Marcatori predittivi dell efficacia di farmaci mirati in pazienti con malattia avanzata. Milo Frattini Istituto cantonale di patologia Locarno

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1 Marcatori predittivi dell efficacia di farmaci mirati in pazienti con malattia avanzata Milo Frattini Istituto cantonale di patologia Locarno Legnano

2 Meyerhardt et al, N Engl J Med 2005;352: Prognosis

Chemotherapeutic treatments Treatment Number of drugs Survival (months) 5-FU/LV 1 11-12

3 Chemotherapeutic treatments Treatment Number of drugs Survival (months) 5-FU/LV Irinotecan/5-FU Oxaliplatin/5-FU ± Irinotecan Meyerhardt et al, N Engl J Med 2005;352:

4 Molecular targets Epidermal growth factor receptor (EGFR) Vascular endothelial growth factor receptor (VEGFR) COX-2 Others Carcino-Embryonic Antigen (CEA) Protein kinase C Matrix Metalloproteinase Ras Cyclin dependent kinase

5 HER (ErbB)-Family cell proliferation tumoral metastasization tumoral invasion angiogenesis Ciardiello and Tortora, N Engl J Med 2008;358:

6 EGFR: mechanism of activation Ligand Monomer Monomer P P Dimer Cross-auto phosphorylation Cell survival Signaling pathways Cell duplication

7 Network of epidermal growth factor receptor (EGFR) interactions with downstream signaling pathways Harari et al, J Clin Oncol 2007;25:

8 EGFR as target therapy Monoclonal Antibodies Cetuximab (Erbitux ) Panitumumab (Vectibix ) Tyrosin Kinase Inhibitors Erlotinib (Tarceva ) Gefitinib (Iressa ) Lapatinib (Tyverb ) Ciardiello and Tortora, N Engl J Med 2008;358:

Anti-EGFR MoAb therapies: when Cetuximab and Panitumumab recognize the extracellular domain of EGFR, lead to receptor internalization and degradation, and therefore block EGFR and its downstream

9 Anti-EGFR MoAb therapies: when Cetuximab and Panitumumab recognize the extracellular domain of EGFR, lead to receptor internalization and degradation, and therefore block EGFR and its downstream cascade activation. Both drugs are effective only in ~10% of metastatic colorectal cancer (mcrc) patients when administered as single agent therapy, and ~ 20-30% when in combination with irinotecan EGFR protein overexpression by immunohistochemistry is required before treatment (Saltz et al, 2004; Cunningham et al, 2004; Van Cutsem, 2007)

11 EGFR protein expression by IHC There is no relationship between the level of EGFR expression as detected by IHC and anti-egfr MoAbs response. (Saltz et al, 2004; Cunningham et al, 2004) 25% of EGFR negative (as detected by IHC) patients benefit from cetuximab. (Chung et al, 2005) EGFR evaluation by IHC depends on: type of fixative used, storage time of unstained tissue sections methods of immnohistochemistry analyses and/or evaluation (Atkins et al, 2004; Langner et al, 2004; Kersting et al, 2006)

12 EGFR protein expression by IHC EGFR evaluation by IHC depends on the primary antibody used: DAKO pharmdx Zymed (EGFRkit) Ventana (3C6 AK) Cut-off level 1% 75% 93% 86% Cut-off level 5% 61% 80% 78% Cut-off level 10% 48% 72% 60% Penault-Llorca et al., Oncol Rep IHC does not seem to represent the gold standard method for patient selection and for anti-egfr therapy efficacy prediction

13 EGFR FISH normale Normal status (disomy)

14 EGFR FISH Chromosome 7 polysomy

15 EGFR FISH EGFR gene amplification

16 EGFR gene status disomy 3/27 (11%) Chromosome 7 polysomy 16/27 (59%) EGFR gene amplification 8/27 (30%) green = chromosome 7 centromere red= EGFR gene PR * NR (PD+SD) * Moroni et al, * - Patients with disomy do not respond - Patients with chromosome 7 marked polysomy and/or EGFR gene amplification may benefit from cetuximab treatment (Frattini et al, Br J Cancer 2007;97: )

17 EGFR gene status Authors and year Moroni et al Cases 31 Type mcrc FISH interpretation criteria Score EGFR gene/ nucleus (CNG). Increased EGFR CNG was defined as the presence of three or more signals per nucleus. Number of cases and % 9/20 (45%) Observations Of the 9 patients with CNG 8 responded and 1 non responded to cetuximab or panitumumab, suggesting a genetic basis of response to anti-egfr treatment Frattini et al mcrc 1) Loss if 1 copy of chr 7 in >50% of cells 2) Disomy if 2 copies of chr 7 in >50% of cells 3) Low polysomy If 3 or 4 copies of chr 7 in >50% of cells 4) Marked polysomy if >4 copies of chr 7 in >50% of cells 5) Amplification if R> 3 in at least 10% of cells 0/27 (0%) 3/27 (11%) 0/27 (0%) 16/27 (59%) 8/27 (30%) Patients whit amplification or marked polysomy have a increased likelihood to response to cetuximab therapy (depending from K-ras and PTEN status) while the disomic one in generally are resistant Sartore- Bianchi et al mcrc Score EGFR gene/nucleus and use the cut off value. FISH + if > 2.5 and/or > 40% chr 7 polysomy FISH - if 2.5 and/or 40% chr 7 polysomy 38-39/ /58 Patients with disomic or low polisomy of chr7 have a reduced likelihood to response to panitumumab Cappuzzo et al mcrc Score EGFR /nucleus and use the cut off value. FISH + if > 2.92 FISH - if /85 (50%) 42/85 (50%) Patients with EGFR CNG have an increased likelihood to response to cetuximab therapy EGFR gene status by FISH may represent a predictive factor of good response to targeted therapies against EGFR. (Martin et al, doi: /jcp )

18 Guidelines FISH SCORING SYSTEM classification Colorado & Working Group NSCLC Locarno revised mcrc FISH status Monosomy - 1 copy in 50% of cells negative Disomy 2 copies in 90% of cells 2 copies in > 60% of cells negative Low trisomy High trisomy 2 copies in 40% of cells, 3 copies in 10-40% of cells, 4 copies in < 10% of cells 2 copies in 40% of cells, 3 copies in 40% of cells, 4 copies in < 10% of cells - negative - negative Low polysomy 4 copies in 10-40% of cells 3 or 4 copies in > 40% of cells High polysomy Gene amplification 4 copies in 40% of cells > 4 copies in > 40% of cells positive Tight EGFR gene clusters and R 2 or 15 copies of EGFR per cell in 10 of analyzed cells Tight EGFR gene clusters and R 2 or 15 copies of EGFR per cell in 10 of analyzed cells positive (Martin et al, doi: /jcp )

EGFR gene status disomy 3/27 (11%) Chromosome 7 polysomy 16/27

centromere red= EGFR gene PR 0 4 6 1* NR (PD+SD) 3 12 2 * Moroni et

chromosome 7 marked polysomy and/or EGFR gene amplification may

19 EGFR gene status disomy 3/27 (11%) Chromosome 7 polysomy 16/27 (59%) EGFR gene amplification 8/27 (30%) green = chromosome 7 centromere red= EGFR gene PR * NR (PD+SD) * Moroni et al, * - Patients with disomy do not respond - Patients with chromosome 7 marked polysomy and/or EGFR gene amplification may benefit from cetuximab treatment (Frattini et al, Br J Cancer 2007;97: )

20 EGFR & downstream signals EGFR out cytoplasm mutated in ~20-30% of sporadic CRC mutated in ~40% of sporadic CRC PI3K K-Ras loss in ~30% of sporadic CRC PTEN Akt BRAF MEK mutated in ~5-10% of sporadic CRC mtor ERK1,2 cell survival angiogenesis changes in gene expression nucleus cell proliferation tumor invasion tumor metastasization

21 EGFR & downstream signals EGFR out cytoplasm mutated in ~20-30% of sporadic CRC mutated in ~40% of sporadic CRC PI3K K-Ras loss in ~30% of sporadic CRC PTEN Akt BRAF MEK mutated in ~5-10% of sporadic CRC mtor ERK1,2 cell survival angiogenesis changes in gene expression nucleus cell proliferation tumor invasion tumor metastasization

22 Patients, treatment regimens and clinical response No. % No. of patients with mcrc* Site of primary disease Colon cancer Rectal cancer Anti-EGFR MoAb ** Cetuximab Cetuximab + Chemotherapy Panitumumab Prior adjuvant chemotherapy # Yes Prior lines of chemotherapy EGFR IHC expression ( 1% cells) Yes Clinical response (RECIST) Responders Non-responders * 45 patients were treated at Oncology Institute of Southern Switzerland (Bellinzona, Switzerland) and 68 were treated at Ospedale Cà Granda (Milan, Italy) (Di Nicolantonio & Martini & Molinari et al, J Clin Oncol 2008;26: )

23 K-Ras status and clinical response Wild-type Mutant K-Ras 79/113 (70%) 34/113 (30%) * Responders 22/79 (28%) 2/34 (6%) Non-responders 57/79 (72%) 32/34 (94%) p<0.05, two-tailed Fisher s exact test K-Ras mutations correlate with resistance to to anti-egfr MoAbs (Di Nicolantonio & Martini & Molinari et al, J Clin Oncol 2008;26: )

24 Mutazioni di K-Ras: meta-analisi Lavoro Moroni et al. Lancet Oncology 2005 Pazienti rispondenti Casi K-Ras mutati 2 8 K-Ras wt 8 13 Pazienti resistenti % di mutazione di K-Ras 32% Lièvre et al. Clin Cancer Res 2006 Casi K-Ras mutati % K-Ras wt 11 6 Di Fiore et al. Casi % Mutazioni di K-Ras associate a resistenza a cetuximab/panitumumab Br J Cancer 2007 K-Ras mutati 0 16 Frattini et al. Br J Cancer 2007 K-Ras wt casi che rispondono, 5 hanno la mutazione di K-Ras (5%) Casi K-Ras mutati 1 9 K-Ras wt % Benvenuti et al. Cancer Res 2007 De Roock et al. ASCO Proc Amado et al J Clin Oncol 2007 Karapetis et al N Engl J Med 2008 Casi K-Ras mutati 1 15 K-Ras wt Casi 8 29 K-Ras mutati 0 15 K-Ras wt 8 14 Casi K-Ras mutati 0 84 K-Ras wt Casi K-Ras mutati 1 80 K-Ras wt % 41% 43% 42%

25 K-Ras status and clinical response Wild-type Mutant K-Ras 79/113 (70%) 34/113 (30%) * Responders 22/79 (28%) 2/34 (6%) Non-responders 57/79 (72%) 32/34 (94%) p<0.05, two-tailed Fisher s exact test?!? K-Ras mutations correlate with resistance to to anti-egfr MoAbs (Di Nicolantonio & Martini & Molinari et al, J Clin Oncol 2008;26: )

26 BRAF status and clinical response on wild-type K-Ras patients Wild-type Mutant BRAF 68/79 (86%) 11/79 (14%) * Responders 22/68 (32%) 0/11 (0%) Non-responders 46/68 (68%) 11/11 (100%) p<0.05, two-tailed Fisher s exact test BRAF mutations are are associated with lack of of response to to MoAbs treatment in in K-Ras wild-type tumors (Di Nicolantonio & Martini & Molinari et al, J Clin Oncol 2008;26: )

27 Role of BRAF mcrc patients treated with panitumumab or cetuximab, n = 113 KRAS mutational status **p<0.05 (p=0.011) Mutant KRAS 34/113 (30%) Wild-Type KRAS 79/113 (70%) Responders 2/34 (6%)* 22/79 (28%)** Non Responders 32/34 (94%)** 57/79 (72%)** K-Ras mutations identify 32/89 (36%) NR patients Mutant KRAS 34/113 (30%) BRAF mutational status on Wild-Type KRAS tumors *p<0.05 (p=0.029) Mutant BRAF 11/79 (14%) Wild-Type BRAF 68/79 (86%) Responders 0/11 (0%)* 22/68 (32%)* Non Responders 11/11 (100%)* 46/68 (68%)* BRAF mutations identify 11/89 (12%) NR patients Mutant BRAF 11/113 (10%) 40% patients show either K-Ras or or BRAF mutations K-Ras and BRAF mutations identify 48% of of NR NR patients (Di Nicolantonio & Martini & Molinari et al, J Clin Oncol 2008;26: )

28 Cellular models of CRC and response to anti-egfr MoAbs BRAF V600E mut The presence of of BRAF V600E allele in in CRC cells impairs their response to to cetuximab or or panitumumab (Di Nicolantonio & Martini & Molinari et al, J Clin Oncol 2008;26: )

CRC cell lines and response to cetuximab +

sinergistically (Di Nicolantonio & Martini

29 CRC cell lines and response to cetuximab + sorafenib Sorafenib restores sensitivity to to cetuximab in in BRAF V600E resistant CRC cells Cetuximab and Sorafenib act act sinergistically (Di Nicolantonio & Martini & Molinari et al, J Clin Oncol 2008;26: )

30 Role of BRAF mcrc patients treated with panitumumab or cetuximab, n = 113 KRAS mutational status **p<0.05 (p=0.011) Mutant KRAS 34/113 (30%) Wild-Type KRAS 79/113 (70%) Responders 2/34 (6%)* 22/79 (28%)** Non Responders 32/34 (94%)** 57/79 (72%)** K-Ras mutations identify 32/89 (36%) NR patients Mutant KRAS 34/113 (30%) BRAF mutational status on Wild-Type KRAS tumors *p<0.05 (p=0.029) Mutant BRAF 11/79 (14%) Wild-Type BRAF 68/79 (86%) Responders 0/11 (0%)* 22/68 (32%)* Non Responders 11/11 (100%)* 46/68 (68%)* BRAF mutations identify 11/89 (12%) NR patients Mutant BRAF 11/113 (10%) 40% patients show either K-Ras or or BRAF mutations K-Ras and BRAF mutations identify 48% of of NR NR patients (Di Nicolantonio & Martini & Molinari et al, J Clin Oncol 2008;26: )?!?

31 EGFR & downstream signals EGFR out cytoplasm mutated in ~20% of sporadic CRC mutated in ~30-40% of sporadic CRC PI3K K-Ras loss in ~30% of sporadic CRC PTEN Akt BRAF MEK mutated in ~10% of sporadic CRC mtor ERK1,2 cell survival angiogenesis changes in gene expression nucleus cell proliferation tumor invasion tumor metastasization

32 PIK3CA status and clinical response Wild-type Mutant PIK3CA 95/110 (86%) 15/110 (14%) * Responders 22/95 (23%) 0/15 (0%) Non-responders 73/95 (77%) 15/15 (100%) p<0.001, two-tailed Fisher s exact test PIK3CA mutations are are associated with lack of of response to to MoAbs treatment in in mcrc (Sartore-Bianchi & Martini & Molinari et al, Cancer Res 2009;69: )

33 (Di Nicolantonio & Martini & Molinari et al, J Clin Oncol 2008;26: ) (Sartore-Bianchi & Martini & Molinari et al, Cancer Res 2009;69: ) K-Ras, BRAF and PIK3CA vs PFS and OS K-Ras BRAF PIK3CA P= Wild-TypePIK3CA MutantPIK3CA PFS Time sincestart of Treatment days) ( OS P= Wild-TypePIK3CA MutantPIK3CA Time sincestart of Treatment days) ( 910 K-Ras mutations are are associated with shorter PFS BRAF mutated tumor have a shorter PFS and OS OS PIK3CA mutations are are associated with shorter PFS

34 EGFR & downstream signals Overexpressed in ~70% of sporadic CRC mutated in ~20-30% of sporadic CRC EGFR out cytoplasm mutated in ~40% of sporadic CRC PI3K K-Ras loss in ~30% of sporadic CRC PTEN Akt BRAF MEK mutated in ~5-10% of sporadic CRC mtor ERK1,2 cell survival angiogenesis changes in gene expression nucleus cell proliferation tumor invasion tumor metastasization

35 PTEN PTEN pos 16/27 (62%) PTEN neg 11/27 (38%) PTEN pos PTEN neg PR 10 0 NR (PD+SD) 6 11 p<0.001 Absence of PTEN expression confers cetuximab resistance (Frattini et al, Br J Cancer 2007;97: )

Conclusions I mcrc mcrc Stato genico stato di EGFR di K-Ras (FISH) sul tumore sul tumore primitivo primitivo disomia mutato alta polisomia del cromosoma 7 Amplificazione Wild-type del gene EGFR no

36 Conclusions I mcrc mcrc Stato genico stato di EGFR di K-Ras (FISH) sul tumore sul tumore primitivo primitivo disomia mutato alta polisomia del cromosoma 7 Amplificazione Wild-type del gene EGFR no cetuximab resistenza PTEN + K-Ras wt risposta risposta BRAF mutato BRAF cetuximab PIK3CA mutato PIK3CA PTEN perso PTEN EGFR disomico PTEN + PTEN - EGFR K-Ras mut K-Ras mut BRAF wt PIK3CA wt EGFR CNG resistenza PTEN + PTEN - K-Ras wt

37 Problem PRIMARY TUMOR EGFR, PTEN (IHC) K-Ras, BRAF (mutations) EGFR (FISH) Do primary tumor and distant metastatic lesions share the same molecular and gene profile?

Primary tumor vs metastasis: literature EGFR gene status (FISH) COLORECTAL CANCER Article Cases Ooi et al, Mod Pathol, 2004 3 (PT+LN) Cappuzzo et al, Ann Oncol, 2007 22 (PT+M) Differences PT vs M

38 Primary tumor vs metastasis: literature EGFR gene status (FISH) COLORECTAL CANCER Article Cases Ooi et al, Mod Pathol, (PT+LN) Cappuzzo et al, Ann Oncol, (PT+M) Differences PT vs M number of cases (%) 0/3 1/22 (4.5%) Concordant EGFR EGFR gene gene status status between primary tumor tumorand metastasis NON-SMALL-CELL LUNG CANCER Italiano et al, Ann Oncol, (PT+M) 7/26 (27%) Different EGFR EGFR gene gene status status between primary tumor tumorand metastasis

Primary tumor vs metastasis: literature K-Ras mutations COLORECTAL CANCER Article Cases (PT+M) Differences PT vs M number of cases (%) Oudejans et al, Int J Cancer, 1991 31 4/31 (13%) Losi L et al,

39 Primary tumor vs metastasis: literature K-Ras mutations COLORECTAL CANCER Article Cases (PT+M) Differences PT vs M number of cases (%) Oudejans et al, Int J Cancer, /31 (13%) Losi L et al, Eur J Cancer, /35 Zauber et al, J Clin Pathol, /42 Etienne-Grimaldi et al, Clin Cancer Res, /48 Concordant K-Ras K-Ras mutational status status between primary tumor tumor and and metastasis Artale et al, J Clin Oncol, /48 (6.25%) Al-Mulla et al, J Pathol, (PT+M+LN) 8/73 (11%) Tortola et al, J Clin Oncol, /13 (54%) NON-SMALL-CELL LUNG CANCER Kalikaki et al, Brit J Cancer, /25 (24%) Different K-Ras K-Ras mutational status status between primary tumor tumor and and metastasis

40 Aim PRIMARY TUMOR AND METASTASIS EGFR, PTEN IHC K-Ras, BRAF EGFR FISH To compare EGFR expression and molecular alterations predictive for anti-egfr therapies response (EGFR gene status, K-Ras and BRAF mutations, and PTEN protein expression) between primary CRC and paired distant metastatic lesions and correlate these data with clinical response

41 Patients and methods 38 mcrc patients (12 cetuximab/panitumumab treated) primary tumor and paired metastasis EGFR and PTEN protein expression immunohistochemistry EGFR gene status FISH K-Ras and BRAF mutations direct sequencing Statistical analysis: Cohen s Kappa test 0.41 k 0.6, moderate agreement 0.61 k 0.8, good agreement (Molinari et al, Br J Cancer Mar 17. [Epub ahead of print])

Results: EGFR gene status (FISH) PRIMARY TUMOR 36 evaluable cases METASTASIS Chr7 Chr7 disomy disomy

Chr7 polysomy polysomy 17 17 cases cases (47%) (47%) Chr7 Chr7 polysomy polysomy 21 21 cases cases

Amplific. 88 cases cases (22%) (22%) Gene Gene Amplific.

88 cases cases (22%) (22%) Chr Chr7 loss loss (L) (L) 11 case case (3%) (3%) Chr Chr7 loss loss (L)

42 Results: EGFR gene status (FISH) PRIMARY TUMOR 36 evaluable cases METASTASIS Chr7 Chr7 disomy disomy (D) (D) cases cases (28%) (28%) Chr7 Chr7 disomy disomy (D) (D) 66 cases cases (17%) (17%) Chr7 Chr7 polysomy polysomy cases cases (47%) (47%) Chr7 Chr7 polysomy polysomy cases cases (58%) CNG (58%) Gene Gene Amplific. Amplific. 88 cases cases (22%) (22%) Gene Gene Amplific. Amplific. 88 cases cases (22%) (22%) Chr Chr7 loss loss (L) (L) 11 case case (3%) (3%) Chr Chr7 loss loss (L) (L) 11 case case (3%) (3%) Discordant EGFR gene status in 8/36 (22%) cases (κ=0.49, P=0.0002) Primary Tumor Metastasis EGFR deregulation (CNG) 6/36 cases (17%) Chr7 disomy (D) CNG Loss of EGFR deregulation 2/36 cases (5.5%) CNG (Molinari et al, Br J Cancer Mar 17. [Epub ahead of print]) Chr7 disomy (D)

Results: K-Ras mutations PRIMARY TUMOR 37 evaluable cases METASTASIS Wild-type 21 21 cases cases (57%) (57%) Wild-type 22 22 cases

3/37 (8%) cases (κ=0.83, P<0.0001) Primary Tumor Metastasis Gain of K-Ras mutation 1/37 cases (2.

43 Results: K-Ras mutations PRIMARY TUMOR 37 evaluable cases METASTASIS Wild-type cases cases (57%) (57%) Wild-type cases cases (60%) (60%) Mutated cases cases (43%) (43%) Mutated cases cases (40%) (40%) Discordant K-Ras mutational status in 3/37 (8%) cases (κ=0.83, P<0.0001) Primary Tumor Metastasis Gain of K-Ras mutation 1/37 cases (2.7%) * Wild-type sequence G12C mutation * Loss of K-Ras mutation 2/37 cases (5.4%) G12C mutation Wild-type sequence (Molinari et al, Br J Cancer Mar 17. [Epub ahead of print])

44 Results: BRAF mutations PRIMARY TUMOR 36 evaluable cases METASTASIS Wild-type cases cases (94%) (94%) Wild-type cases cases (94%) (94%) Mutated 2 cases cases (6%) (6%) Mutated 2 cases cases (6%) (6%) No differences in BRAF mutational status between primary tumor and metastasis (κ=1, P<0.0001) BRAF mutated cases are K-Ras wild-type, confirming that BRAF and K-Ras mutations are mutually exclusive (Molinari et al, Br J Cancer Mar 17. [Epub ahead of print])

Results: PTEN expression (IHC) PRIMARY TUMOR 38 evaluable cases METASTASIS Positive 30 30 cases cases (79%) (79%) Positive

protein expression in 4/38 (10%) cases (κ=0.73, P<0.

45 Results: PTEN expression (IHC) PRIMARY TUMOR 38 evaluable cases METASTASIS Positive cases cases (79%) (79%) Positive cases cases (68%) (68%) Negative 8 cases cases (21%) (21%) Negative cases cases (32%) (32%) Discordant PTEN protein expression in 4/38 (10%) cases (κ=0.73, P<0.0001) Primary Tumor Metastasis Reduction of PTEN expression 4/38 cases (10%) Positive PTEN expression Reduction of PTEN expression (Molinari et al, Br J Cancer Mar 17. [Epub ahead of print])

46 Results: Clinical response and molecular profile 12 patients were treated with Cetuximab or Panitumumab Anti-EGFR MoAbs response: PR: 2/12 (17%) (RECIST criteria) NR: 10/12 (83%) cases EGFR IHC EGFR FISH K-Ras BRAF PTEN IHC clinical T M T M T M T M T M response CNG CNG WT WT WT WT + + PR CNG CNG WT WT WT WT + + PR D D WT WT V600E V600E + + NR D D WT WT WT WT + + NR CNG CNG G12S G12S WT WT + + NR CNG CNG G12A G12A WT WT + + NR CNG CNG WT WT WT WT - - NR D CNG G12A G12A WT WT - - NR D CNG G12D G12D WT WT + + NR CNG CNG G12A G12A WT WT + - NR CNG CNG WT WT V600E V600E + - NR CNG CNG WT WT WT WT + - NR The absence of PTEN expression limited to the metastasis may explain the resistance to anti-egfr MoAbs (Molinari et al, Br J Cancer Mar 17. [Epub ahead of print])

47 Conclusions II Primary CRC and paired metastasis may show different gene and protein expression profiles in a consistent fraction of patients. A trend toward gene or protein expression deregulation most likely reflect a malignant progression of metastatic cells. Loss of deregulation in metastasis compared to paired primary tumor may be explained by a different metastatic clone with respect to the main clone of the primary tumor. Differences between primary tumor and paired metastatic site of EGFR and K-Ras gene status and PTEN protein expression, may impair anti-egfr therapies efficacy. Future clinical trials based on anti-egfr therapies should be designed taking into account the coupled analysis of molecular predictive markers both on primary tumor and paired metastatic site.

48 Summary KRAS mut: SI BRAF mut: se KRAS wt PIK3CA mut: se KRAS wt PTEN IHC: non ancora EGFR IHC: NO EGFR FISH:

49 Francesca Molinari Thanks to... ICP - Locarno Luca Mazzucchelli Stefano Crippa Elena Zanellato Vittoria Martin IOSI - Bellinzona Piercarlo Saletti Sara De Dosso Osp. Ca Granda Milano Salvatore Siena Andrea Sartore-Bianchi Silvio Veronese Salvatore Artale Michele Nichelatti IRCC Candiolo (TO) Alberto Bardelli Federica Di Nicolantonio Miriam Martini

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