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Retinoblastoma gene product expression in oesophageal cancer
A454
AGA ABSTRACTS
QUANTITATIVE LIVER FUNCTION BY MONOETHY/.~LYCINEXYLIDIDE (MEGX) TEST IN LIVER METASTASES. ILC~nni~'~ro.I.gobicux*,R.Sorio*, E.BorsalliA, M.C'anita~, L.D¢ Apolloaxia*, F.Bortolm:zi, S. Tumolo*,D. Crivellari*, M. Valentini. Depts. of &astromterology,*Onoology,*Biodannis~and ^Nuclear Medicine, Centre di Oncologico 33081Aviano (PN), Italy. Formation of lidocaine metabolite Monoethytglycinexylidide (MEGX) has been usedas a rapid and sensitive~ v e indexof lactic liwr fimotic~a.To apply' recent poss~ilit/es of ~ in liver metastases, the a s s e s ~ of quaaaml~ liver functioa may provide useful k£~,matioa in order to cvaluate prosmsis in suda p a t i o s . Aimof tl~ stadywas to evaluatetl~ ~t'cct of various d~ of sce~laxy liv©ri n v o l ~ , en MF~X fonnad~ 67 patimts (pts), (mean age 60, range 32-81, 59 females) affected by breast cancer (34), uterus and ovary (18), stemada and colin (6), and miscellaneous cancers (9) wore inserted in the study. P ~ w/th ~ carcinoma, ~'laosis, acute or daronic viral hepatitis, b'diary duas compr~si~, ~ cardiac, respiratory or rcml failure were excluded. ~ ~ b y was performed in every patient and the ~l~ation of % livervolumereplaced by tumor was evaluated, as a u~mumumt of liwr size, numberof lesioas, maximumand minimuad/ameter of lcsioas aad m a n ~ of hepatic s~mmts involved(Group h 0%; 2: 1-25%; 3: 26-75%; 4: >75%). MEGX test was performedas follows:the patients received aa intravenousdose of I mg/Kgof lido~J~ plasmasampleswere drawn before, 45 and 60 minutes after injection. MEGX eonee~-ation was measured by Fleoresceace Polarization lmtmoassay(TDx).No significamdiffereace betwem MEGX values at 45 and 60 minuteswas foend by paired t-test; we chose to use the 45-minute value for forthesanalysis.No sewre adverseeffects were observed aflea"lidocaine injection. MEGX value in 37 ~ of group 1 was 74.+_30 I.t/l (~n+gO); in 11 of group 2: 68.+.31; in 9 of group 3: 82.+_24;in l0 of grnnp 4: 42.+.38. MEGX values in patiemsin group4 we~ significantiylowerthan in the 3 othergroups(Anova;p=0.04). W'ahinthe 10 patiesu in group4, two had normal MEGX (100 and 105) and six ve~ low MEGX values (6 to 33): the lattor patimts died in I to 14 weeksflora hepatic failu~ tbs fonn~two did not develop hepatic failure. One of ~ is still ally©ai~- 20 mmxli~tbs other dicd aflor ~ mmah from toxic neetropmia and septi~mia. We ~ MEGX test is a safe aad simplequantitative test of liver fimetica in ~nces padmts: it is ~onificnntly redoced in pafien~ with ~ liver ~ aad may perhapsprovide usoful prognosticinformaticns.
• INVOLVEMENT OF MISMATCH REPAIR SYSTEM IN G2 GROWTH ARREST CAUSED BY N - M E T H Y L . N ' . N I T R O - N NITROSOGUANIDINE (MNNG) TREATMENT IN CANCER CELLS. J.M. C~rethers, M. Koi, M.T. Hawn, D.P. Chauhan, G. Marra, and C.R. Boland. University of Michigan Medical Center and VAMC, A n n Arbor, MI. MNNG is an alkylating agent which methylates the 06 position of guanine nucleotides causing the mispairing of bases on complementary strands of DNA. Tolerance to the effects of MNNG has been attributed to cells with mismatch repair (MMR) deficiency allowing altered bases to be replicated in daughter strands. MNNG resistant clones derived from cells sensitive to MNNG have been described which escape an observed G2 cell cycle arrest in parental cell lines. We examined the effect of MNNG on the cell cycle using the MMR deficient colon cancer call line HCT116, and HCT116 calls corrected for MMR and microsatellite instability (MSI) by chromosome 3 transfer to determine if G2 arrest is associated with, or possibly requires, an intact MMR system. Methods: Exponentially growing cells were exposed to equimolar concentrations (2.5 or 5 taM) of MNNG for 45 rain at 37oC in serum-free media. Cell viability was determined by trypan blue exclusion, and cell cycle analysis of isolated nuclei was performed at 12 hr intervals for 2 days, followed by 24 hr intervals for 3 days. HCTll6 cells which received a normal copy of the hMLH1 gene on chromosome 3 (HCT116+ch3-6) have previously been shown in our laboratory to be corrected for MSI and MMR, whereas HCT116 cells which received a normal copy of chromosome 2 (HCT116+ch2-1) remained MMR deficient and exhibited MSI. Results: A dosage-dependent inhibition of cell growth occurred on parental HCTll6 ceils, as well as HCT116+ch2-1 and HCTll6+ch3-6 cells. Equimolar treatment of cells with MNNG caused a transient S phase delay within the first 24 hr and successive accumulation of calls in the G2 phase of the cell cycle. MMR deficient cell lines (HCTll6, HCTll6+ch2-1, LoVe) and a cell line with MSI (2774) traversed the G2 phase within 48 hrs without arrest and exhibited normal growth. In contrast, MMR proficient call lines (HCT116+ch3-6, SW480) arrested at the G2 phase after passing the first S phase after treatment. In HCTll6+eh3-6 ceils, the G2 arrest lasted more than 5 days without cell death, whereas SW480 cells exhibited cell death by 120 hrs after MNNG exposure. Conclusions: Our findings suggest involvement of the MMR system in mediating DNA postreplieative cell cycle arrest, possibly in an attempt to restore normal base pairing or replace rulsincorporated nucleotides on the replicated DNA strand. This suggests that progression into mitosis is linked to the completion of DNA repair in MMR proficient cells.
GASTROENTEROLOGY, VoI. IO8, No. 4
• IN VITRO EFFECT OF 5-FLUOROURACIL ON CELLS WITH AND W I T H O U T P R O F I C I E N C Y IN DNA MISMATCH REPAIR. LM. Ccrethers. M. Koi, M.T. Hawn, G. Marra and C.R. Boland. Univ. of Michigan Medical Center and VAMC, Ann Arbor, MI. The pyrimidine analog 5-fluorouracil (5FU) is currently the most widely used anticancer agent in the treatment of colorectal cancer. 5FU inhibits thymidylate synthetase, and is incorporated into both RNA and DNA. The incorporation of 5FU into RNA has been associated with cytotoxicity, and has major effects on the processing and function of RNA, whereas the significance of 5FU incorporation into DNA is less clear. Recently cell lines which show microsatellite instability (MSI), the phenotype exhibited by tumors in hereditary nonpolyposis colon cancer (HNPCC), have been shown deficient in DNA mismatch repair (MMR). We examined treatment with 5FU on HCTll6 cells, which are MMR deficient and show MSI, and HCTll6 cells corrected for MSI and MMR by chromosome 3 transfer, to discern whether survival of these cells would be affected by presence of a competent MMR system. Methods: Cell lines were maintained in culture media containing FBS, and continuously exposed to 1, 2.5, and 5 pM 5FU. Fresh media and 5FU were exchanged every three days. Cloning efficiency on plastic culture plates was then calculated after ten days of exposure. HCT116 cells which received a normal copy of the hMLH1 gene on chromosome 3 (HCT116+ ch3-6) have previously been shown in our laboratory to be corrected for MSI and MMR, whereas HCTll6 cells which received a normal copy of chromosome 2 (I--ICT116+ch2-1) remained MMR deficient and had MSI. Results: Relative Colony Forming Ability of 5FU Treated Cells Cell Ling MSI/MMR Deficient %Relative Surviving Fraction (5uM) HCTll6 yes 14.2 HCT116+ch2-1 yes 8.6 HCT116+ch3-6 no 0.1 SW480 no 0.3 2774 yes 25.9 LoVe yes 60.5 Conclcsions: Colony forming ability, determined 10 days after treatment with 5FU, was reduced an average of 94 fold in MMR proficient ceils when compared to MMR deficient cell lines. These data suggest that DNA incorporation of 5FU may contribute to its cytotoxicity in addition to its effects on RNA in MMR proficient cells, and suggests that altered pyrimidine nucleotides are tolerated in cells exhibiting MMR deficiency. A clinical trial of 5FU may be needed to compare response rates of tumors in HNPCC and non-HNPCC patients, or more generally, patients whose tumors exhibit MSI.
RETINOBLASTOMA GENE OESOPHAGEAL CANCER
PRODUCT
EXPRESSION
IN
D. Carroll, E. Ryan, E. Mulligan, M. Duggan, R. Mcrriman, P. MacMathuna, D. Graham t, J. Lennon, J. Crowe. Gastrointestinal Unit and Department of Pathology ~, Mater Misericordiae Hospital, University College Dublin. Introduction: Lesions at various genetic loci have been identified in human oesophageal cancer including frequent loss of heterozygosity at the retinoblastoma (Rb) locus. However, Rb protein (pRb) expression has not been investigated and the role of this gene with regard to survival has not been examined. Consequently, the aim of this study was to assess pRb expression in oesophageal squamous cell carcinomas and adenocarcinomas and to correlate the findings with survival. Methods: Oesophageal cancer samples from 32 patients (14 adenocarcinomas and 18 squamous cell carcinomas) were immunohistochemieally assessed for nuclear pRb expression using the mouse monoclonal antibody PMG2.345. pRb expression was classified as Rb positive (pRb+) or altered (pRb/~,) depending on the percentage of tumour cells which expressed the protein. Bladder tumour sections known to stain positively for pRb were used as positive controls. Survival data was available on all patients with a median follow up of 16 months ranging from 1 to 57 months. Prognostic significance of pRb expression was assessed from a Kaplan-Meier survival curve and log-rank analysis. Results: Twenty (63%) of the 32 tumour samples were pRb+ and 12 (37%) were pRbA. pRb expression did not correlate with survival (p=0.6) Of the 14 adenocarcinomas, 8/14 (57%) and 6/14 (43%) were pRb+ and pRbAtumours respectively. Of 18 squamous cell carcinomas, 12/18 (67%) and 6/18 (33%) were pRb+ and pRb *"tumours respectively. Conclusion: Survival analysis studies indicate the Rb protein expression does not bear prognostic significance in these cancers, however our results support the hypothesis that the Rb gene is involved in the pathogencsis of oesophageal cancer.
AGA ABSTRACTS
QUANTITATIVE LIVER FUNCTION BY MONOETHY/.~LYCINEXYLIDIDE (MEGX) TEST IN LIVER METASTASES. ILC~nni~'~ro.I.gobicux*,R.Sorio*, E.BorsalliA, M.C'anita~, L.D¢ Apolloaxia*, F.Bortolm:zi, S. Tumolo*,D. Crivellari*, M. Valentini. Depts. of &astromterology,*Onoology,*Biodannis~and ^Nuclear Medicine, Centre di Oncologico 33081Aviano (PN), Italy. Formation of lidocaine metabolite Monoethytglycinexylidide (MEGX) has been usedas a rapid and sensitive~ v e indexof lactic liwr fimotic~a.To apply' recent poss~ilit/es of ~ in liver metastases, the a s s e s ~ of quaaaml~ liver functioa may provide useful k£~,matioa in order to cvaluate prosmsis in suda p a t i o s . Aimof tl~ stadywas to evaluatetl~ ~t'cct of various d~ of sce~laxy liv©ri n v o l ~ , en MF~X fonnad~ 67 patimts (pts), (mean age 60, range 32-81, 59 females) affected by breast cancer (34), uterus and ovary (18), stemada and colin (6), and miscellaneous cancers (9) wore inserted in the study. P ~ w/th ~ carcinoma, ~'laosis, acute or daronic viral hepatitis, b'diary duas compr~si~, ~ cardiac, respiratory or rcml failure were excluded. ~ ~ b y was performed in every patient and the ~l~ation of % livervolumereplaced by tumor was evaluated, as a u~mumumt of liwr size, numberof lesioas, maximumand minimuad/ameter of lcsioas aad m a n ~ of hepatic s~mmts involved(Group h 0%; 2: 1-25%; 3: 26-75%; 4: >75%). MEGX test was performedas follows:the patients received aa intravenousdose of I mg/Kgof lido~J~ plasmasampleswere drawn before, 45 and 60 minutes after injection. MEGX eonee~-ation was measured by Fleoresceace Polarization lmtmoassay(TDx).No significamdiffereace betwem MEGX values at 45 and 60 minuteswas foend by paired t-test; we chose to use the 45-minute value for forthesanalysis.No sewre adverseeffects were observed aflea"lidocaine injection. MEGX value in 37 ~ of group 1 was 74.+_30 I.t/l (~n+gO); in 11 of group 2: 68.+.31; in 9 of group 3: 82.+_24;in l0 of grnnp 4: 42.+.38. MEGX values in patiemsin group4 we~ significantiylowerthan in the 3 othergroups(Anova;p=0.04). W'ahinthe 10 patiesu in group4, two had normal MEGX (100 and 105) and six ve~ low MEGX values (6 to 33): the lattor patimts died in I to 14 weeksflora hepatic failu~ tbs fonn~two did not develop hepatic failure. One of ~ is still ally©ai~- 20 mmxli~tbs other dicd aflor ~ mmah from toxic neetropmia and septi~mia. We ~ MEGX test is a safe aad simplequantitative test of liver fimetica in ~nces padmts: it is ~onificnntly redoced in pafien~ with ~ liver ~ aad may perhapsprovide usoful prognosticinformaticns.
• INVOLVEMENT OF MISMATCH REPAIR SYSTEM IN G2 GROWTH ARREST CAUSED BY N - M E T H Y L . N ' . N I T R O - N NITROSOGUANIDINE (MNNG) TREATMENT IN CANCER CELLS. J.M. C~rethers, M. Koi, M.T. Hawn, D.P. Chauhan, G. Marra, and C.R. Boland. University of Michigan Medical Center and VAMC, A n n Arbor, MI. MNNG is an alkylating agent which methylates the 06 position of guanine nucleotides causing the mispairing of bases on complementary strands of DNA. Tolerance to the effects of MNNG has been attributed to cells with mismatch repair (MMR) deficiency allowing altered bases to be replicated in daughter strands. MNNG resistant clones derived from cells sensitive to MNNG have been described which escape an observed G2 cell cycle arrest in parental cell lines. We examined the effect of MNNG on the cell cycle using the MMR deficient colon cancer call line HCT116, and HCT116 calls corrected for MMR and microsatellite instability (MSI) by chromosome 3 transfer to determine if G2 arrest is associated with, or possibly requires, an intact MMR system. Methods: Exponentially growing cells were exposed to equimolar concentrations (2.5 or 5 taM) of MNNG for 45 rain at 37oC in serum-free media. Cell viability was determined by trypan blue exclusion, and cell cycle analysis of isolated nuclei was performed at 12 hr intervals for 2 days, followed by 24 hr intervals for 3 days. HCTll6 cells which received a normal copy of the hMLH1 gene on chromosome 3 (HCT116+ch3-6) have previously been shown in our laboratory to be corrected for MSI and MMR, whereas HCT116 cells which received a normal copy of chromosome 2 (HCT116+ch2-1) remained MMR deficient and exhibited MSI. Results: A dosage-dependent inhibition of cell growth occurred on parental HCTll6 ceils, as well as HCT116+ch2-1 and HCTll6+ch3-6 cells. Equimolar treatment of cells with MNNG caused a transient S phase delay within the first 24 hr and successive accumulation of calls in the G2 phase of the cell cycle. MMR deficient cell lines (HCTll6, HCTll6+ch2-1, LoVe) and a cell line with MSI (2774) traversed the G2 phase within 48 hrs without arrest and exhibited normal growth. In contrast, MMR proficient call lines (HCT116+ch3-6, SW480) arrested at the G2 phase after passing the first S phase after treatment. In HCTll6+eh3-6 ceils, the G2 arrest lasted more than 5 days without cell death, whereas SW480 cells exhibited cell death by 120 hrs after MNNG exposure. Conclusions: Our findings suggest involvement of the MMR system in mediating DNA postreplieative cell cycle arrest, possibly in an attempt to restore normal base pairing or replace rulsincorporated nucleotides on the replicated DNA strand. This suggests that progression into mitosis is linked to the completion of DNA repair in MMR proficient cells.
GASTROENTEROLOGY, VoI. IO8, No. 4
• IN VITRO EFFECT OF 5-FLUOROURACIL ON CELLS WITH AND W I T H O U T P R O F I C I E N C Y IN DNA MISMATCH REPAIR. LM. Ccrethers. M. Koi, M.T. Hawn, G. Marra and C.R. Boland. Univ. of Michigan Medical Center and VAMC, Ann Arbor, MI. The pyrimidine analog 5-fluorouracil (5FU) is currently the most widely used anticancer agent in the treatment of colorectal cancer. 5FU inhibits thymidylate synthetase, and is incorporated into both RNA and DNA. The incorporation of 5FU into RNA has been associated with cytotoxicity, and has major effects on the processing and function of RNA, whereas the significance of 5FU incorporation into DNA is less clear. Recently cell lines which show microsatellite instability (MSI), the phenotype exhibited by tumors in hereditary nonpolyposis colon cancer (HNPCC), have been shown deficient in DNA mismatch repair (MMR). We examined treatment with 5FU on HCTll6 cells, which are MMR deficient and show MSI, and HCTll6 cells corrected for MSI and MMR by chromosome 3 transfer, to discern whether survival of these cells would be affected by presence of a competent MMR system. Methods: Cell lines were maintained in culture media containing FBS, and continuously exposed to 1, 2.5, and 5 pM 5FU. Fresh media and 5FU were exchanged every three days. Cloning efficiency on plastic culture plates was then calculated after ten days of exposure. HCT116 cells which received a normal copy of the hMLH1 gene on chromosome 3 (HCT116+ ch3-6) have previously been shown in our laboratory to be corrected for MSI and MMR, whereas HCTll6 cells which received a normal copy of chromosome 2 (I--ICT116+ch2-1) remained MMR deficient and had MSI. Results: Relative Colony Forming Ability of 5FU Treated Cells Cell Ling MSI/MMR Deficient %Relative Surviving Fraction (5uM) HCTll6 yes 14.2 HCT116+ch2-1 yes 8.6 HCT116+ch3-6 no 0.1 SW480 no 0.3 2774 yes 25.9 LoVe yes 60.5 Conclcsions: Colony forming ability, determined 10 days after treatment with 5FU, was reduced an average of 94 fold in MMR proficient ceils when compared to MMR deficient cell lines. These data suggest that DNA incorporation of 5FU may contribute to its cytotoxicity in addition to its effects on RNA in MMR proficient cells, and suggests that altered pyrimidine nucleotides are tolerated in cells exhibiting MMR deficiency. A clinical trial of 5FU may be needed to compare response rates of tumors in HNPCC and non-HNPCC patients, or more generally, patients whose tumors exhibit MSI.
RETINOBLASTOMA GENE OESOPHAGEAL CANCER
PRODUCT
EXPRESSION
IN
D. Carroll, E. Ryan, E. Mulligan, M. Duggan, R. Mcrriman, P. MacMathuna, D. Graham t, J. Lennon, J. Crowe. Gastrointestinal Unit and Department of Pathology ~, Mater Misericordiae Hospital, University College Dublin. Introduction: Lesions at various genetic loci have been identified in human oesophageal cancer including frequent loss of heterozygosity at the retinoblastoma (Rb) locus. However, Rb protein (pRb) expression has not been investigated and the role of this gene with regard to survival has not been examined. Consequently, the aim of this study was to assess pRb expression in oesophageal squamous cell carcinomas and adenocarcinomas and to correlate the findings with survival. Methods: Oesophageal cancer samples from 32 patients (14 adenocarcinomas and 18 squamous cell carcinomas) were immunohistochemieally assessed for nuclear pRb expression using the mouse monoclonal antibody PMG2.345. pRb expression was classified as Rb positive (pRb+) or altered (pRb/~,) depending on the percentage of tumour cells which expressed the protein. Bladder tumour sections known to stain positively for pRb were used as positive controls. Survival data was available on all patients with a median follow up of 16 months ranging from 1 to 57 months. Prognostic significance of pRb expression was assessed from a Kaplan-Meier survival curve and log-rank analysis. Results: Twenty (63%) of the 32 tumour samples were pRb+ and 12 (37%) were pRbA. pRb expression did not correlate with survival (p=0.6) Of the 14 adenocarcinomas, 8/14 (57%) and 6/14 (43%) were pRb+ and pRbAtumours respectively. Of 18 squamous cell carcinomas, 12/18 (67%) and 6/18 (33%) were pRb+ and pRb *"tumours respectively. Conclusion: Survival analysis studies indicate the Rb protein expression does not bear prognostic significance in these cancers, however our results support the hypothesis that the Rb gene is involved in the pathogencsis of oesophageal cancer.