Esteller, New England Journal of Medicine, 2008

Cancer of Unknown Primary Fernandez et al., Genome Research, 2012

DNA Methylation Microarray Tumoral Types DNA Methylation Microarray Methylation Characteristics Primary Tumors Algorithm Generation Training Cohort n=2,790 PEBC: 692 TCGA: 2,098 Validation Cohort n=7,687 PEBC: 1,944 TCGA: 5,743 CUPs Cohort (n=216)

CUPs Cohort Location at diagnosis Germany Italy USA Australia Badalona Barcelona Badalona Lleida Germany Italy Australia USA Zaragoza Madrid Santiago

Validation & Discovery Cohorts (n=10,481) 99.6% Specificity Sensitivity

EpiCup Prediction in CUPs Cohort Compatible with Histology 96.1%

Efficacy of Epigenetic Profiling to Classify Cancer of Unknown Primary A Necropsy of CUP case predicted as Sarcoma by EPICUP B Histopathologic characterization of CUP case predicted as Sarcoma by EPICUP H&E Vimentin S100 C PET image of CUP case predicted as Pancreatic Carcinoma by EPICUP CK7 CK20 Mammoglobin D IHC of CUP case predicted as Breast Carcinoma by EPICUP CDX2 E IHC of CUP case predicted as Colon Carcinoma by Moran et al., The Lancet Oncology 2016

Site-specific therapy for CUPs confers improved survival: Prospective studies Hainsworth et al., J Clin Oncol 2013 Yoon et al., Annals of Oncology 2016 Varadhachary et al., Clin Cancer Res 2011

Months Since Diagnosis Hazard Ratio for OS (95% CI) Efficacy of Epigenetic Profiling to Classify Cancer of Unknown Primary A Overall Survival, according to therapy C IHC and molecular characterization of CUP case predicted as NSCLC by EPICUP H&E CK7 CK20 1.0 Specific therapy (n = 31; 13.6 m) 0.8 0.6 Empiric therapy (n = 61; 6.0 m) Overall Survival 0.4 TTF-1 C-MET amplification Log-rank; p-value = 0.003 HR (95% CI) = 3.24 (1.42 – 7.38) 0.2 0.0 2 Months Since Diagnosis 4 6 8 10 Patients at risk: Therapy 31 29 25 24 Specific 61 59 46 38 34 30 Empiric B Multivariate Analysis D IHC and molecular characterization of CUP case predicted as NSCLC by EPICUP H&E CK7 CK20 Patients features (N= 92) HR (95% CI) Benefit Risk Gender 1.09 (0.55 – 2.18); p=0.799 Age 0.64 (0.32 – 1.28); p= 0.209 Diagnostic method 0.96 (0.69 – 1.33); p=0.809 Histology at diagnosis 1.15 (0.72 – 1.85); p= 0.555 Biopsy site 0.99 (0.87 – 1.10); p=0.976 TTF-1 EGFR mutation in exon 18 Multiple metastasis sites 2.03 (0.84 – 4.92); p=0.117 Specific therapy 3.14 (1.35 – 7.30); p=0.008 0.5 1 2.5 5 10 Hazard Ratio for OS (95% CI) 95% of Confidence Interval (95% CI) Hazard Ratio (HR) Moran et al., The Lancet Oncology 2016

Aim: Predict tumor of origin for Cancer of Unknown Primary patients customerservice@ferrerincode.com Tumoral Types Methylation Characteristics

EPICUP® Biopsy Sites (n=158)

EPICUP® Diagnoses (n=158)

CNV using MethylationEPIC Illumina MethylationEPIC 853.307 CpGs Avg. density = 1 marker / 3.8 Kb Normalized Intensity Intensity = Fluorescence Methylated + Unmethylated channels

(Stomach; NSCLC; NSCLC) CNV EPICUP® (n=48) Gene Samples with Gains (CN ≥ 4) EGFR ERBB2 2 (Bladder) ERBB3 FGFR1 1 (Breast) KIT C-MET (Stomach; NSCLC) C-MYC 3 (Stomach; NSCLC; NSCLC) L-MYC (NSCLC) N-MYC PDGFR

Esteller et al., NEJM 2000 Heyn and Esteller, Nature Reviews Genetics, 2012

Identification Primary Site Diagnostic Procedures CUP sample Identification Primary Site Actionable Target Effective Drug Non-Small cell lung carcinoma ALK rearrangement c-MET amplification EGFR mutation NTRK rearrangement RET rearrangement ROS1 rearrangement Alectinib, Ceritinib, Crizotinib Crizotinib Afatinib, Erlotinib, Gefitinib Entrectinib, LOXO-101 Alectinib, Cabozantinib, Vandetanib Head & Neck squamous cell carcinoma FGFR rearrangement PI3KCA mutation FGFR inhibitors PI3KCA inhibitors Breast carcinoma BRCA1 methylation BRCA1/2 germline mutation HER2 amplification PARP inhibitors, Platins HER2 inhibitors, Lapatinib, Trastuzumab Colon carcinoma MLH1 methylation Pembrolizumab (putative treatment) Hepatocelular carcinoma TCS2 loss Tivantinib Everolimus Ovarian carcinoma Endometrial carcinoma PTEN Loss Ridaforolimus, Temsirolimus Sarcoma ASPSCR1/TFE3 rearrangement COL1A1/PDGFB rearrangement TSC2 loss Crizotiniib Sunitinib Imatinib Stomach carcinoma MET inhibitors Trastuzumab Esophageal carcinoma Skin cutaneous melanoma BRAF mutation KIT amplification KIT mutation NRAS mutation Dabrafenib, Vemurafenib KIT inhibitors MEK inhibitors Physical exploration Imaging Immunohistochemistry Expression microarray EPICUP Diagnostic Procedures Moran et al., Nature Reviews in Clinical Oncology 2017

EPICUP Advance® NGS Library-Prep 815 Amplicons to cover EPICUP® CpGs MiSeq DX