1. Genomic Signatures
of Specific Sites of Metastases
Laura J. van ‘t Veer
Helen Diller Family
Comprehensive Cancer Center
University of California San Francisco
ABC1
Lisbon, Portugal, 2011

2. Breast cancer metastasis:
metastatic capacity and organ specificity?
through blood or lymph vessels

3. Metastasis Models
I------------------primary tumor---------------I----metastasis-----I
Subpopulations
All cells
Dynamic heterogeneity
Clonal dominance
Genometastasis
Nat Rev Cancer 5, p 591-602, 2005

4. Breast Cancer
Risk of Metastasis and Survival
Kaplan-Meier Survival Curve Stage 1/2
~30% die of breast cancer
survival
~70% survive breast cancer
time (years)

5. 70 Gene Prognosis Profile MammaPrint
70 significant prognosis genes
Tumor samples
van´t Veer et al., Nature 415, p. 530-536, 2002
threshold set with 10% false negatives
91 % sensitivity, 73% specificity

6. IGFBP5, TGFB3, FGF18, ESM1, RARRES3, PITRM1, EXT1, EXTL3, SCUBE2,
EBF4,CDC42BPA, CDCA7, CDCA7L, GMPS, MELK, RFC4, WISP1, HRASLS,
proliferation
BBC3, DTL, FBXO31, EGLN1, GNAZ, MTDH, FLT1, ECT2, DIAPH3, NUSAP1,
AKAP2, NDC80, PRC1, ORC6L, CENPA, DCK, CCNE2, MCM6, QSOX2, STK32B
COL4A2, FLT1, FGF18, MMP9
angiogenesis
adhesion to extracellular matrix
MMP9, COL4A2
FLT1, TGFB3, IGFBP5, FGF18, RARRES3,
COL4A2, FLT1, MMP9, TGFB3, MTDH, DIAPH3,
intravasation, survival, extravasation
local invasion
CDCA7L, WISP1, DIAPH3, AKAP2, CDC42BPA,
PALM2, DCLK2, NMU, NMUR1, NMUR2
PALM2, DCLK2, NMU, NMUR1, NMUR2
adhesion to extracellular matrix
MMP9, COL4A2
IGFBP5, TGFB3, FGF18, ESM1, RARRES3, PITRM1, EXT1, EXTL3, SCUBE2,
proliferation
EBF4,CDC42BPA, CDCA7, CDCA7L, GMPS, MELK, RFC4, WISP1, HRASLS,
BBC3, DTL, FBXO31, EGLN1, GNAZ, MTDH, FLT1, ECT2, DIAPH3, NUSAP1,
AKAP2, NDC80, PRC1, ORC6L, CENPA, DCK, CCNE2, MCM6, QSOX2, STK32B
COL4A2, angiogenesis MMP9
FLT1, FGF18,
Nature, 2002

7. Breast Cancer – node negative
Stage 1/2 Survival by profiling
Distinguish in: 40% good profile, 60% poor profile
metastases-free
overall survival
time (years) time (years)
151 patients, <53, LN0
NEJM 347, p1999-2009, 2002 10 year survival curve

8. Breast Cancer – 1-3 node positive
Stage 2 Survival by profiling
Good profile (n=99)
Poor profile (n=142)
Distant metastases as first
event Overall survival
95% 95%
77% 73%
HR 4.1 HR 5.4
(95%CI 1.7 – 10.0), p<0.002 (95%CI 2.1 – 13.8), p<0.001
Mook et al, BCRT 2010

9. Molecular portraits of breast cancer
Capacity to metastasize
Perou & Sorlie et al,
Nature 2000
Survival analysis was done on the 51 doxorubicin treated patients only

10. Molecular Profiles of the Primary Tumor
can predict risk of metastasis:
- MammaPrint 70-gene profile
- Intrinsic subtypes (e.g., basal, luminal)
- Oncotype 21-gene recurrence score
- etc
and are clinically useful
to guide the choice of adjuvant therapy

11. Profiles hard-wired and maintained
throughout metastatic process?
Nat Rev Cancer 5, p 591-602, 2005

12. Primary breast tumors – distant metastases
Hierarchical clustering
8 primary breast tumors and distant metastases of the same patient
PNAS 100, p 15901-15905, 2003

13. Profiles maintained throughout
metastatic process
70-gene prognosis profile
primary tumor 8
good
signature metastasis 8
primary tumor 4
primary tumor 7
metastasis 1
primary tumor 5
primary tumor 1
metastasis 5
metastasis 4
metastasis 7
primary tumor 3 primary
poor tumor 6
signature
metastasis 6
metastasis 3
Cancer Res 65, 2005

14. Profiles maintained throughout
metastatic process
primary tumor – lymph node metastasis pairs
primary tumor – distant metastasis pairs
primary tumor – brain metastasis pair
primary tumor – distant metastasis pairs autopsy
Collaboration
C. Perou
Cancer Res 65, 2005

15. Profiles hard-wired and maintained
throughout metastatic process
Nat Rev Cancer 5, p 591-602, 2005

16. Organ-specific breast cancer metastasis
Prognostic profiles
for metastasis
such as the
70 gene MammaPrint,
76 gene Rotterdam
are maintained, but
can not predict
site of relapse
through blood or lymph vessels

17. Organ-specific breast cancer metastasis
Site of relapse
intrinsic subtypes,
model system signatures
through blood or lymph vessels

18. Intrinsic Subtypes of breast cancer
Foekens et al
HER2
ER
Is there a relationship with the site
of relapse? KRT5
Rotterdam data set:
Affymetrix U133A chip
luminal B luminal A HER2 normal basal 344 untreated lymph
node-negative patients
Smid et al; Cancer Res 68, May 2008

19. Association with site of relapse
Foekens et al
118 patients of the 344 had a distant metastasis:
- 81 patients with metastasis to single organ
- 37 patients with multiple relapse sites (30 with 2 organs)
- in total 165 relapses
misc: 20
bone: 71
pleura: 12
brain: 14
liver: 18 lung: 30
Smid et al; Cancer Res 68, May 2008

20. Non-random distribution X² statistic 42.78
p 0.0003
Foekens et al
Subtype
Smid et al; Cancer Res 68, May 2008

21. Intrinsic Subtypes of breast cancer (IHC)
ERpos / Luminal
Higher likelihood for Bone Metastases
HER2pos and Triple negative
Higher likelihood for Brain Metastases
Kennecke, JCO 2010

22. Lung metastasis profile ‘experimental model’
applied to human breast cancer
Massague et al
Memorial Sloan-Kettering Netherlands Cancer Institute
Erasmus Medical Center

23. Lung metastasis profile ‘experimental model’
applied to human breast cancer
Massague et al
Evaluated for
first distant
failure

24. Lung metastasis profile ‘experimental model’
applied to human breast cancer
Massague et al
for primary tumors >3cm
Netherlands Cancer Institute
Erasmus Medical Center

25. Organ-specific breast cancer metastasis
Intrinsic subtypes and
model system signatures
show differential preference
Clinical useful?
through blood or lymph vessels

26. Organ-specific breast cancer metastasis
Bone specific profile
clinically helpful
to direct
biphosphonate therapy?
through blood or lymph vessels

27. Site of relapse
NKI 295 breast cancer dataset
88 patients developed distant metastases as first site
(- A: 26 bone as first event)
(- B: 15 bone concurrent with other site)
-C: 53 bone at any time
Hierarchical clustering 84 samples by 25.000
expressed genes
Kreike et al, EBCC

28. B
A
Testing
Training samples
samples
Kreike et al, EBCC
specific genes NKI295
Gene index
Classification by 223 bone site
Bone-metastasis status (A)
Bone-metastasis status (B)
Bone-metastasis status (C)
ER Status
>90% bone mets

29. Profile for bone metastasis
286 patients, 107 relapses (Lancet, 2005)
Training Validation
72 patients: 35 patients:
- 46 x bone - 23 x bone
- 26 x non-bone - 12 x non-bone
SAM and PAM analysis
31 - gene set
Smid et al, JCO 24, 2006

30. Performance of the 31-gene predictor
Validation set of 35 patients
Sensitivity: 100% (23/23)
Specificity: 50% (6/12)
Genes higher
SAM Fold expressed in
Probe-id Gene Symbol Score Change bone FDR (%) PAM Gene Title
205009_at TFF1 -4,92 3,1 * 1,9 yes trefoil factor 1
204623_at TFF3 -4,23 2,6 * 1,9 yes trefoil factor 3 (intestinal)
209173_at AGR2 -4,06 1,9 * 1,9 anterior gradient 2 homolog
214440_at NAT1 -4,04 2,5 * 1,9 yes N-acetyltransferase 1
205081_at CRIP1 -3,80 1,9 * 1,9 yes cysteine-rich protein 1 (intestinal)
214774_x_at TNRC9 -3,72 1,9 * 1,9 yes trinucleotide repeat containing 9
214858_at --- -3,60 2,0 * 1,9 yes Pp14571
219197_s_at SCUBE2 -3,59 2,1 * 1,9 signal peptide, CUB domain, EGF-like 2
215108_x_at TNRC9 -3,57 1,9 * 1,9 yes trinucleotide repeat containing 9
206754_s_at CYP2B6 -3,57 2,1 * 1,9 cytochrome P450, family 2, subfamily B, polypeptide 6
210056_at RND1 -3,48 1,7 * 1,9 yes Rho family GTPase 1
Smid et al, JCO 24, 2006
205186_at DNALI1 -3,45 2,0 * 1,9 dynein, axonemal, light intermediate polypeptide 1
203130_s_at KIF5C -3,42 2,0 * 1,9 kinesin family member 5C

31. Organ-specific breast cancer metastasis
Bone metastases signatures
interesting, but
need clinical validation
through blood or lymph vessels

32. Breast cancer metastases Management
targeted therapies – multiple sites
Targeted therapies
match the therapy to the
biomarker status of the
metastases
Hardwiring of prognostic
profiles seen,
how about
specific mutations from
primary site to metastases?
through blood or lymph vessels

33. Recurrent Mutations in Primary and Metastasis
one basal breast cancer example
Whole Genome Sequencing – Red highlighted text = mutation
many more in the metastasis
Primary Tumor Metastasis
Ding, Ellis, Mardis et al, Nature, 2010 others: new mutations occur, not all maintained

34. Progression to metastases model
prognostic hardwiring maintained,
heterogeneity for (single) gene mutations

35. Breast cancer metastases Management
targeted therapies – multiple sites
Different metastases may
have different ‘mutations’
Future management:
1) assess biology of primary
tumor
and
2) re-assess by fine needle
aspirates (multiple lesions)
biomarker mutation status
relevant for targeted therapies
(also ER, PR, HER2)
through blood or lymph vessels

36. Acknowledgements
The Netherlands Cancer Institute
Amsterdam, NL (Bas Kreike, Britta Weigelt, Marc van de Vijver, Stella Mook, Marleen Kok,
Lodewyk Wessels, Hans Peterse, Rene Bernards)
University of North Carolina
Chapel Hill, US (Chuck Perou, Zhiyuan Hu, Cheng Fan)
Erasmus Medical Center
Rotterdam, NL (Marcel Smid, John Martens, Jan Klijn, Els Berns, John Foekens)
University California San Francisco
San Francisco, US (Denise Wolf, Hope Rugo, Michelle Milesko, Pamela Munster, Joe Gray)