Can molecular targeted therapies win the war against cancer?

Transcription

1 Can molecular targeted therapies win the war against cancer? DR. PETÁK ISTVÁN PHD KPS MOLEKULÁRIS DIAGNOSZTIKAI KÖZPONT MTA-SE PATHOBIOKÉMIAI MUNKACSOPORT Anti- GEFITINIB An - M+ KRAS Schwab Richárd, Peták István, Pintér Ferenc.Kopper László, Kéri György, Pap Ákos J Clin Oncol Oct 20;23(30):

2 N EnglJ Med May 20;350(21): Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG, Louis DN, Christiani DC, SettlemanJ, Haber DA. 1 month treatment of mutant patient with egfr inhibitor lazarus effect

3 12,7 MILLION 7,6 MILLION 1:4 GLOBOCAN 2008, WHO 2003?

4 Cancer Cancer Cancer Somatic Germline Tissue Molecular Symbol Name GeneID Chr Chr Band Mut Mut Tumour Types (Somatic Mutations) Tumour Types (Germline Mutations) Cancer Syndrome Type Genetics Mutation Type Translocation Partner AKT1 v-akt murine thymoma viral oncogene homolog q32.32 yes breast, colorectal, ovarian, NSCLC E Dom Mis AKT2 v-akt murine thymoma viral oncogene homolog q13.1-q13.2 yes ovarian, pancreatic E Dom A ALDH2 aldehyde dehydrogenase 2 family (mitochondrial) q24.2 yes leiomyoma M Dom T HMGA2 ALK anaplastic lymphoma kinase (Ki-1) p23 yes yes ALCL, NSCLC, Neuroblastoma, Breast, neuroblastoma Colon cc APC adenomatous polyposis of the colon gene q21 yes yes colorectal, pancreatic, desmoid, hepatoblastoma, glioma, other CNS colorectal, pancreatic, desmoid, hepatoblastoma, glioma, other CNS ATRX alpha thalassemia/mental retardation syndrome X-linked 546 X Xq21.1 yes Pancreatic neuroendocrine tumors E Rec Mis, F, N Familial neuroblastoma L, E, M Dom T, Mis, A NPM1, TPM3, TFG, TPM4, ATIC, CLTC, MSN, ALO17, CARS, EML4 Adenomatous polyposis E, M, coli; O Turcot Rec syndrome D, Mis, N, F, S v-raf murine sarcoma viral oncogene homolog B q34 yes melanoma, colorectal, papillary thyroid, borderline ov, Non small-cell lung cancer (NSCLC), cholangiocarcinoma, E Dom pilocytic Mis, astrocytoma T, O AKAP9, KIAA1549 BRCA1 familial breast/ovarian cancer gene q21 yes yes ovarian breast, ovarian Hereditary breast/ovarian E cancerrec D, Mis, N, F, S BRCA2 familial breast/ovarian cancer gene q12 yes yes breast, ovarian, pancreatic breast, ovarian, pancreatic, leukemia (FANCB, Hereditary FANCD1) breast/ovarian L, EcancerRec D, Mis, N, F, S CDKN2A -p16(ink4a) cyclin-dependent kinase inhibitor 2A (p16(ink4a)) gene p21 yes yes melanoma, multiple other tumour types melanoma, pancreatic CDKN2A- p14arf cyclin-dependent kinase inhibitor 2A-- p14arf protein p21 yes yes melanoma, multiple other tumour types melanoma, pancreatic Familial malignant melanoma L, E, M, O Rec D, Mis, N, F, S Familial malignant melanoma L, E, M, O Rec D, S CTNNB1 catenin (cadherin-associated protein), beta p22-p21.3 yes colorectal, cvarian, hepatoblastoma, others, pleomorphic salivary adenoma E, M, O Dom H, Mis, T PLAG1 DAXX death-domain associated protein p21.3 yes Pancreatic neuroendocrine tumors E Rec Mis, F, N EP kd E1A-Binding protein gene q13 yes colorectal, breast, pancreatic, AML, ALL, DLBCL L, E Rec T, N, F, Mis, O MLL, RUNXBP2 ERBB2 v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma q21.1 derived yes oncogene homolog (avian) breast, ovarian, other tumour types, NSCLC, gastric E Dom A, Mis, O ERCC2 excision repair cross-complementing rodent repair deficiency, 2068 complementation 19 19q13.2-q13.3 group 2 (xeroderma yes pigmentosum D) skin basal cell, skin squamous cell, melanoma Xeroderma pigmentosum E (D) Rec Mis, N, F, S ERCC3 excision repair cross-complementing rodent repair deficiency, 2071 complementation 2 2q21 group 3 (xeroderma yes pigmentosum group B complementing) skin basal cell, skin squamous cell, melanoma Xeroderma pigmentosum E (B) Rec Mis, S ERCC4 excision repair cross-complementing rodent repair deficiency, 2072 complementation 16 16p13.3-p13.13 group 4 yes skin basal cell, skin squamous cell, melanoma Xeroderma pigmentosum E (F) Rec Mis, N, F ERCC5 excision repair cross-complementing rodent repair deficiency, 2073 complementation 13 13q33group 5 (xeroderma yes pigmentosum, complementation group G (Cockayne skin basal syndrome)) cell, skin squamous cell, melanoma Xeroderma pigmentosum E (G) Rec Mis, N, F FBXW7 F-box and WD-40 domain protein 7 (archipelago homolog, Drosophila) q31.3 yes colorectal, endometrial, T-ALL E, L Rec Mis, N, D, F FGFR2 fibroblast growth factor receptor q26 yes gastric. NSCLC, endometrial E Dom Mis IL6ST interleukin 6 signal transducer (gp130, oncostatin M receptor) q11 yes hepatocellular ca E Dom O KRAS v-ki-ras2 Kirsten rat sarcoma 2 viral oncogene homolog p12.1 yes pancreatic, colorectal, lung, thyroid, AML, others L, E, M, O Dom Mis MADH4 Homolog of Drosophila Mothers Against Decapentaplegic 4 gene q21.1 yes yes colorectal, pancreatic, small intestine gastrointestinal polyps Juvenile polyposis E Rec D, Mis, N, F MAP2K4 mitogen-activated protein kinase kinase p11.2 yes pancreatic, breast, colorectal E Rec D, Mis, N MDM2 Mdm2 p53 binding protein homolog q15 yes sarcoma, glioma, colorectal, other M, O, E, LDom A MLH1 E.coli MutL homolog gene p21.3 yes yes colorectal, endometrial, ovarian, CNScolorectal, endometrial, ovarian, CNS Hereditary non-polyposis E, Ocolorectal Reccancer, D, Turcot Mis, syndrome N, F, S MSH2 muts homolog 2 (E. coli) p22-p21 yes yes colorectal, endometrial, ovarian colorectal, endometrial, ovarian Hereditary non-polyposis E colorectal Reccancer D, Mis, N, F, S MSH6 muts homolog 6 (E. coli) p16 yes yes colorectal colorectal, endometrial, ovarian Hereditary non-polyposis E colorectal Reccancer Mis, N, F, S MSI2 musashi homolog 2 (Drosophila) q23.2 yes CML L Dom T HOXA A CENSUS OF HUMAN CANCER GENES SANGER DATABASE LEUKEMIAS LYMPHOMAS 500 cancer genes 1% of allgenes 90% somatic mutations 20% germlinemutations 10% show both somatic and germline mutations 100 CANCER GENES IN EPITHELIAL CANCERS Nat Rev Cancer Mar;4(3): FutrealPAet al. Cancer Genome Project, Human Genome Analysis Group and PfamGroup, WellcomeTrust Sanger Institute, 2008 first individual human genome sequenced COST OF SEQUENCING JAMES WATSON

5 2012 CANCER ATLAS POJECT 33 GENES IN COLON CC 40 GENES IN BREAST CC 2013 CANCER GENOME LANDSCAPES Total of 138 driver genes (20/20 rule ) 1-8 driver genes / tumor Cancer genome landscapes. Vogelstein B, Papadopoulos N, VelculescuVE, Zhou S, Diaz LA Jr, KinzlerKW. Science Mar 29;339(6127):

6 GENOME LANDSCAPE IN COLON CANCER PIK3CA P53 KRAS APC Laura D. Wood, et al.science 318, 1108 (2007); More than 1 reason for multigene tumor profiling! We know 138 driver genes and each tumor contains 1-8 of them. Only tumors with 1 driver mutation respond well to targeted monotherapy Cancer genome landscapes. Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA Jr, Kinzler KW. Science Mar 29;339(6127): Strategies to overcome clinical, regulatory, and financial challenges in the implementation of personalized medicine. Tsimberidou AM, Ringborg U, Schilsky RL. Am Soc Clin Oncol Educ Book. 2013;2013: WE NEED COMBINATIONS OF TARGETED THERAPIES BASED ON THE WHOLE MOLECULAR PROFILE!

7 DRIVER GENE ACTIVATION RECEPTOR/LIGAND OVEREXPRESSION Anti-GFR MABs COPY NUMBER GAIN POINT MUTATIONS TKI TKI Which are the druggable gene mutation? Growth factor Growth Factor Receptor Tyrosin kinases A Mutant Gene Cancer Cell Division Nucleus Faulty signal transduction protein Targeted drug

8 NORMAL CELL AMP EGF KRAS CANCER CELL AMP EGF EGF Anti- KRAS

9 CANCER CELL Anti- KRAS CANCER CELL Anti- KRAS

10 CANCER CELL Anti- MET MET KRAS MET CANCER CELL Anti- AMP EGF KRAS

11 CANCER CELL Anti- AMP EGF KRAS THE exclusive club within the targeted therapies: Causative therapies of ONCOGENIC DRIVERS BCR/ABL KIT M+ M+ M+ EML4/ALK M+ IMATINIB NILOTINIB DASATINIB BOSUTINIB IMATINIB SUNITINIB GEFITINIB ERLOTINIB AFATINIB AFATINIB CRIZOTINIB PLX4032 CML ALL GIST MELANOMA NSCLC SCLC OVARIAN, GASTRIC, BREAST NSCLC NSCLC NEUROBL. MELANOMA (COLON) RR= % PFS=~ 1 year/years

12 FROM TARGET TO MARKET: 2 YEARS? OPPORTUNITY BUT FAST FOLLOWERS! OLD MODELL NEW MODELL ~100 TARGETS ~400 DRUGS WHICH DRUG? WHICH PATIENTS?

13 5000< CLINICAL TRIALS FOR BREAST CANCER ( MORE AND MORE BIOMARKER DRIVEN LOOKING FOR SMALL FRACTION OF PATIENTS DRUGABLE DRIVER MUTATIONS IN LUNG CANCER gefitinib erlotinib 5-15% (M+) 5-40% (2 X ) AMP HEREG TGFa afatinib 4% (M+) 22% (2 X ) 5-20 (M+) NRAS 5% (M+) 1% (M+) GSK % PLANO (2 X ) 3% ADENO TKI258 crizotinib FGFR 4-7% (break) KRAS HRAS GSK'436 vemurafenib ALK/EML4 PIK3CA AKT/PKB MTOR C-MET 5% (M+) BEZ235 1% (M+) PTEN everolimus 3% (M+) 12% (2 X ) 2% (M+) KIT PDGFR MET-MAB crizotinib imatinib sunitinib

14 DEPENDENT TUMORS M+, FISH, EGF+ 50% TARGETS IN LUNG ADENOCARCINOMA M+ PIK3CA M+ 5% MET+ 5% ALK M+ 5% 5% 1% M+ 5% M+ 10% FISH+ AMP+ 40% KRAS M+ (20-30%) RR 10% INHIBITORS INH INH MET PI3K INH INH INH INH SD 30-40% PD 50% INH G I LINE PFS 12 MONTHS ERLOTINIB ONLY REINBURSED IN KRAS WT OR M+ IN HUNGARY ERLOTINIB II line (PFS 6 MONTHS) DRUGGABLE DRIVERS IN COLON CANCER Anti- MABs 15% EGF 0% M+ 10% CNG NRAS 3% M+ 5-8% CNG 40% (M+) 8% (M+) KRAS M+ 5% (M+) KRAS WT WNT FZD APC 50% (M+) WNT5a ROR2 C-MET 3% M+ 10% CNG PIK3CA AKT/PKB 15% (M+) HGF PTEN IGFR IRS2 7% IGF2 3% (M+) 22% CDC25 ERK B-Catenin 6% (M+) MTOR

15 ONCOGENIC DRIVERS IN COLON CANCER -DRIVEN COLON CC 20-25% ( CNG, +LIGANDS) M+ 5% ALK/EML! 2,5% PIK3CA M+ MET+ 10% NRAS 2,3,4 M+ 10% 5-10% KRAS 2,3,4 exons M+ 10% M+ 5% KRAS 1 exons M % ANTI- MABs Panitumumab ALK INH? MET INH? INH? PI3K INH? INH? INH? INH? Cetuximab WT M KRAS M12 KRAS M13 PIK3CA M NRAS M

16 IDENTIFICATION OF RESPONDERS WITH MULTIPE GENE TESTING NOT TREATED NON RESPONDERS RESPONDERS KRAS 1 GENE KRAS, NRAS,, PIK3CA 4 GENES COLON ADENO CC (KRAS/NRAS//PIK3CA WT) ANTI- (CETUXIMAB) TREATMENT

17 DRUGABLE DRIVER MUTATIONS IN breast CANCER trastuzumab 20-30% (CNG) pertuzumab 16% (CNG) 2-12% (M+) lapatinib 16-27% (CNG) FGFR ALK/EML4 C-MET 2-3% (M+) KRAS PIK3CA 25-40% (M+) HRAS NRAS AKT/PKB MTOR PTEN KIT PDGFR 4% (M+) 2-7% (M+) 50% (del) 1% (M+) ER AR PR lapatinib everolimus EMLŐDAGANATOK MOLEKULÁRIS SPEKTRUMA DRUGABLE DRIVER MUTATIONS IN breast CANCER M+ 1-10% CNG 10% CNG 15% CNG +PK3CA/ PTEN 15% PIK3CA+ 20% AKT 4% 1% MET+?% FGFR 20% 5% KRAS 3% ALK 2,5% HER FISH+ + PIK3CA/ PTEN INHIBITORS INHIBITORS / INHIBITORS PIK3CA INH mtor INH AKT INH INH MET INH FGFR INH INH ALK INH INH

18 1 DRIVER: (KINASE DOMAIN MUTATIONS) More sensi ve than wild type Lapa nib Cell line IC50 = 496nM Less sensi ve than wild type Trastuzumab Lapa nib Cell line IC50 = 286 nm Resistant Lapa nib Cell line IC50 = 249nM Lapa nib Cell line IC50 = 96 nm Trastuzumab Lapa nib Cell line IC50 =1433 nm Lapa nib Cell line IC50 = 461nM Lapa nib Cell line IC50 = 3339 nm Lapa nib Cell line IC50 =894 nm Washington University Next Generation Sequencing 1.6 percent, 4,000 women per year in the US alone (same as CML) Lapa nib Cell line IC50 = 407nM Lapa nib Cell line IC50 =670 nm Most are lapatinib resistant! Trastuzumab Trastuzumab Lapa nib Cell line IC50 = 3273 nm All neratinib sensitive (study in USA) Lapa nib Cell line IC50 =1056 nm Lapa nib Cell line IC50 = 1127nM Lapa nib Cell line IC50 = 2861nM Lapa nib Cell line IC50 = 253 nm Lapa nib Cell line IC50 = 1154nM MULTIPLE DRIVER BREAST CANCERS ( is not alone) PTEN PIK3CA MET ALK KRAS Single Driver

19 2 DRIVERS: AND AMP EGF TGFA HEREG AMP EGF An - HER-3 An - trastuzumab Pertuzumab Lapatinib (Neratinib) KRAS PI3K AKT MTOR GhoshR, NarasannaA, Wang SE, Liu S, ChakrabartyA, BalkoJM, González-AnguloAM, Mills GB, PenuelE, Winslow J, SperindeJ, DuaR, PidaparthiS, Mukherjee A, LeitzelK, KostlerWJ, Lipton A, Bates M, ArteagaCL. Trastuzumabhas preferential activity against breast cancers driven by HER2homodimers. Cancer Res Mar 1;71(5): DRIVERS: AND PIK3CA An - lapatinib KRAS PI3K AKT MTOR

20 2 DRIVERS: AND PIK3CA ~50% ~50% PIK3CA PTEN PIK3CA PTEN WT M+ Trastuzumab 66,7% P=0,02 18,2% Lapatinib 41,7% P=0,05 81,3% JCO 2011/01 20/2 HETEROGENECITY AND SYNCHRON TUMORS DRIVER A DRIVER A +B DRIVER C

21 Can I give this drug to my patient? OR Which is the best option to my patient? Registered drug 1 Registered drug 1 DRUG (registered, in trial) PATIENT Trial drug 2 Trial drug 1 AN EXAMPLE WITH REGISTERED DRUGS IN LUNG CANCER CAN I GIVE ERLOTINIB?, KRAS? CAN I GIVE CRIZOTINIB? ALK? (4-7%) WHICH DRUG IS THE BEST FOR MY PATIENT? gefi nib erlo nib 5-15% (M+) 5-40% (2 X ) AMP HEREG TGFa afa nib 4% (M+) 22% (2 X ) 5-20 (M+) NRAS 5% (M+) 1% (M+) GSK % PLANO (2 X ) 3% ADENO TKI258 crizo nib FGFR 4-7% (break) KRAS HRAS ALK/EML4 PIK3CA C-MET 5% (M+) BEZ235 AKT/PKB PTEN GSK'436 1% (M+) vemurafenib MTOR everolimus 3% (M+) 12% (2 X ) 2% (M+) KIT PDGFR DCR erlotinibin ALK + 0%* DCR crizotinib IN ALK 90%** MET-MAB crizo nib ima nib suni nib *Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK. Shaw AT, YeapBY, Mino-KenudsonM, DigumarthySR, Costa DB, Heist RS, Solomon B, Stubbs H, AdmaneS, McDermott U, SettlemanJ, Kobayashi S, Mark EJ, RodigSJ, ChirieacLR, KwakEL, Lynch TJ, IafrateAJ. J Clin Oncol Sep 10;27(26): **Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. KwakEL, Bang YJ, CamidgeDR, Shaw AT, Solomon B, Maki RG, OuSH, DezubeBJ, JännePA, Costa DB, Varella-Garcia M, Kim WH, Lynch TJ, Fidias P, Stubbs H, EngelmanJA, SequistLV, Tan W, Gandhi L, Mino-KenudsonM, Wei GC, ShreeveSM, RatainMJ, SettlemanJ, Christensen JG, Haber DA, WilnerK, SalgiaR, Shapiro GI, Clark JW, IafrateAJ. N EnglJ Med Oct 28;363(18):

22 AN EXAMPLE WITH REGISTERED DRUGS IN COLON CANCER CAN I GIVE BEVACIZUMAB? NO TEST! CETUXIMAB PANITUMUMAB 3% M+ 5-8% CNG 15% EGF 0% M+ 10% CNG WNT FZD WNT5a ROR2 HGF C-MET IGF2 3% M+ 10% CNG IGFR 22% 40% (M+) KRAS WT IRS2 7% CAN I GIVE CETUXIMAB? NRAS KRAS M+ APC 8% (M+) 50% (M+) 5% (M+) PIK3CA 15% (M+) AKT/PKB PTEN 3% (M+) B-Catenin MTOR KRAS TEST CDC25 ERK 6% (M+) CAN I GIVE PANITUMUMAB? KRAS, NRAS TEST WHICH DRUG IS THE BEST FOR MY PATIENT? KRAS M+ = BEVACIZUMAB KRAS WT = BEVACIZUMAB/ CETUXIMAB KRAS/NRAS WT = PANITUMUMAB IMPACT (Initiative for Molecular Profiling and Advanced Cancer Therapy) in the University of Texas MD Anderson Cancer Center, USA* 2,282 patients with solid tumors ( ) 12 genes (seqeuncing, immunohystochemistry, FISH) 52,2% with 1 or more mutations 440 patients (19,3%) were enrolled into Phase I trials based onthe molecular profile and 442 into molecularly not mached trials Patient benefit significantly more from molecular profile based trials RR was significantly higher (39% versus 15% P<0,0001) PFS was significantly better (4,9 months versus 2,2 months, P<0,0001) OS was significantly longer (11,2 months versus 8,6 months, P<0,006) Median number of previous line of therapy: 3! Apostolia M. Tsimberidou Patients benefit significantly more molecular profile based Phase I trials than from the previous line of registered chemotherapy protocol (TTF 4,0 versus 3,1 months, P<0,0008). In contrary, in unmatchedtrials patients had worse response in the trials (2,0 versus 3,2 months, P<0,0001). *Tsimberidou AM et al. Strategies to overcome clinical, regulatory, and financial challenges in the implementation of personalized medicine. Soc Clin Oncol Educ Book. 2013;2013: Tsimberidou AM et al. Personalized medicine in a phase I clinical trials program: the MD Anderson Cancer Center initiative. Clin Cancer Res Nov 15;18(22): doi: / CCR Epub 2012 Sep 10.,

23 The Integrated Molecular Profiling in Advanced Cancers Trial (IMPACT) at the Princess Margaret Cancer Centre (PMCC), Toronto, Canada* 678 patients with solid tumors (03/ /2013) 23 genes (280 mutations, Sequenom) (89%, 3,6 ng, 5,3 weeks) 48 genes (MiSeq) 42%/78% with 1 or more mutations % treatment based on molecular profile (After a median follow up of 7.0 months!) 53% of treatments in clinical trials Median number of previous line of therapy: 2 (ECOG 0,1,2 37%/61%/2%) OR 29%, SD 70% (N=21) The average response rate observed in Phase I clinical trials is 5-10% ** *Philippe L. Bedard, MD Integrated Molecular Profiling in Advanced Cancers Trial (IMPACT) at the Princess Margaret Cancer Centre(PMCC)using genotyping and targeted next-generation sequencing (NGS). Abstract No: 11002, ASCO 2013 **Patient selection for oncology phase I trials: a multi-institutional study of prognostic factors. Olmos D, A'hern RP, Marsoni S, Morales R, Gomez-Roca C, Verweij J, Voest EE, Schöffski P, Ang JE, Penel N, Schellens JH, Del Conte G, Brunetto AT, Evans TR, Wilson R, Gallerani E, Plummer R, Tabernero J, Soria JC, Kaye SB. J Clin Oncol Mar 20;30(9): Molecular screening for cancer treatment optimization MOSCATO-01, Institute GustaveRoussy, Villejuif, France* 129 patients with solid tumors (lung, head and neck, bladder, prostate, ovarian, breast, colorectal, esophagus, gastric, pancreas, uterus, biliary track, renal, CUP, and other rare tumors) 50 genes (Ion AmpliseqCancer Hotspot Panel, Life Technologies) Median number of previous line of therapy: 3 (ECOG 1) 25%<? 47%! 47% with more than 1 target 29% of all patients were treated based on the molecular profile RR 21%, SD 48%, PD 21% PFS ratio in comparison to previous chemotherapy was >1.3 among 9 out of 19 evaluable patients (47%) *Antoine Hollebecque et al. Institut Gustave Roussy, Villejuif, France; Institut de Cancerologie Gustave Roussy, Villejuif, France Molecular screening for cancer treatment optimization (MOSCATO 01): A prospective molecular triage trial Interim results. Abstract No: 2512 J Clin Oncol 31, 2013 (suppl; abstr 2512)

24 The personalized medicine platform Molecular Oncologist Molecular Pharmacologist Molecular Diagnostics Molecular Oncologist Have a strong understanding of molecular medicine. Use only solid scientific information published in peer reviewedscientific journals listed in PUBMED. Share the vision that there is no alternative or classic medicine, only science based. Share the vision that molecular evidence has to be incorporated into the treatment strategy of cancer patients. Share the vision that patients have the right to be informed about all scientifically proven treatment and diagnostic options. Share the vision that if there is no curative standard treatmentavailable in clinical use, the patient has to be referred to a clinical development program, which is most likely provide additional chance even if that clinical trial site is at another centrum or even in a different country.

25 PARTNER MOLECULAR ONCOLOGISTS ALL OVER THE WORLD Targeted Drugs in Use and Development DRUGS IN CLINICAL USE: (32) AXITINIB, BEVACIZUMAB, BORTEZOMIB, BOSUTINIB, CABOZANTINIB, CETUXIMAB, CRIZOTINIB, DAENIB, DASATINIB, ERLOTINIB, EVEROLIMUS, GEFITINIB, IMATINIB, LAPATINIB, METFORMIN, NILOTINIB, PANITUMUMAB, PAZOPANIB, PERTUZUMAB, PONATINIB, REGORAFENIB, RUXOLITINIB, SORAFENIB, SUNITINIB, TEMSIROLIMUS, TRAMETINIB, TRASTUZUMAB, TDM-1, VANDETANIB, VEMURAFENIB, VISMODEGIB, ZIV-AFLIBERCEPT DRUGS IN CLINICAL TRIALS: (173) AKN-028, AMG 208, AMG 319, AMG 337, AP26113, ARQ 092, ARQ 736, ASP-3026, AT13148, AT9283, AUY922, AV-412, AXL1717, AZD0424, AZD1480, AZD2014, AZD4547, AZD5363, AZD8330, BAY , BEZ235, BGJ398, BGT226, BKM120, BMN 673, BMS , BMS , BRIVANIB, BYL719, CC-223, CEDIRANIB, CH , CO-1686, CRENOLANIB, DALOTUZUMAB, DACOMITINIB, DEMCIZUMAB, DKN-01, DOVITINIB, DS-7423, E-3810, E6201, EMD , EMD , ERISMODEGIB, FORETINIB, GANITUMAB, GDC-0068, GDC-0623, GDC-0941, GDC-0973, GDC-0980, GOLVATINIB, GSK , GSK A, GSK , GSK , GSK , GSK , GSK343, GSK , HM61713, IMC-CS4, INIPARIB, JNJ , KU 55933, LDK-378, LENVATINIB, LESTAURTINIB, LGX818, LINSITINIB, LJM716, LY , LY , LY , LY , LY287445, MASITINIB, MEDI- 573, -162, MGAH22, MGCD265, MIDOSTAURIN, MK-0752, MK-2206, MK-8242, MLN1117, MM-302, MOMELOTINIB, NECITUMUMAB, NERATINIB, NINTEDANIB, NS-398, OLAPARIB, OLARATUMAB, ONARTUZUMAB, OSI-027, P7170, PACRITINIB, PD , PD , PD173074, PF , PF , PF , PF , PF , PIMASERTIB, PKI-179, PRI-724, PX-866, R , RABUSERTIB, RAF265, RAMUCIRUMAB, REBASTINIB, REFAMETINIB, RIDAFOROLIMUS, RILOTUMUMAB, RO , RO , RUCAPARIB, SAR125844, SARACATINIB, SARIDEGIB, SCH , SELUMETINIB, SEMAGACESTAT, SGX523, SR13668, SU-1127, TAE684, TAK-733, TAK-960, TG101348, TILMACOXIB, TIVANTINIB, TIVOZANIB, TREBANANIB, TRICIRIBINE, VELIPARIB, VOLASERTIB, VX-509, WX-554, WHI-P154, X- 396, X-82, XL019, XL147, XL281, XL765, ZM39923, ZSTK474

26 STEP 1. BUILT A DATABASE OF TARGETS AND DRUGS NCBI - National Center of Biotechnology Information KEGG Kyoto Encyclopedia of Genes and Genomes NCI National Cancer Institute Selleckchem Genomics of Drug Sensitivity in Cancer FDA U. S. Food and Drug Administration EMEA European Medicines Agency OEP Országos Egészségbiztosítási Pénztár Kohánka Andrea STEP2. BUILT a DATABASE OF MUTATIONS Kirsch Klára

27 STEP 3. DEVELOP A PROGRAM TO FIND MUTATIONS Pongor Lőrinc HeurAA Heuristic Amplicon Analyzer HeurAA: accurate and fast detection of genetic variations with a novel heuristic amplicon aligner program for next generation sequencing. PongorLS, PintérF, PetákI. PLoS One. 2013;8(1):e STEP4. BUILT PROGRAM FOR AUTOMATIC INTERPRETATION Binder Zsófi

28 TARGETED RESEQUENCING PANELS STEP5. DEVELOP THE DIAGNOSTIC PANEL Illumina HiSeq 2000 platform for 2574 exons representing 176 genes captured using Agilent SureSelect 50% acionable mutations MGH's Diagnostic Molecular Pathology lab SNaPshot assay, hotspot mutations in 14 oncogenes 50 to 60 patients per week NEXT: NGS 50 different targets Genomweb September 19, in combination with fluorescence in situ hybridization 58 genes ABL1, AKT1, ALK, APC, ATM,, CDH1, CDKN2A, CSF1R, CTNNB1,, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, VHL.+ egyedi gének + FISH:,, FGFR, ALK, MET YOU NEED A MOLECULAR DIAGNOTICS LAB Separated PRE and POST PCR ROOM! Watch out for contamination! External Quality Control Ring Trials! Internal Quality Control!

29 SOMATIC MUTATIONS ARE MORE DIFFICULT TO ANALYZE THAN HEREDITARY MUTATIONS! Hereditary mutations and SNPs: a) present in all cells b) the ratio of mutant allele 0% 50% 100% Somatic mutations: a) only present in cancer cells b) the ratio of mutant allele can anything between 0-100%!!! The % of a mutant allele depends on: a) normal tumor cell ratio b) heterogeneity II Normal cell II Tumor cell II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II IIIII II II II II II II II II II IIIII II II II IIIII II II II II II II USE LCM FOR SMALL SAMPLES OR LOW TUMOR RATIO 100% LCM OPTIMIZED DIRECT PCR MULTIPLEX PCR

30 SANGER SEQUENCING TECHNOLOGIES qpcr NGS The Gold standard ~100% Specificity ~100% mutations ~15-30% limit sensitivity IVD kits available Fast Selected mutations ~1% limit sensitivity High capacity ~100% Specificity ~100% mutatioms ~0,01-5% digital quantitative data CLONAL SEQUENCING WITH NGS Load beads into PicoTiter Plate Load Enzyme Beads Centrifuge Step 44 μm

31 Results (N=72, follow up 6 months) Molecular profile matched registered drug found in 23% Molecular profile matched drug in clinical use found in 67% Molecular profile matched drug in clinical development found in 94% Patients entered molecular profile matched clinical trials: 22% 3 cross-country patients 61 years old man with ER+/-mamma cc. Chemotherapy and hormone therapy resistant With FGFR1 amplification (PIK3CA-, MET-) 35 years! old lady, triple negative breast cancer Multiple line of chemotherapy FGFR3-F384L Multicentric, Phase 1/b, for solid tumors FGFR amplfified or mutant trastuzumab 16-27% (CNG) 20-30% (CNG) FGFR pertuzumab 16% (CNG) 2-12% (M+) lapa nib ALK/EML4 C-MET 2-3% (M+) KRAS PIK3CA 25-40% (M+) HRAS NRAS 4% (M+) 2-7% (M+) AR AKT/PKB PTEN 50% (del) 1% (M+) ER PR MTOR lapa nib KI T PDGFR everolimus

32 51 ÉVES NEM-DOHÁNYZÓTÜDŐADENOCARCINOMA ALK+ NEM-KISSEJTES TÜDŐRÁKOS BETEG gefi nib erlo nib 5-15% (M+) 5-40% (2 X ) AMP HEREG TGFa afa nib 4% (M+) 22% (2 X ) 5-20 (M+) NRAS 5% (M+) 1% (M+) GSK KRAS 20% PLANO (2 X ) 3% ADENO TKI258 crizo nib FGFR 4-7% (break) HRAS ALK/EML4 PIK3CA C-MET 5% (M+) BEZ235 AKT/PKB PTEN GSK'436 1% (M+) vemurafenib MTOR everolimus 3% (M+) 12% (2 X ) 2% (M+) KIT PDGFR MET-MAB crizo nib ima nib suni nib MIVEL IS TÖLTÖM AZ EGÉSZ NAPJAIMAT???? ESTÉNKÉNT ELSÉTÁLOK A TENGERPARTRA ÁLTALÁBAN 6-9IG OTT VAGYOK.. HÉTVÉGÉN A CSALÁDDAL BESZÉLEK NETEN FACEBOOKON,.ÉS KÖZBE IKTATOM A VIZSGÁLATOK NAPJAIT, ÉS MÉG MINDIG HIMZEK, UTÁNNA PEDIG HORGOLNI FOGOM A TERITŐK SZÉLEIT.. MALAGA, SPANYOLORSZÁG