- Email: [email protected]
P-090 Epigenetic regulation of the cyclooxgenase-2 (COX-2) gene in lung cancer cell lines
Posters/Basic science/Ceil and molecular biology [P~8~ COX-2 expression during early lung squamous call cardnomN on¢ogenesls C MescaL= ~ B Martin ~. J Verdebout ~. V Ninane 3. J Soulier ~ ~FNRS
(Nabona/ Fund of Scientific Research), Brussels, Belgium, 2tns~tut Jules Border, Brusee/s, Belgium, 3 CHU Saint-Pierrs, Brussels, Belgium Cyolooxygenase 2 (COX-2) is an inducible enzyme that is implicated in onoogonesJs of many cancers and COX 2 inhibitors are effective in preventing the development of tumours like colon cancer. COX 2 expres=on Is increased In non small cell lung cancer (NSCLC) and assoaated w ~ poor prognosis. Inhibiting COX 2 may be of interest to prevent the development of lung cancer. We assessed COX 2 expression In normal bronchtal epithelium es well as In all the putative precursors of squamous cell carcinomas (SQCC). COX 2 expression was studied by JmmunohJstochemJslz'y (IHC) In 106 blppsJes collected during autofluorescence brenchoscopy in consecutive patients at high~'isk for lung cancer Fourteen lesions of each histological type were analysed, excepted invaslve carcinomas where only 6 samples wore available All biopsies corresponding to normal epithelium or low grade lesions (hyperplasia. metaplasia, mild and moderate dysplasia) did not show increased COX-2 expression Lesions were positive b r COX-2 in 8/14 severe dysplasia. 8/14 carcinomas in situ and ,518 invaslve carcinomas Wa found a strong stat]shcally significant c|fference (,o<0.001) in COX 2 expression, between normal epithelium or low grade lesions and high grade lesions (severe dysplasia or worse). The positive and negative predictive values of COX 2 expression with regard to high grade lesions were 100% and 82.35%. respectively. In conclusion, bronchial precursors of SQCC showed increesod COX 2 expression with a differential expression in low versus high grade vatha high positive predictive value. COX 2 thus appears as a potential early marker of SQCC ~P~87I Search for suscapUblllty alleles to lung cancer by scresnlng familial cases for constitutional mutaUons In the Xeroderma
Plgmentosum Complementatlon genes A. Matakidou. T. Eisen. H. Bndlo. R. Houlston. Golceps Consortium. Institute
ot Cancer Research, Sutton, UK Background: Nuoleotide excision repair (NER) is involved in removing a wide range of lesions, including tobacco carcinogen induced bulky DNA adducts Relatives of patients with Xoroderma Pigmontosum have heon reported to have an increased risk of lung cancer (Swift M and Chase C Cancer in families with Xeroderma Pigmentosum JNCI 1979:62 (6): 1415 1421) We hypothesised that sequence vadants in the Xeredorma Pigmontosum Complomentation Group genes confer susceptibility to lung cancer Methods: The eight Xeroderma Pigrnentosum Complemontation genes are screened in 92 patients who wore diagnosed wtth lung cancer before ago of 55 and reported at Ioest one frst,:logroe relative with lung cancer. Detection of sequence changes is being undertaken by denatunng high performance liquid chromatography (DHPLC) and samples with aberrant elut]on probes are sequenced. Results: To date. mutation analys~s of the XPA and XPC genes has boon completed and a series of known polymorphisms have been detected Analysis of the XPC gone revealed 5 novel int]-onie varialJons and a novel hotoroz'ygous C to T t]'ansition at nuelool~do 2011 ofthe XPC geno was detected in one of the cases screened This results in an Arginine to Cystelne substitution within the conserved domain (position 671) of the protein Investigation of the remaining Xoroderma Pigmontosum genes is ongoing and espacted to be completed by April 200,5 Results will be presented at the meeting Conclusions: To our knowledge this is the first oomprehons~ve sequence analysis of the Xorederma Plgmentosum genes in familial lung cancer and wll provide insights into the nolo of subpolymorphic nuoleotide excision repair gone vanation and lung cancer suscept]bil~.
[ P • "MI ere CT E Matsui G/fu Research Institute for Environmental Medicine, Japan
Background: The advance of CT technology provide fully 3 0 imago of pulmonary nodules wtth a high spatial resolution, which ranges between 300p.m and 500 ~.m. However. these spatial resolutions have limitation for the quantitalJve diagnosis of fine lung st]'ucturee So we developed micro-computed tomography (micro-CT) systems to assess the relationship between preexisting lung structures and localized lesions Materials & Methods: The basic components of the micro CT systems consist of a microfcous X-ray source, a specimen manipulator, and an image intensifier detector coupled to charge-coupled device(CCD)camera or a fiat panel detector Three-cimensional(3D) image reconst]'u~on was per~rmed slice by slice. A standard far. beam convolution and backproJeotion algorithm was used to reoonsb'uct the canter plane intersecting the X~-ay source. The preprocassing of the 3D image reconst~'uet]on included the correction of the goornetncal distortions and the shading artifact int]-oduced by the image intensifier. The main advances of the systems are to obtain a high resolution
$137
which ranges between 4v.m to 25 p.m. Materials are surgically roseetod, inflated and r=ed lung specimens of 3 peripheral pulmonary carcinoma cases Results: We compared the CT images with soft X-ray and histological findings of the specimens Experimental results reveal microstructures of the lung. such as alveolar walls, intedobolar septa, bmnchiolee, and intTalesional architectures Conclusions: From the results, the micro CT systems are expeoted to have interesting potentials for experimental and dinical invostlgalJons The microCT imagos reveal fine lung structures such as alveolar walls, bronehiolos, and int]'alesional architeotures. [P-~
prognosUc score (GPS) and the general blochernlcal upaet In patients with aclvanca¢l lung and gastrolntestlnel cancer
An Inflamrnatlon b a ~ d
R MilroyI . D Brown 2. 1" Preston 3. D Mcmillan 4 lStobhi//Hospital, Glasgow,
UI~ 2St. Cetumbas Hospt~, Edtnburgll, UP( 3SUERC East ~lbnde, Glas#ow, UI~ 4 Um versity Sept. of Surge~ Royal tnf/rmaty Glasg~v, Glasg~v, UK Background: Progressive malignant disease is frequently associated with a complex biochemical and metabolic upset which results in the syndrome of cancer cachexia only some of which appears related to tumour borden. There is increasing evidence that C reactive protein (CRP) has prognostic value (Scott et al.. 2002). Moreover. recently a oumulat]vo score (Glasgow Prognostic Score. GPS) (a combination of C~'eaet]vo protein and albumin) hes been shown to have additional prognoslJc value in patients with advanced lung cancer (Forrest et al. 2003) This study further assesses the systemic inflammatory response in patients with advanced lung and GI cancer Methods: Patients with a clagnosis of malignancy not amenable to curative t]'eat]'nent were recruited and compared to a group of age and sex matched healthy volunteers Fifty advanced cancer palJents (38 lung. 12 GI) and 13 healthy subjects were compared The majority of patients had metastatic clseaso. All study participants had their weight, performance status and blood counts measured All study parl]clpants also had detailed biochemical studies. inoluding measurement of albumin. CR,R. liver function tests and eleotz'olyto and trace elements. Results:. The cancer group were significantly lighter (P <0.01). had lower albumin and haemoglobin concent]'at]ons and had a poorer performance status (P<0.001). In oontz'ast the cancer group had higher wilto cell counts (P<0001) and higher platelet counts ( P < 0 0 5 ) and higher CRP concentrations (P <0 001) As a result the cumulative progneslJc score of CRP and albumin (GPS) was significantly elevated in the cancer group All palJents with hypoalbuminaemia (n 13) had an elevated CRP concent]-ation Cancer patients ware grouped according to the GPS (0. 1 or 2) Ago. sos. and BMI ware similar in the GPS groups However. with increasing GPS there were significant reductions in performance status (p <0 01) haemoglobin (p <0 05). and sodium & zinc (p<0.01). Porformanee status, haemoglobin, white cell count, zinc. alkaline phosphataso and gamma glutamyl tz'ansferase wore all correlated with CRP and albumin. Conclusion: A host of biochemical qosots have been ident]hed in patients w ~ advanced lung and GI cancer and are correlated wtth the malignancy" associated inflammatory response. [P~]
Eplgenetl¢ regulation of the ¢yc|oc~xgenase-Z (COX-2) gone In
lung cancer cell lines K. Miyaiima 1'2. S. Toyooka 2. R. Maruyama 2. M. S u z ~ i 2. H. Silgomatsu 2. M Tsuboi 1. N Ikeda1. 1" Hirano 1. H Kate I . A Gazdar2 1First Department
of Surgery, Tokyo Medical University, Tokyo, Japan, 2Hamon Center for Therapeubc Oncology Research, University or Texas Sou~hwesfam Medical Center, Dallas, Texas, USA Background: Immunostaining studies indicate frequent up regulation of COX-2 expression in non small cell lung cancer (NSCLC) which is related to increased invaslveness and lymph node metastases. By contrast, expression in small cell lung cancer (SCLC) is weak or absent. We studied the mechanism for the clfferent]al esprossion of COX 2 gone using lung cancer cell lines. Methods: COX 2 mL~IA expression essays wore examined by reverse tz'anscnpt]en PCR (RT PCR) and semi quantttat]vo real time RTJJCR. Aberrant methylat]on of the promoter region of COX 2 was studied by mothylat]on specific polymerase chain reaction (MSP) and by bisulfite DNA sequencing of doned DNA ofPCR amplicons The effects of the demethylating agent `5'-aza2-deox'ycytidine (5-A,za-CdR) or the histone deaeatylase inhibitor 1Tichostatln A (TSA) were tested in expression negative call lines Results: COX-2 expression was present in all 18. NSCLC cell lines tested. and. compared to normal bronchial epithelial cells, was up regulated in about hall However. espression was weak or absent in 16 (73%) of 22 SCLC cell lines. Compared to normal brenctlal calls, mean RNA expression was 7 fold increesod in NSCLC lines and 10 fold decreased in SCLC lines. Thus. the mean expression level in NSCLC lines was about 70 fold greater than in SCLC lines. By MSP essay, methylat]on was absent in all of the NSCLC cell lines but was present in 33% of the SCLC lines. In SCLC cell lines three patterns
(Nabona/ Fund of Scientific Research), Brussels, Belgium, 2tns~tut Jules Border, Brusee/s, Belgium, 3 CHU Saint-Pierrs, Brussels, Belgium Cyolooxygenase 2 (COX-2) is an inducible enzyme that is implicated in onoogonesJs of many cancers and COX 2 inhibitors are effective in preventing the development of tumours like colon cancer. COX 2 expres=on Is increased In non small cell lung cancer (NSCLC) and assoaated w ~ poor prognosis. Inhibiting COX 2 may be of interest to prevent the development of lung cancer. We assessed COX 2 expression In normal bronchtal epithelium es well as In all the putative precursors of squamous cell carcinomas (SQCC). COX 2 expression was studied by JmmunohJstochemJslz'y (IHC) In 106 blppsJes collected during autofluorescence brenchoscopy in consecutive patients at high~'isk for lung cancer Fourteen lesions of each histological type were analysed, excepted invaslve carcinomas where only 6 samples wore available All biopsies corresponding to normal epithelium or low grade lesions (hyperplasia. metaplasia, mild and moderate dysplasia) did not show increased COX-2 expression Lesions were positive b r COX-2 in 8/14 severe dysplasia. 8/14 carcinomas in situ and ,518 invaslve carcinomas Wa found a strong stat]shcally significant c|fference (,o<0.001) in COX 2 expression, between normal epithelium or low grade lesions and high grade lesions (severe dysplasia or worse). The positive and negative predictive values of COX 2 expression with regard to high grade lesions were 100% and 82.35%. respectively. In conclusion, bronchial precursors of SQCC showed increesod COX 2 expression with a differential expression in low versus high grade vatha high positive predictive value. COX 2 thus appears as a potential early marker of SQCC ~P~87I Search for suscapUblllty alleles to lung cancer by scresnlng familial cases for constitutional mutaUons In the Xeroderma
Plgmentosum Complementatlon genes A. Matakidou. T. Eisen. H. Bndlo. R. Houlston. Golceps Consortium. Institute
ot Cancer Research, Sutton, UK Background: Nuoleotide excision repair (NER) is involved in removing a wide range of lesions, including tobacco carcinogen induced bulky DNA adducts Relatives of patients with Xoroderma Pigmontosum have heon reported to have an increased risk of lung cancer (Swift M and Chase C Cancer in families with Xeroderma Pigmentosum JNCI 1979:62 (6): 1415 1421) We hypothesised that sequence vadants in the Xeredorma Pigmontosum Complomentation Group genes confer susceptibility to lung cancer Methods: The eight Xeroderma Pigrnentosum Complemontation genes are screened in 92 patients who wore diagnosed wtth lung cancer before ago of 55 and reported at Ioest one frst,:logroe relative with lung cancer. Detection of sequence changes is being undertaken by denatunng high performance liquid chromatography (DHPLC) and samples with aberrant elut]on probes are sequenced. Results: To date. mutation analys~s of the XPA and XPC genes has boon completed and a series of known polymorphisms have been detected Analysis of the XPC gone revealed 5 novel int]-onie varialJons and a novel hotoroz'ygous C to T t]'ansition at nuelool~do 2011 ofthe XPC geno was detected in one of the cases screened This results in an Arginine to Cystelne substitution within the conserved domain (position 671) of the protein Investigation of the remaining Xoroderma Pigmontosum genes is ongoing and espacted to be completed by April 200,5 Results will be presented at the meeting Conclusions: To our knowledge this is the first oomprehons~ve sequence analysis of the Xorederma Plgmentosum genes in familial lung cancer and wll provide insights into the nolo of subpolymorphic nuoleotide excision repair gone vanation and lung cancer suscept]bil~.
[ P • "MI ere CT E Matsui G/fu Research Institute for Environmental Medicine, Japan
Background: The advance of CT technology provide fully 3 0 imago of pulmonary nodules wtth a high spatial resolution, which ranges between 300p.m and 500 ~.m. However. these spatial resolutions have limitation for the quantitalJve diagnosis of fine lung st]'ucturee So we developed micro-computed tomography (micro-CT) systems to assess the relationship between preexisting lung structures and localized lesions Materials & Methods: The basic components of the micro CT systems consist of a microfcous X-ray source, a specimen manipulator, and an image intensifier detector coupled to charge-coupled device(CCD)camera or a fiat panel detector Three-cimensional(3D) image reconst]'u~on was per~rmed slice by slice. A standard far. beam convolution and backproJeotion algorithm was used to reoonsb'uct the canter plane intersecting the X~-ay source. The preprocassing of the 3D image reconst~'uet]on included the correction of the goornetncal distortions and the shading artifact int]-oduced by the image intensifier. The main advances of the systems are to obtain a high resolution
$137
which ranges between 4v.m to 25 p.m. Materials are surgically roseetod, inflated and r=ed lung specimens of 3 peripheral pulmonary carcinoma cases Results: We compared the CT images with soft X-ray and histological findings of the specimens Experimental results reveal microstructures of the lung. such as alveolar walls, intedobolar septa, bmnchiolee, and intTalesional architectures Conclusions: From the results, the micro CT systems are expeoted to have interesting potentials for experimental and dinical invostlgalJons The microCT imagos reveal fine lung structures such as alveolar walls, bronehiolos, and int]'alesional architeotures. [P-~
prognosUc score (GPS) and the general blochernlcal upaet In patients with aclvanca¢l lung and gastrolntestlnel cancer
An Inflamrnatlon b a ~ d
R MilroyI . D Brown 2. 1" Preston 3. D Mcmillan 4 lStobhi//Hospital, Glasgow,
UI~ 2St. Cetumbas Hospt~, Edtnburgll, UP( 3SUERC East ~lbnde, Glas#ow, UI~ 4 Um versity Sept. of Surge~ Royal tnf/rmaty Glasg~v, Glasg~v, UK Background: Progressive malignant disease is frequently associated with a complex biochemical and metabolic upset which results in the syndrome of cancer cachexia only some of which appears related to tumour borden. There is increasing evidence that C reactive protein (CRP) has prognostic value (Scott et al.. 2002). Moreover. recently a oumulat]vo score (Glasgow Prognostic Score. GPS) (a combination of C~'eaet]vo protein and albumin) hes been shown to have additional prognoslJc value in patients with advanced lung cancer (Forrest et al. 2003) This study further assesses the systemic inflammatory response in patients with advanced lung and GI cancer Methods: Patients with a clagnosis of malignancy not amenable to curative t]'eat]'nent were recruited and compared to a group of age and sex matched healthy volunteers Fifty advanced cancer palJents (38 lung. 12 GI) and 13 healthy subjects were compared The majority of patients had metastatic clseaso. All study participants had their weight, performance status and blood counts measured All study parl]clpants also had detailed biochemical studies. inoluding measurement of albumin. CR,R. liver function tests and eleotz'olyto and trace elements. Results:. The cancer group were significantly lighter (P <0.01). had lower albumin and haemoglobin concent]'at]ons and had a poorer performance status (P<0.001). In oontz'ast the cancer group had higher wilto cell counts (P<0001) and higher platelet counts ( P < 0 0 5 ) and higher CRP concentrations (P <0 001) As a result the cumulative progneslJc score of CRP and albumin (GPS) was significantly elevated in the cancer group All palJents with hypoalbuminaemia (n 13) had an elevated CRP concent]-ation Cancer patients ware grouped according to the GPS (0. 1 or 2) Ago. sos. and BMI ware similar in the GPS groups However. with increasing GPS there were significant reductions in performance status (p <0 01) haemoglobin (p <0 05). and sodium & zinc (p<0.01). Porformanee status, haemoglobin, white cell count, zinc. alkaline phosphataso and gamma glutamyl tz'ansferase wore all correlated with CRP and albumin. Conclusion: A host of biochemical qosots have been ident]hed in patients w ~ advanced lung and GI cancer and are correlated wtth the malignancy" associated inflammatory response. [P~]
Eplgenetl¢ regulation of the ¢yc|oc~xgenase-Z (COX-2) gone In
lung cancer cell lines K. Miyaiima 1'2. S. Toyooka 2. R. Maruyama 2. M. S u z ~ i 2. H. Silgomatsu 2. M Tsuboi 1. N Ikeda1. 1" Hirano 1. H Kate I . A Gazdar2 1First Department
of Surgery, Tokyo Medical University, Tokyo, Japan, 2Hamon Center for Therapeubc Oncology Research, University or Texas Sou~hwesfam Medical Center, Dallas, Texas, USA Background: Immunostaining studies indicate frequent up regulation of COX-2 expression in non small cell lung cancer (NSCLC) which is related to increased invaslveness and lymph node metastases. By contrast, expression in small cell lung cancer (SCLC) is weak or absent. We studied the mechanism for the clfferent]al esprossion of COX 2 gone using lung cancer cell lines. Methods: COX 2 mL~IA expression essays wore examined by reverse tz'anscnpt]en PCR (RT PCR) and semi quantttat]vo real time RTJJCR. Aberrant methylat]on of the promoter region of COX 2 was studied by mothylat]on specific polymerase chain reaction (MSP) and by bisulfite DNA sequencing of doned DNA ofPCR amplicons The effects of the demethylating agent `5'-aza2-deox'ycytidine (5-A,za-CdR) or the histone deaeatylase inhibitor 1Tichostatln A (TSA) were tested in expression negative call lines Results: COX-2 expression was present in all 18. NSCLC cell lines tested. and. compared to normal bronchial epithelial cells, was up regulated in about hall However. espression was weak or absent in 16 (73%) of 22 SCLC cell lines. Compared to normal brenctlal calls, mean RNA expression was 7 fold increesod in NSCLC lines and 10 fold decreased in SCLC lines. Thus. the mean expression level in NSCLC lines was about 70 fold greater than in SCLC lines. By MSP essay, methylat]on was absent in all of the NSCLC cell lines but was present in 33% of the SCLC lines. In SCLC cell lines three patterns