiwamu Tanaka, Goshi Shiota, Hironaka Kawasaki, Tottori Univ, Yonago, Japan; Tottori Univ, Yonaga, Japan. Aims. Genomic imprinting is epigenetic modification leading to the functional inequality of paternal and maternal alleles in somatic cells and loss of imprinting (LOl) has been shown to play an important role in carcinogenesis. Recently, the transcript, called LIT! (long QT intoronic transcript I), has been identified within the KvLQT! locus and expressed preferentially from the paternal allele and produced in most human tissues. LOI of LIT! was found in Beckwith-Wiedemann syndrome (BWS) patients and reported to be associated with the etiology of BWS. However, there is no information about genomic imprinting of LIT! in adult cancers. To clarify genomic imprinting of LIT! in colorectal cancer, we examined LOl in surgically resected tissues. Methods. 44 patients who underwent surgical resection of colorectal cancer were enrolled in this study. We first searched for informative cases using DNA samples judged by PCR-restriction fragment length polymorphism. Next, we studied the status of LOl using RNA samples of those informative cases in the tumor tissues and the normal tissues. Results. Of 44 patients with colorectal cancer, 10 patients were informative case. LOl of LIT I was observed in 4 of 10 (40%) patients, although it was not seen in noncancerous tissues. Conclusion. Our results suggest that LOI of LIT! is one of the causes which promote colorectal carcinogenesis. 6421 MUTATION ANALYSIS IN BELGIAN FAMILIAL COLORECTAL CANCER KINDREDS: HIGH PROPORTION OF NOVEL MUTA· TIONS IN THE MISMATCH REPAIR GENES. Sabine Tejpar, Marijke Spaepen, Bruno Vankeirseblick, Eric Van Cutsem, Karel Geboes. Gert Matthijs, Eric Legius, Jean-Pierre Fryns, Ctr for Human Genetics KULeuven, Leuven, Belgium; Dept of Gastroenterology, KU Leuven, Leuven, Belgium; Dept of Pathology KU Leuven, Leuven, Belgium. Background: Colorectal Cancer(CRC) families complying to the modified Amsterdam Criteria 1 are predicted to have mutations in the mismatch repair genes, in upto 45 % The modified Amsterdam criteria are: At least three relatives with CRC or extra-intestinal cancers, one of which should be a first degree relative of the other two, two succesive generations affected. at least one CRC diagnosed before the age of 50. Methods: Forty Amsterdam criteria positive families and 32 families not complying to the criteria but with familial clustering of CRC, were selected for mutation analysis of hMSH2 and hMLHI, using the DGGE technique. Results: - In the Amsterdam criteria positive group II mutations were identified, 7 mutations in MSH2 and 4 in MLHI. - Nine of these mutations are novel, previously undescribed mutations. - In some of the mutation positive families unusual phenotypes were observed. - No mutations were identified in the Amsterdam criteria negative group. Discussion: The clinical phenotype of Amsterdam criteria positivity, does indeed select well for patients with mutations in Mismatch Repair genes, whereas Amsterdam criteria negative families rarely have mutations. Altough a high proportion of novel mutations were identified in our study, these mutations still behaved according to the predicted model. Some unusual phenotypes were found in our mutation positive families, highlighting the difficulties in clinical selection and counselling of these families. 'Vasen et al. Gastroenterology, 1999, 116;1453-1456 2Wijnen et al. The new England Journal of Medicine, 1998, 339(8); 511-518