1. Esteller, New England Journal of Medicine, 2008

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

3. 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)

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

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

6. EpiCup Prediction in CUPs Cohort
Compatible with Histology
96.1%

7. 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

8. 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

9. 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

10. Aim: Predict tumor of origin for Cancer of Unknown Primary patients
Tumoral Types
Methylation Characteristics

11. EPICUP® Biopsy Sites (n=158)

12. EPICUP® Diagnoses (n=158)

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

14. (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

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

16. 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

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