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PD-027 Abnormal HLA expression as an immune escape mechanism inlung cancer cell lines
$74
Poster Discussions t Basic science/Ceil and rnoiecu/ar biology
not necessanly upregulated or have the same prognostic value in each growth pattem
•T
Expression of High Mobility Group A proteins In lung cancer correlates with survival
observed according to histology, sex. ago. tumor s~ze. type of surgery and MVD Conclusions: Expression of PAR-1 and/or PAR-4 in both AC and SCC tumor samples emerged as a negative prognostic factor in surgically resected stage IB NSCLC. and there~re could be helpful in selecting subgroups of patients at higher nsk of recurrence
Y. Sarhadi ~. H. Wikman ~. K. Salmenklvi ~. E. Kuosma 3. ~ S~ons4. J. Salo ¢. A Kar]alanen ~. S. Knuut]la ~. S. Anttlla ~. ~DepL r~ Occupat/ona/M~d/c/ne,
Finnish ths~tute of Occupational Health, Helsinki, Fir~and, ~Dept of Pathology, Haartman Institute and HUSLAB, Helsinki, Finland, ~Dept Eptdermology and Btostattst~cs, Fmmsh Institute of Occupational Health, Helsin~d, Finland, ~Dept of Thoracic and Cardiovascular Surge~ Helsinkf Un/vers~ty Central Hospital, F/nland, 5Dept. ot Pathology, Laboratory of Cytomotecu/ar Genetics, Haartman tns~tute and HUSLAB, Helsm/~, FtWand Background: High mobility group proteins HMGA1 and 2 are small non~istone chromosomal protoins associated with many important I:xological processes like rogulation of t]'anscnption, cifforentlatlon and neoplastic translbrmatlon. Although incroa~sed expression of theso proteins has been reported Ibr many different types of cancers, very little is known about their role in lung cancer. Methods: Wo stodiod the expression of beth tho isoforms ofHMGA1 (a & b) and that of HMGA2 gone in different histological types of lung cancer by real time semi-cluantltative RT~CR and their protein expression in tumor tissue by immunohistochemistry (IHC) on tissue micro arrays Results: We found a very high e0¢pression of HMGA1 and HMGA2 at both mRNA and protein level in lung cancar tissue of all histological types Increased nuclear expression of these genes correlated with poor survival, especially in the adeneearclnoma group (HMGAI. p 0 006: HMGA2. p 0 05) In addition to nudear staining, strong cytoplasmic staining for HMGA1 protein was also noted that was associated with s~geificantly good prognosis in all histological types of lung cancer (p = 0.02). A better prognosis of lung cancer assoclatod with cytoplasmic localization of HMGA1 suggests an unknown, yet important feature of HMGA1 protein. Cofl~u~lons: As HMGA protoins were found to be o v e r , pressed in most of the tumors and assoclatod with survival, they can bo very usoful markers Ibr lung cancer diagnosis and survival.
~sT
Prognostic role of Protease-Actlvatsd Receptor I (PAR-l) and 4 (PAR-4) In Stags IB non-small cell lung cancer (NSCLC)
G. SeivaQ(:liI . P. Lausi 2 . R Ghio I . S. Novelle I . S. Cappia 3. M. Papotti~. P Borasio 2. G Scagiiotti ~ ~University ~ Turin, Thoracic Oncology,
Orbassano, Italy. 2 Untversrty of Tunn, Thoractc Surgery, Orbassano, ita/f, ~University ~ Turin, Pathology, Orbassano, Italy Bad(ground: PAR-1 and PAR-4 seem to play a major role in exlTacellular mat]ix invasion by tumor cells and in the neoangiogenesis process We analyzed 60 cases of stage IB (pT:2~10) NSCLC for the expression of PAR1. PAR 4. and micrevossei dons~ (MVD) and evaluated potential prognostic implications. All cases underwent radical resection in our institution (52 Iobectomios. 6 pneumonoctomies and 3 wedge resections). Methods: Histology was 30 adenocarc~nomas (AC) and 30 squamous cell carclnomes (SCC); median age was 67.5 years in both groups (range ,50-82). with 38% of patients 70 years or older; 54 were males and 6 were females (10%. all AC). Tumor samples wore stained using immunohlstochemislry (IHC) with antibodies anti-PAR-1 (done WEDE 15) and anti-PAR-4 (goat policlonal antibody): we evaluated the percentage of positive cells in each tumor sample For PARd posltivity we adopted a cutoff at 20% positive cells or above MVD was quantified by the 3 most vascular+zed areas by CD34 staining (clone Qbendl O) Results: Twenty-five patients (42%) expressed PAR-1 and 39 patients (6,5%) e0¢pressed PAR-4 Significantly more AC than SCC cases were positive ~r PAR 1 (17 vs 8) and PAR 4 (26 vs 13). In 18 cases (14 AC. 4 SCC) both PAR 1 and PARA. were expressed on the same sample (30%). Average intensity of espress~en of PAR 1 and PAR 4 (% of pesltlve cells) was significantly higher in AC samples than in SCC samples (37% vs 18% p < 0.01; 39% vs 15% p < 0 002. respectively) Average MVD was 51 micreveasel/m 2 in AC samples and 2,5 micrevossel/m 2 in SCC samples (p < 0.001). No correlation was found between PAR 1 and PAR 4 espresslen and MVD levels, regardless of AC or SCC histology Median follow up was 38 months: 41 patients (68%) are still alive at 3 years. 19 patients (32%) died (12 AC and 7 SCC) Actuadal overall 3-year survival was 43% At univariate analysis overall 3-year survival was significantly shorter in patients e0¢pressing PAR-4 compared to patients with no e0¢pression of PAR4 (29% vs 60% - p 0 002) In 46 patients with concomitant espression of PAR-1 and PAR-4 or posifivity b r either one receptor 3-year survival was 30% vs 68% in 14 patients (13 SCC. 1 AC) with no PAR expression (p= 0.002). A trend toward a shorter 3year survival was also seen in PAR 1 posWvo patients compared to PAR 1 negatlvo cases (34% vs 46% p <0.06). Multivariate analys~s identified oxpression of PAR 1 and/or PAR4 as indopen dont prognostic factors for reduced survival. No difference in survival was
The analysis of serum DNA concentration by means of hI~_RT quantlflceUon: A useful prognostic factor In advanced non-small cell lung cancer (NSCLC) R. Sirera ~. C. Camps ~. R. Bremnes 2, V Albercla~. V. Rodenas I . M. Safont ~. A Blasee ~. M Taron4. J Sanohez ~. R Rosell 4 1Hospital Genera/
Universitario De Valencia, Valencia, Spain, 2University ~ Trorns~, Norway, 3Hospta/ Amau De Vt/anova, Valancta, Spatn: 4lnstttut Catala D'On~tog/a, Hospital Germans Trias Pujol, Badalona, Spain, ~Universidad Autenoma De Madrid, Madrid, Spain Background: Analys~s of circulating DNA in blood is a premising non~nvaslve clagnostic tool. We stucled the association between the amount of free circulating DNA and clinical vanables in advanced NSCLC patients Methods: 100 stage IIIB and stage IV patients, homogeneously tTeated with gemcitabine/clsplatln, were examined Blood samples were collected before chemotherapy, and circulating DNA was extracted From the serum using commercial adsorption columns The amount of free circulating DNA in the serum was assayed by titTation of the eataly'dc subunlt of telomerase (hTERT). quantified using RT-PCR Results: Median age was 61 years (35-82) and 91% were males. 86% had performance status 0-1. 71% wore stage IV and 29% stage IIIB. The histological subtypes wore: 38% squamous cell caranoma. 37% adencoa~noma. 5% anaplastlc large cell. and 20% undifferentiated Patients received a median of 6 cycles of chemotherapy (1-7). 4% achieved complete response (CR). 38% partial response (PR). 25% had stable disease (SD) and 30% progressive disease (PD). hTERT levels Ibr stage IIIB patients wore 21 1 [49 1,5202] versus 1803 [29 8488] for stage IV patients (P 053) There was no association between hTERT values and therapy response. 17 8 (2g 845 8) in the CR+PR group vs 221 (,52 ,534 7) in the SD+PD group (P 0 23) hTERT values were not related to metastatic site The multivanate analysis showed that hTERT was an independent predic~ve vanable for time to progression(HR1 1 [95%C1:1 1 1 2].P 002)andforeverallsurvival(HR1 1 [g5%c1:1 1 12].P 0004) Conclusions: The quantification of free c~rculatlng DNA in serum, by moans of hTERT, is an affordable and accurate method due to the low intra assay vanabillty and its high specificity. In advanced NSCLC. high serum hTERT levels may be an indicator of poor prognosis for time to progression and survival.
•
Abnorrnal HLA expression as an Immune escape mecllanlsm in lung cancer ceil lines
T So. T Haeagin. M Mizukami. Y Ichiki. M Sugaya. M Takenoyama. K Sugio. K Yasumoto University of Occupational and Environmental Health,
/~takyushu, Japan Background: A number of reports suggest that eancer cells possess several escape mechanisms from immune surveillance system. Abnormal espress~en of HLA class I is one of major obstades for tumor-specific immunotherapy In this study, we focused on a hablotype loss of HLA class I from a viewpoint of cellular immunity Matedals and Results: We have established 21 lung cancer call lines from the surgically resected 87,5 tumor tlssuea In the DNA genotybe analysis. 7" out of 21 call lines (33 3%) had a haplotype loss of HLA class I A large cell carcinoma call line A904L had the haplotype of HLA-A°2402. B*0702. CW*0702. but lest HLA A'2603. B'3901. Cw°0702 We newly established the HLA A~2603 and B*3901 transfoctod A904L vanants (A904L A26 and AgO4L B39. respact]vely). Autologous lymphocytes were stimulated by A904L A26 or A904LB39 independently, and then we induced the HLAB*3901~-esthcted CTL clones. One of them. CTL G3 lysed Ag04LB39. but not either the parental A904L or NK sensitive K562. Using a cDNA espression cloning method, we isolated the antlgon~ncoding gone. and then identtfied the antigenic paptlde presented on the HLA~.*3901 molecules The gone SGT1 has been reported to play a key role for cell cycle regulation In order to investigate whether the HLA haplotype loss happened in in wvo or not. 13 subclones of A904L were established from the primary culture CTL G3 did not produce IFN-';' in response to either the parental h,904L or those subclones, suggesting that the HLA haplotype loss had not occurred in in vitro culture, but in in vivo Conclusion: In this study, the findings suggest that lung cancer cells with haplotype loss of HLA class I can escape from the CTL attack, and these immuno selected vanants become clinically developed cancer. Furthermore. CTL response can be recovered by a t]'ansfection of the lost HLA class I gone to tumor cells. HLA reconst]-uction in tumor cells should be a new stz'atogy to improve tumor specific immunothorapy.
Poster Discussions t Basic science/Ceil and rnoiecu/ar biology
not necessanly upregulated or have the same prognostic value in each growth pattem
•T
Expression of High Mobility Group A proteins In lung cancer correlates with survival
observed according to histology, sex. ago. tumor s~ze. type of surgery and MVD Conclusions: Expression of PAR-1 and/or PAR-4 in both AC and SCC tumor samples emerged as a negative prognostic factor in surgically resected stage IB NSCLC. and there~re could be helpful in selecting subgroups of patients at higher nsk of recurrence
Y. Sarhadi ~. H. Wikman ~. K. Salmenklvi ~. E. Kuosma 3. ~ S~ons4. J. Salo ¢. A Kar]alanen ~. S. Knuut]la ~. S. Anttlla ~. ~DepL r~ Occupat/ona/M~d/c/ne,
Finnish ths~tute of Occupational Health, Helsinki, Fir~and, ~Dept of Pathology, Haartman Institute and HUSLAB, Helsinki, Finland, ~Dept Eptdermology and Btostattst~cs, Fmmsh Institute of Occupational Health, Helsin~d, Finland, ~Dept of Thoracic and Cardiovascular Surge~ Helsinkf Un/vers~ty Central Hospital, F/nland, 5Dept. ot Pathology, Laboratory of Cytomotecu/ar Genetics, Haartman tns~tute and HUSLAB, Helsm/~, FtWand Background: High mobility group proteins HMGA1 and 2 are small non~istone chromosomal protoins associated with many important I:xological processes like rogulation of t]'anscnption, cifforentlatlon and neoplastic translbrmatlon. Although incroa~sed expression of theso proteins has been reported Ibr many different types of cancers, very little is known about their role in lung cancer. Methods: Wo stodiod the expression of beth tho isoforms ofHMGA1 (a & b) and that of HMGA2 gone in different histological types of lung cancer by real time semi-cluantltative RT~CR and their protein expression in tumor tissue by immunohistochemistry (IHC) on tissue micro arrays Results: We found a very high e0¢pression of HMGA1 and HMGA2 at both mRNA and protein level in lung cancar tissue of all histological types Increased nuclear expression of these genes correlated with poor survival, especially in the adeneearclnoma group (HMGAI. p 0 006: HMGA2. p 0 05) In addition to nudear staining, strong cytoplasmic staining for HMGA1 protein was also noted that was associated with s~geificantly good prognosis in all histological types of lung cancer (p = 0.02). A better prognosis of lung cancer assoclatod with cytoplasmic localization of HMGA1 suggests an unknown, yet important feature of HMGA1 protein. Cofl~u~lons: As HMGA protoins were found to be o v e r , pressed in most of the tumors and assoclatod with survival, they can bo very usoful markers Ibr lung cancer diagnosis and survival.
~sT
Prognostic role of Protease-Actlvatsd Receptor I (PAR-l) and 4 (PAR-4) In Stags IB non-small cell lung cancer (NSCLC)
G. SeivaQ(:liI . P. Lausi 2 . R Ghio I . S. Novelle I . S. Cappia 3. M. Papotti~. P Borasio 2. G Scagiiotti ~ ~University ~ Turin, Thoracic Oncology,
Orbassano, Italy. 2 Untversrty of Tunn, Thoractc Surgery, Orbassano, ita/f, ~University ~ Turin, Pathology, Orbassano, Italy Bad(ground: PAR-1 and PAR-4 seem to play a major role in exlTacellular mat]ix invasion by tumor cells and in the neoangiogenesis process We analyzed 60 cases of stage IB (pT:2~10) NSCLC for the expression of PAR1. PAR 4. and micrevossei dons~ (MVD) and evaluated potential prognostic implications. All cases underwent radical resection in our institution (52 Iobectomios. 6 pneumonoctomies and 3 wedge resections). Methods: Histology was 30 adenocarc~nomas (AC) and 30 squamous cell carclnomes (SCC); median age was 67.5 years in both groups (range ,50-82). with 38% of patients 70 years or older; 54 were males and 6 were females (10%. all AC). Tumor samples wore stained using immunohlstochemislry (IHC) with antibodies anti-PAR-1 (done WEDE 15) and anti-PAR-4 (goat policlonal antibody): we evaluated the percentage of positive cells in each tumor sample For PARd posltivity we adopted a cutoff at 20% positive cells or above MVD was quantified by the 3 most vascular+zed areas by CD34 staining (clone Qbendl O) Results: Twenty-five patients (42%) expressed PAR-1 and 39 patients (6,5%) e0¢pressed PAR-4 Significantly more AC than SCC cases were positive ~r PAR 1 (17 vs 8) and PAR 4 (26 vs 13). In 18 cases (14 AC. 4 SCC) both PAR 1 and PARA. were expressed on the same sample (30%). Average intensity of espress~en of PAR 1 and PAR 4 (% of pesltlve cells) was significantly higher in AC samples than in SCC samples (37% vs 18% p < 0.01; 39% vs 15% p < 0 002. respectively) Average MVD was 51 micreveasel/m 2 in AC samples and 2,5 micrevossel/m 2 in SCC samples (p < 0.001). No correlation was found between PAR 1 and PAR 4 espresslen and MVD levels, regardless of AC or SCC histology Median follow up was 38 months: 41 patients (68%) are still alive at 3 years. 19 patients (32%) died (12 AC and 7 SCC) Actuadal overall 3-year survival was 43% At univariate analysis overall 3-year survival was significantly shorter in patients e0¢pressing PAR-4 compared to patients with no e0¢pression of PAR4 (29% vs 60% - p 0 002) In 46 patients with concomitant espression of PAR-1 and PAR-4 or posifivity b r either one receptor 3-year survival was 30% vs 68% in 14 patients (13 SCC. 1 AC) with no PAR expression (p= 0.002). A trend toward a shorter 3year survival was also seen in PAR 1 posWvo patients compared to PAR 1 negatlvo cases (34% vs 46% p <0.06). Multivariate analys~s identified oxpression of PAR 1 and/or PAR4 as indopen dont prognostic factors for reduced survival. No difference in survival was
The analysis of serum DNA concentration by means of hI~_RT quantlflceUon: A useful prognostic factor In advanced non-small cell lung cancer (NSCLC) R. Sirera ~. C. Camps ~. R. Bremnes 2, V Albercla~. V. Rodenas I . M. Safont ~. A Blasee ~. M Taron4. J Sanohez ~. R Rosell 4 1Hospital Genera/
Universitario De Valencia, Valencia, Spain, 2University ~ Trorns~, Norway, 3Hospta/ Amau De Vt/anova, Valancta, Spatn: 4lnstttut Catala D'On~tog/a, Hospital Germans Trias Pujol, Badalona, Spain, ~Universidad Autenoma De Madrid, Madrid, Spain Background: Analys~s of circulating DNA in blood is a premising non~nvaslve clagnostic tool. We stucled the association between the amount of free circulating DNA and clinical vanables in advanced NSCLC patients Methods: 100 stage IIIB and stage IV patients, homogeneously tTeated with gemcitabine/clsplatln, were examined Blood samples were collected before chemotherapy, and circulating DNA was extracted From the serum using commercial adsorption columns The amount of free circulating DNA in the serum was assayed by titTation of the eataly'dc subunlt of telomerase (hTERT). quantified using RT-PCR Results: Median age was 61 years (35-82) and 91% were males. 86% had performance status 0-1. 71% wore stage IV and 29% stage IIIB. The histological subtypes wore: 38% squamous cell caranoma. 37% adencoa~noma. 5% anaplastlc large cell. and 20% undifferentiated Patients received a median of 6 cycles of chemotherapy (1-7). 4% achieved complete response (CR). 38% partial response (PR). 25% had stable disease (SD) and 30% progressive disease (PD). hTERT levels Ibr stage IIIB patients wore 21 1 [49 1,5202] versus 1803 [29 8488] for stage IV patients (P 053) There was no association between hTERT values and therapy response. 17 8 (2g 845 8) in the CR+PR group vs 221 (,52 ,534 7) in the SD+PD group (P 0 23) hTERT values were not related to metastatic site The multivanate analysis showed that hTERT was an independent predic~ve vanable for time to progression(HR1 1 [95%C1:1 1 1 2].P 002)andforeverallsurvival(HR1 1 [g5%c1:1 1 12].P 0004) Conclusions: The quantification of free c~rculatlng DNA in serum, by moans of hTERT, is an affordable and accurate method due to the low intra assay vanabillty and its high specificity. In advanced NSCLC. high serum hTERT levels may be an indicator of poor prognosis for time to progression and survival.
•
Abnorrnal HLA expression as an Immune escape mecllanlsm in lung cancer ceil lines
T So. T Haeagin. M Mizukami. Y Ichiki. M Sugaya. M Takenoyama. K Sugio. K Yasumoto University of Occupational and Environmental Health,
/~takyushu, Japan Background: A number of reports suggest that eancer cells possess several escape mechanisms from immune surveillance system. Abnormal espress~en of HLA class I is one of major obstades for tumor-specific immunotherapy In this study, we focused on a hablotype loss of HLA class I from a viewpoint of cellular immunity Matedals and Results: We have established 21 lung cancer call lines from the surgically resected 87,5 tumor tlssuea In the DNA genotybe analysis. 7" out of 21 call lines (33 3%) had a haplotype loss of HLA class I A large cell carcinoma call line A904L had the haplotype of HLA-A°2402. B*0702. CW*0702. but lest HLA A'2603. B'3901. Cw°0702 We newly established the HLA A~2603 and B*3901 transfoctod A904L vanants (A904L A26 and AgO4L B39. respact]vely). Autologous lymphocytes were stimulated by A904L A26 or A904LB39 independently, and then we induced the HLAB*3901~-esthcted CTL clones. One of them. CTL G3 lysed Ag04LB39. but not either the parental A904L or NK sensitive K562. Using a cDNA espression cloning method, we isolated the antlgon~ncoding gone. and then identtfied the antigenic paptlde presented on the HLA~.*3901 molecules The gone SGT1 has been reported to play a key role for cell cycle regulation In order to investigate whether the HLA haplotype loss happened in in wvo or not. 13 subclones of A904L were established from the primary culture CTL G3 did not produce IFN-';' in response to either the parental h,904L or those subclones, suggesting that the HLA haplotype loss had not occurred in in vitro culture, but in in vivo Conclusion: In this study, the findings suggest that lung cancer cells with haplotype loss of HLA class I can escape from the CTL attack, and these immuno selected vanants become clinically developed cancer. Furthermore. CTL response can be recovered by a t]'ansfection of the lost HLA class I gone to tumor cells. HLA reconst]-uction in tumor cells should be a new stz'atogy to improve tumor specific immunothorapy.