GSK Medicine: Study Number: Title: Rationale: Study Period: Objectives: Indication: Study Investigators/Centers: Research Methods:

Transcription

1 The study listed may iclude approved ad o-approved uses, mulatios or treatmet regimes. The results reported i ay sigle study may ot reflect the overall results obtaied o studies of a product. Bee prescribig ay product metioed i this Register, healthcare professioals should cosult prescribig imatio the product approved i their coutry. GSK Medicie: Not applicable Study Number: (ONCO-RD-011) Title: Tissue Biomarker Study: Shapig Treatmet i Melaoma Ratioale: This study aimed at defiig ew therapeutic strategies better treatmet of melaoma patiets by mappig some key tissue biomarkers i melaoma with promisig cacer immuotherapeutic cadidate. This was reached by examiig the Gee Sigature (GS) ad the expressio of MAGE-A3, other tumor atiges ad kow biomarkers idividually or i combiatio, i differet stages of the disease ad to determie their progostic value. The study was termiated early o 30 April 2014 followig assessmet of the lack of efficacy of the MAGE-A3 ASCI study product Phase 3 Mage 3 studies. Pre-specified testigs that had ot bee permed by the, were ot goig to be doe aymore. Study Period: Formali Fixed Paraffi Embedded (FFPE) samples were recruited from 1994 to Objectives: To determie the level of expressio of 1) MAGE[Melaoma AtiGE]-A3, 2) other tumor atiges {PRAME [Preferetially expressed Atige of Melaoma], NY- ES0-1) ad 3) prove biomarkers {B-RAF) ad if sufficiet material is available N-RAS ad C- KIT, either idividually or i combiatio, i primary ski tumors ad distat metastasis. To evaluate the tumor atige promoter methylatio status: Examie the feasibility of the curret MAGE-A3 Reverse Trascriptio-Polymerase Chai Reactio (RT-PCR) screeig assay tumor samples derived from setiel lymph odes (SLN). Correlate the demethylatio assay betwee paired fresh froze ad FFPE tumor samples. Optimize demethylatio MAGE-A3 assay SLN ad blood. To evaluate the predictive GS foud i primary ski lesios ad tumor ifiltrated lymph odes*. No categorizatio was made betwee primary ad secodary objectives i the study protocol. As such, all objectives were classified as primary this results summary. *Objective ot permed due to early termiatio of the study. Idicatio: Melaoma Treatmet Study Ivestigators/Ceters: 1 Ceter i Spai Research Methods: from macro-dissected samples: all selected blocks were 1 slide of 5mm staied with H&E to defie. Pictures were take ad set to GSK Bio to cofirm selected. A of at least 50 mm2 was macro-dissected ad picture take from each samples usig as may slides as suggested by GSK Bio. was extracted ad quatified the amout extracted (aodrop). was permed usig QIAamp FFPE tissue kit (Qiage) followig maufacturer s istructios. Raw Data was set to GSK Bio. If quality was sufficiet, aalysis the detectio of mutatios BRAF, NRAS ad c-kit was permed. Iitially it was decided to look somatic mutatios o samples BRAF (exo 11 ad 15), NRAS (exos 1 ad 2) ad C-KIT (exos 9, 11, 13, 17 ad 18) ad also perm Multiplex Ligatio-depedet Probe Amplificatio (MLPA) aalysis usig P175 kit from MRC-HOLLAND. Testig strategy was first sequecig exo 15 from BRAF ad if it was positive, go o with MLPA ad the remaiig exos ad gees. Results evideced a lack of amplificatio too may samples, so ew strategies i order to obtai PCR products were developed. Some samples were diluted i some cases ad was amplified. Takig i cosideratio the difficulty of CKIT amplificatio, the small percetage of mutatios foud i that gee ad the total amout of it was decided to focus o BRAF ad NRAS sequecig.

2 If there was eough, a fluorescet allele-specific PCR followed by capillary electrophoresis was permed to detect the BRAF V600E mutatio, after BRAF ad NRAS sequecig, to cofirm the results foud durig the sequecig process. from fresh froze tumor samples: from selected samples was obtaied usig QIAamp Mii Kit. quatificatio ad purificatio was permed usig aodrop ad was kept at -80ºC. Data Source: Tissue samples obtaied from patiets with pathologically prove stage II, Ill ad IV melaoma specimes ad correspodig patiet data were obtaied from bio bak of the Hospital Cliic of Barceloa. Specifically, access to the followig was used: tumor tissue samples ad a restricted set of specific cliical data of the patiets. Study Desig: This was a retrospective study based upo the aalysis of melaoma specimes ad correspodig patiet-related data already available. There was o study treatmet, ad o study-specific procedure was to be carried out o patiets. Study Populatio: Male ad female melaoma patiets diagosed with stage II, III ad IV, with relapse ad free of relapse, respectively. Owig the retrospective ature of the study, all data ad samples were take from those already stored at the ivestigatio site this reaso may patiets may o loger be alive, or o loger i cotact with the ivestigatio site. Strict aoymity of all data was established ad maitaied. Study Exposures, Outcomes: Tissues samples were classified i Primary tumor, SLN ad metastasis groups. The samples were obtaied from patiets with pathologically prove stage II, III ad IV melaoma specimes. The followig subgroups were defied accordig to the stagig: Stage II free of relapse, Stage II to IV, Stage III free of relapse ad Stage III to IV Please refer to the objectives of this documet, all study outcome variables were defied i this paragraph. No categorizatio was made betwee primary ad secodary outcomes i the study protocol. As such, all outcomes were classified as primary this results summary. Data Aalysis Methods: I order to assess whether some characteristics of the sample could affect to the sequecig success, differet parameters were evaluated (% of tumor i, % of tumoral cells i, % of immue,, % of other, / (mm2), ad ) i all samples selected ad accordig to: Stagig (stage II free of relapse, stage III free of relapse, Stage II o IV ad Stage III to IV), sample type (Primary tumor, SLN, metastasis), age of the block (<5 years, 5 to 10 years, >10 years), success (OK, o OK), sequecig process success (BRAF ad NRAS sequeced, oly oe gee sequeced, oe of the gees sequeced) ad mutatioal status of the samples (BRAF/NRAS Wild Type [WT], MUTATED). The variables % of tumor i, % of tumoral, % of immue,, % of other, / (mm2), ad did ot follow a Normal distributio. Thus, oparametric tests (Kruskal Wallis) were used i order to aalyze data. The Pearso correlatio betwee ad ad the other cotiuous variables was also assessed. The 2-sided Exact Fisher test was used categorical variables. All p-values were corrected usig the False Discovery Rate method. Limitatios: Noe Baselie characteristics: Primary tumors SLN Metastasis Total Samples assessed eligibility Stage II free of relapse Stage II to IV Stage III free of relapse Stage III to IV Excluded samples 21 Not eough tumor i block 5 failed 16 Samples available BRAF/NRAS

3 mutatioal screeig Stage II free of relapse Stage II to IV Stage III free of relapse Stage III to IV Primary Outcome Results: Study Samples overview - Types of samples icluded i the study. Samples Type of sample N Selected Blocks ot available (either exhausted or ot located) More blocks available H&E slides evaluated OK OK Primary melaoma FFPE (61%) 67 (67%) Froze Distat metastasis FFPE (76%) 38 (76%) SLN FFPE (86%) 51 (100%) SLN= setiel lymph odes N= umber of plaed subjects H&E slides : Hematoxyli ad Eosi slides Note: samples availability has bee defied as 20 FFPE tumor blocks (puch biopsies 3mm 3, to be comparable to what is goig be used durig the study): 15 FFPE slides of 10µm + 1 slide of 5µm H&E staiig to cofirm %tumor issue ad defie. 50 mm 2 would be required to allow optimal MAGE-A3 testig. More slides could be requested if ot sufficiet Primary Outcome Results: Imatio about samples set accordig to the stagig Subgroup Samples N OK OK Stage II free of relapse Primary (52%) 17 (68%) melaoma Stage III free of relapse Primary melaoma (68%) 16 (64%) SLN (84%) 25 (100%) Stage II to IV Primary melaoma (56%) 14 (56%) Metastasis (72%) 18 (72%) Stage III to IV Primary melaoma (68%) 20 (80%) SLN (88%) 26 (100%) Metastasis (80%) 20 (80%) N= umber of plaed subjects Primary Outcome Results: Results - stage II free of relapse Primary Tumor Samples N Selected Block ot More blocks available OK BRAF 15 SEQ OK NRAS SEQ OK available Primary melaoma (52%) 11 (44%) 11 (40%) N= umber of plaed subjects Primary Outcome Results: Results- stage III free of relapse Primary Tumor ad SLN

4 Samples N Selected Block ot available More blocks available OK BRAF 15 SEQ OK NRAS SEQ OK Primary * 5 17 (68%) 15 (60%) 12 (48%) melaoma SLN ** 0 21 (84%) 19 (76%) 19 (76%) SLN= setiel lymph odes N= umber of plaed subjects * 1 patiet was retired from the study ** I 1 case two SLN from the same patiet were studied Primary Outcome Results: Results - stage II to IV Primary Tumor ad Metastasis Samples N Selected Block ot More blocks available OK BRAF 15 SEQ OK NRAS SEQ OK available Primary melaoma (56%) 12 (48%) 9 (36%) Metastasis 25 26* (72%) 15 (60%) 11 (44%) N= umber of plaed subjects * I 5 cases two metastasis from the same patiet were studied Primary Outcome Results: Results - stage III to IV Primary Tumor, SLN ad Metastasis Samples N Selected Block ot available More blocks available OK BRAF 15 SEQ OK NRAS SEQ OK Primary melaoma * 7 17 (68%) 15 (60%) 14 (56%) SLN 25 41** 8* 5 22 (88%) 22 (88%) 22 (88%) Metastasis 25 44*** 15* 7 20 (80%) 18 (72%) 15 (75%) SLN= setiel lymph odes N= umber of plaed subjects * 1 patiet was retired from the study, ** I 4 cases two SLN from the same patiet were studied, *** 4 patiets had more tha oe metastasis to aalyze

5 Primary Outcome Results: BRAF ad NRAS status i all primary tumors. BRAF 15 status i all tumors N= 53 % V600E V600M 1 2 WT NRAS status i all tumors N=45 % Q61R 3 7 Q61K 1 2 Q61H 1 2 WT N= umber of primary melaomas evaluated = umber of samples i each category %=percetage i each category Primary Outcome Results: Number of samples with Stage II free of relapse melaomas carryig the V600E mutatio i BRAF/NRAS gees Primary melaoma V600E BRAF 6 NRAS WT 5 NRAS NA 1 ALL WT 3 NRAS WT 3 BRAF V600E + NRAS mutated 1 NRAS mutated 1 BRAF WT +NRAS mutated 1 NRAS mutated 1 NRAS NA: o test NRAS status Primary Outcome Results: Number of samples with Stage III free of relapse melaomas carryig the V600E mutatio i BRAF/NRAS gees Primary melaoma SLN V600E BRAF 9 V600E 6 (66.6%) All WT 1 NA 2 All WT 4 V600E 1 All WT 2 (40%) NA 1 V600E + NRAS mutated 1 NRAS mutated 1 BRAF WT 1 V600E 1 NA 7 V600E 3 All WT 4 % expresses the correlatio betwee samples from the same patiet NA: Data ot available Primary Outcome Results: Number of samples with Stage II to IV melaomas carryig the V600E mutatio i BRAF/NRAS gees Primary melaoma Metastasis V600E BRAF 3 V600E 3** (66.7%) NA 1 V600M BRAF 1 NA 1 NRAS mutated 1 All WT 1 BRAF WT 7 BRAF WT 3 (43%) NA 4 NA 9 V600E BRAF 4^ BRAF WT % expresses the correlatio betwee samples from the same patiet **2/3 metastasis belog to the same patiet ^ Same patiet with 2 discordat metastasis 2 patiets with 2 metastasis both with the V600E mutatio T metastasis belog to the same patiet Primary Outcome Results: Number of samples with Stage III to IV melaomas carryig the V600E mutatio i BRAF/NRAS gees Primary melaoma SLN Metastasis V600E BRAF 8 V600E 4 (50%) V600E 1 (12.5%) NA 3 WT 1 NA 1 NA 3 V600E 2 (25%)

6 NA 1 BRAF WT 6 BRAF WT 4 (66.7%) BRAF WT 2 (33.3%) NA 2 V600E 1 NA 1 NA 1 V600E 1 NRAS mutated 1 WT 2* Ukow 1 NA 22 V600E 1 V600E 1^ WT 1^ V600E + NRAS mutated 3 All gees WT 1 NA 3* NRAS mutated 1 NRAS mutated 1** BRAF WT 5* V600E 1 NRAS mutated 1 NA 2 NA 3 BRAF WT 4* NRAS mutated 2* % expresses the correlatio betwee samples from the same patiet * 2 samples belog to the same patiet * * Differet mutatios ^ Same patiet with 2 discordat metastasis Primary Outcome Results: Descriptive aalysis of the parameters i All samples. i tumoral area / N (%) (86.9) 150 (93.75) Missig (%) (13.1) 10 (6.25) Mea Media St deviatio Miim value Maxim value Primary Outcome Results: Evaluatio of the parameters i Primary tumors grouped by Stagig i tumoral area / Stage II free of relapse N (%) 18 (25.7) 18 (25.7) 18 (25.7) 18 (25.7) 18 (25.7) 18 (25.7) 14 (23) 17 (25.7) Missig Mea Media St deviatio Miim value Maxim value Stage III free of relapse N (%) 17 (24.3) 17 (24.3) 17 (24.3) 17 (24.3) 17 (24.3) 17 (24.3) 17 (27.8) 16 (24.2) Missig Mea Media St deviatio Miim value Maxim value Stage II to IV N (%) 14 (20) 14 (20) 14 (20) 14 (20) 14 (20) 14 (20) 14 (23) 14 (21.2) Missig Mea Media

7 St deviatio Miim value Maxim value Stage III to IV N (%) 21 (30) 21 (30) 21 (30) 21 (30) 21 (30) 21 (30) 16 (26.2) 19 (28.9) Missig Mea Media St deviatio Miim value Maxim value N Total (87.1) 66 (94.3) Adj. p-value N(%)= umber (percetage) of samples i the category Percetage=100*N/N Total Adjusted p-values were calculated usig the False Discovery Rate method. Primary Outcome Results: Evaluatio of the parameters accordig to sample type i tumoral area / Primary tumor N (%) 70 (43.8) 70 (43.8) 70 (43.8) 70 (43.8) 70 (43.8) 70 (43.8) 61 (43.9) 66 (44) Missig Mea Media St deviatio Miim value Maxim value SLN N (%) 53(33.1) 53(33.1) 53(33.1) 53(33.1) 53(33.1) 53(33.1) 43 (30.9) 49 (32.6) Missig Mea Media St deviatio Miim value Maxim value Metastasis N (%) 37 (23.1) 37 (23.1) 37 (23.1) 37 (23.1) 37 (23.1) 37 (23.1) 35 (25.2) 35 (23.3) Missig Mea Media St deviatio Miim value Maxim value N Total (86.9) 150 (93.8) Adj. p-value < < <0.001 < <0.001 N(%)= umber (percetage) of samples i the category Percetage=100*N/N Total Adjusted p-values were calculated usig the False Discovery Rate method. Primary Outcome Results: Evaluatio of the parameters accordig to the age of the blocs i tumoral area / < 5 years N (%) 38 (23.8) 38 (23.8) 38 (23.8) 38 (23.8) 38 (23.8) 38 (23.8) 34 (24.5) 37 (24.7) Missig

8 Mea Media St deviatio Miim value Maxim value to 10 years N (%) 83 (51.9) 83 (51.9) 83 (51.9) 83 (51.9) 83 (51.9) 83 (51.9) 71 (51.1) 77 (51.3) Missig Mea Media St deviatio Miim value Maxim value >10 years N (%) 39 (24.3) 39 (24.3) 39 (24.3) 39 (24.3) 39 (24.3) 39 (24.3) 34 (24.4) 36 (24.0) Missig Mea Media St deviatio Miim value Maxim value N Total (86.9) 150 (93.8) Adj. p-value N(%)= umber (percetage) of samples i the category Percetage=100*N/N Total Adjusted p-values were calculated usig the False Discovery Rate method. Primary Outcome Results: Evaluatio of the parameters accordig to the success of i tumoral area / OK N (%) 139 (89.7) 139 (89.7) 139 (89.7) 139 (89.7) 139 (89.7) 139 (89.7) (89.3) Missig ^ 5 Mea Media St deviatio Miim value Maxim value NO OK N (%) 16 (10.3) 16 (10.3) 16 (10.3) 16 (10.3) 16 (10.3) 16 (10.3) - 16 (10.7) Missig Mea Media St deviatio Miim value Maxim value N Total 155* 155* 155* 155* 155* 155* (96.7) Adj. p-value N(%)= umber (percetage) of samples i the category. Percetage=100*N/N Total. Adjusted p-values were calculated usig the False Discovery Rate method. * 5 samples were evaluated but ot extracted, thus we have ot icluded them i this aalysis ^I oe sample measure was ot available although the sample was successfully sequeced This aalysis was ot permed as oly few samples failed. Primary Outcome Results: Evaluatio of the parameters accordig to the availability of the sequecig results cells I tumoral cocetrati

9 area / o All gees N (%) 108 (75) 108 (75) 108 (75) 108 (75) 108 (75) 108 (75) 107 (77) 105 (75.5) Missig Mea Media St deviatio Miim value Maxim value Oly oe gee N (%) 16 (11.1) 16 (11.1) 16 (11.1) 16 (11.1) 16 (11.1) 16 (11.1) 16 (11.5) 14 (10.1) Missig Mea Media St deviatio Miim value Maxim value Noe N (%) 20 (13.9) 20 (13.9) 20 (13.9) 20 (13.9) 20 (13.9) 20 (13.9) 16 (11.5) 20 (14.4) Missig Mea Media St deviatio Miim value Maxim value N Total 144* 144* 144* 144* 144* 144* 139 (96.5) 139 (96.5) Adj. p-value The Sequecig success was defied as havig a sequece that could be clearly evaluated. N(%)= umber (percetage) of samples i the category. Percetage=100*N/N Total. Adjusted p-values were calculated usig the False Discovery Rate method. * Total of samples evaluated i which PCR was permed Primary Outcome Results: Evaluatio of the parameters accordig to the BRAF sequecig results % other cells I / (mm2) WT N (%) 61 (50.4) 61 (50.4) 61 (50.4) 61 (50.4) 61 (50.4) 61 (50.4) 61 (50.8) 59 (50.9) Missig Mea Media St deviatio Miim value Maxim value MUTATED N (%) 60 (49.6) 60 (49.6) 60 (49.6) 60 (49.6) 60 (49.6) 60 (49.6) 59 (49.2) 57 (49.1) Missig Mea Media St deviatio Miim value Maxim value N Total (99.2) 116 (95.9) Adj. p-value N(%)= umber (percetage) of samples i the category. Percetage=100*N/N Total.

10 Adjusted p-values were calculated usig the False Discovery Rate method. Primary Outcome Results: Evaluatio of the parameters accordig to the NRAS sequecig results cells I % other / (mm2) WT N (%) 98 (88.3) 98 (88.3) 98 (88.3) 98 (88.3) 98 (88.3) 98 (88.3) 97 (88.2) 95 (88.0) Missig Mea Media St deviatio Miim value Maxim value MUTATED N (%) 13 (11.7) 13 (11.7) 13 (11.7) 13 (11.7) 13 (11.7) 13 (11.7) 13 (11.8) 13 (12.0) Missig Mea Media St deviatio Miim value Maxim value N Total Adj. p-value N(%)= umber (percetage) of samples i the category. Percetage=100*N/N Total. Adjusted p-values were calculated usig the False Discovery Rate method. Primary Outcome Results: Correlatio test results betwee / ad the other parameters Correlatio test P-value Coefficiet R / < / / < /% of tumor i < /% of tumor tumor area / / < /% of tumor i < /% of other tumor area /% of stroma tumor area /% of other tumor area P-value from Pearso correlatio test associatio betwee paired samples. Coefficiet R = Pearso Correlatio coefficiet. Coclusio(s): Overall, 165 FFPE samples from 98 melaoma patiets (71 primary melaoma, 53 setiel lymph odes (SLN) ad 41 metastasis) were recruited from 1994 to After evaluatio, was obtaied from 144 samples ad BRAF ad NRAS were sequeced. Complete sequecig results were obtaied from 75% (108/144) of the samples, ad at least oe gee was sequeced i 89% (128/144) of them. BRAF was mutated i 55% (29/53) ad NRAS i 11% (5/45) of the primary melaomas evaluated. A correlatio with was foud the tumor area used (mm2) (adj p-value<0.01, r=0.73). No differeces were detected i the sequecig success based o the age of the block. Date Updated: 14-March-2016