An interactive presentation of new discoveries about early precursor lesions and chromosome alterations in colorectal cancer.

1. New insights into the colorectal carcinogenesis: from
early precursor lesion to the role of aneuploidy
Isabel Quintanilla
Thesis defense
20th July 2017

2. 2
Colorectal cancer: Population facts
Introduction
Data source: Globocan 2012
ASR (W), age standardized rate (weighed)

3. 3
Stepwise colorectal carcinogenesis
≈ 10 years
Perfect scenario for prevention
Identification:
- Early precursor lesions
- Prognosis biomarkers
Normal colonic
mucosa
Early adenoma Advanced
adenoma
Progressed
adenoma
Introduction
Carcinoma
Carcinoma
Normal colonic
mucosa
Serrated lesions
Serrated pathway: 20%
Classical pathway: 80%
Aberrant crypt foci
(ACF)

4. 4
Aberrant crypt foci
Dying technique:
Chromoendoscopy
Clusters of colonic crypts raised above the normal
mucosa:
- Thicker epithelia
- Altered luminal openings
- Larger size than normal colonic crypts
- Hyperplastic or dysplastic
- ACF appear after treatment with colon-
specific carcinogens
ANIMALS
- Number
- Size
- Morphology
- ACF exhibit molecular alterations commonly
found during colorectal carcinogenesis
Increased risk of
colorectal cancer
(CRC)
HUMANS
DATA REMAIN CONTROVERSIAL
Introduction
Aberrant crypt foci
(ACF)

5. 5
Colorectal carcinogenesis: molecular pathogenesis
Normal colonic
mucosa
Early adenoma Intermediate
adenoma
Advanced
adenoma
Carcinoma
Invasive carcinoma
BRAF (7q34)
KRAS (12q12.1)
Introduction
Wnt pathway CDC4 (12p13.31)
Chromosomal instability (CIN)
Microsatellite instability (MSI)
APC (5q21-q22) SMAD4 (18q21.1)
CDC4 (12p13.31)
TP53 (17p13.1)
Other
genes
KRAS (12q12.1)
Mismatch repair gene inactivation
Chromosome alterations
Adapted from Walther, et al. 2009
Altered epigenetics: aberrant DNA methylationCpG island methylator phenotype (CIMP)

6. 6
Aberrant DNA methylation in cancer
Exon 1 Exon 2
TF
Exon 1 Exon 2
TF
Unmethylated Cytosine
Methylated Cytosine
NORMAL CELL CANCER CELL
Repetitive and
transposable
sequences (LINEs)
CpG island
promoters (MGMT,
hMLH1..)
Global hypomethylation Genomic instability
Hypermethylation promoters of
specific genes
Altered gene
expression
TF Transcription factor
Introduction
Field defect
Global DNA methylation levels

7. 7
Chromosome alterations in cancer
NORMAL CELL CANCER CELL
Numerical chromosome alterations or
Copy number alterations (CNAs)
Structural chromosome alterations
ANEUPLOIDY
Introduction

8. 8
Numerical chromosome alterations
Problems during mitosis:
Microtubule (MT)-Kinetochore
(KT) attachments
Normal mitosis
Merotelic (lagging chromosomes)
Introduction
Non-disjunction
Chromosome
missegregation into
a micronucleus
2N+1 2N-1
2N+MNi 2N-1
Adapted from London, et al. 2014
Chromosomes
Spindle pole
Centrosome
Microtubules
Amphitelic
Supernumerary centrosomes

9. 9
Whole genome duplication events
Centrosome
DNA
Introduction
2N
2N
Binucleated tetraploid
cell
4N
Mononucleated
tetraploid cell with extra
centrosomes
Mitosis
Loss of extra
centrosomes
Centrosome
clustering
2N
2N
Cytokinesis
failure
Cell fusion
2N
2N
Mitotic
slippage

10. 10
Zack, et al. 2013; Carter, et al. 2012
Tetraploidy is present in several
precancerous lesions and precedes
aneuploidy in cancer progression
WGD, whole genome duplication.
Whole genome duplication in cancer
Introduction

11. 11
Structural chromosome alterations
Anaphase/Chromosome bridge
Double strand breaks (DSBs)
Telomere dysfunction
Break during cytokinesis
Mitosis
Missegregate
Cause cytokinesis failure
Introduction
Replication stress
Mitosis
CentrosomeKinetochore/Centromere
Acentric fragments
Joint DNA
molecules
Recombination
Dicentric chromosomes

12. 12
Cellular responses
Replication stress response DNA damage response
MCM
complex
RPA
P
ATR
P
CHK1
P
gH2AX 53BP1
ATM
CHK2
P
DNA repair
Cell cycle
progression
DSB
Origin firing
(replication)
Cell cycle
progression Replication fork
stabilization
Introduction
DSB, double strand breaks.

13. 13
The clonal evolution of cancer
Intratumor heterogeneity
Branched evolution
Introduction
Deep-sequencing and single cell analysis

14. 14
Intratumor heterogeneity
Jamal-Hanjani, et al. 2017; Uchi, et al. 2016
Lung cancer Multiregion sampling and
deep-sequencing
Subclonal
alterations
Early clonal
alterations
Genome
doubling
Evolutionary tree
Introduction
Colorectal cancer
Gene mutation
Chr. gain
Chr. lossAPC
KRAS
TP53
+7p
+8q
+13q
+20q
-18q

15. 15
1. Determine whether ACF are predictive biomarkers for CRC by analyzing morphological and
molecular features described for colorectal carcinogenesis in a wide cohort of ACF samples
from different risk groups, including healthy individuals, adenoma and CRC patients.
2. Evaluate whether LINE-1 methylation represents a field defect for CRC using the same
cohort of healthy individuals, adenoma and CRC patients.
3. Analyze progressed adenomas in order to assess chromosomal heterogeneity and define
the specific populations and chromosomal alterations involved in the adenoma-to-
carcinoma transition.
4. Characterize tetraploidy in vitro as it is considered an intermediate stage in the progression
of many cancers, including CRC, and a way to tolerate CIN.
Objectives

16. 16
1. Determine whether ACF are predictive biomarkers for CRC by analyzing
morphological and molecular features described for colorectal carcinogenesis in a
wide cohort of ACF samples from different risk groups, including healthy individuals,
adenoma and CRC patients.
2. Evaluate whether LINE-1 methylation represents a field defect for CRC using the same
cohort of healthy individuals, adenoma and CRC patients.
3. Analyze progressed adenomas in order to assess chromosomal heterogeneity and define
the specific populations and chromosomal alterations involved in the adenoma-to-
carcinoma transition.
4. Characterize tetraploidy in vitro as it is considered an intermediate stage in the progression
of many cancers, including CRC, and a way to tolerate CIN.
Objectives

17. 17
High-definition chromoendoscopy (methylene blue or NBI)
67 healthy
individuals
50 adenoma
patients
50 colon cancer
patients
CONTROL CASE
Endoscopic characterization of ACF (1,103)
Histological evaluation of ACF (686)
Molecular characterization of ACF (294)
100 healthy
individuals
40 adenoma
patients
41 colon cancer
patients
128 ACF
166 ACF
- Mutations of APC, KRAS and BRAF (Sanger sequencing)
- Microsatellite instability (The MSI analysis system)
- MGMT promoter methylation (Pyrosequencing)
ACF, aberrant crypt foci.
- Number ACF per patient
- Size
- Morphology
- Vascular pattern intensity
- Hyperplastic
- Dysplastic
- Serrated
1-3 ACF per patient
Experimental design
Materials and Methods I

18. 18
Endoscopic features of ACF as a surrogate marker for CRC risk
Control
N = 100
Case
N = 100
P value
Number of ACF per patient:
<5/5-15/>15 (%)
23/48/29
(23.0/48.0/29.0)
20/46/34
(20.0/46.0/34.0)
n.s
Patients with:
- At least 1 large ACF (%) 62 (62.0) 78 (78.0) 0.046
- More than 1 large ACF (%) 41 (41.0) 66 (66.0) 0.002
- More than 4 large ACF (%) 6 (6.0) 24 (24.0) 0.003
- One irregular FCA 26 (26.0) 35 (35.0) n.s
ACF with:
- Large Size (%)
(Large ACF/ ACF evaluated)
32.1
(159/496)
45.0
(273/607)
0.001
- Irregular morphology (%)
(Irregular ACF/ ACF evaluated)
7.3
(34/464)
11.9
(63/528)
n.s
- Intensive vascular pattern (%)
(Hipervascular ACF/ ACF evaluated)
2.2
(8/369)
3.8
(15/399)
n.s
Individuals and ACF features predictive of CRC risk adjusted per patient’s age and gender.
Only the number of large ACF correlates with CRC risk
ACF, aberrant crypt foci.
Results I

19. 19
ACF histology as a surrogate marker for CRC risk
Hyperplastic Dysplastic Serrated P value
Large Size (%)
(Large ACF/histologically confirmed ACF)
59.6
(236/396)
50.5
(33/56)
58.9
(51/101)
n.s
Irregular shape (%)
(Irregular ACF/histologically confirmed ACF)
10.1
(39/385)
20.4
(11/54)
20.6
(20/97)
0.006
Asteroid-like or irregular shape (%)
(Asteroid-like or irregular ACF/histologically confirmed
ACF)
20.8
(80/385)
25.9
(14/54)
35
(34/97)
0.012
Intensive vascular pattern (%)
(Hipervascular ACF/histologically confirmed ACF)
3.9
(12/306)
5.1
(2/39)
5.6
(4/71)
n.s
Control group (%)
(ACF from control group/histologically confirmed ACF)
41.7
(165/396)
42.9
(24/56)
45.5
(46/101)
n.s
Case group (%)
(ACF from study group/ histologically confirmed ACF)
58.3
(231/396)
57.1
(32/56)
54.5
(55/101)
n.s
Individuals and ACF features predictive of CRC risk adjusted per patient’s age and gender.
Irregular ACF morphology is associated with dysplastic and serrated lesions
ACF histology does not correlate with the risk of CRC
ACF, aberrant crypt foci.
Results I
Moderate-to-good intra-explorer concordance
Fair inter-explorer concordance

20. 20
Molecular characterization of ACF
Control Case P value
MSI (%)
(ACF with MSI/ACF analyzed)
0
(0/124)
0
(0/152)
n.s
APC mutations (%)
(ACF with APC mutated/ACF
analyzed)
0
(0/127)
3.2
5/158
n.s
BRAF mutations (%)
(ACF with BRAF mutated/ACF
analyzed)
2.3
(3/128)
1.9
(3/162)
n.s
KRAS mutations (%)
(ACF with KRAS mutated/ACF
analyzed)
13.7
(17/124)
18.2
(30/165)
n.s
Hyperplastic Dysplastic Serrated P value
MSI (%)
(ACF with MSI/ACF analyzed)
0
(0/186)
0
(0/35)
0
(0/55)
n.s
APC mutations (%)
(ACF with APC mutated/ACF analyzed)
1.1
(2/190)
2.9
(1/35)
3.3
(2/60)
n.s
BRAF mutations (%)
(ACF with BRAF mutated/ACF analyzed)
1.6
(3/193)
2.9
(1/35)
3.2
(2/62)
n.s
KRAS mutations (%)
(ACF with KRAS mutated/ACF analyzed)
16.9
(33/195)
5.7
(2/35)
20.3
(12/59)
n.s
ACF molecular features related to CRC risk group.
ACF molecular features related to histology.
Is KRAS related with MGMT
promoter hypermethylation?
ACF, aberrant crypt foci.
Results I
ACF only show a few molecular alterations and they do not correlate with the histology or CRC risk
ACF KRAS wt ACF KRAS mut
0
5
10
15
20
25
CaseswithMGMTpromoter
hypermethylation(%)
P < 0.05

21. 21
1. Determine whether ACF are predictive biomarkers for CRC by analyzing morphological and
molecular features described for colorectal carcinogenesis in a wide cohort of ACF samples
from different risk groups, including healthy individuals, adenoma and CRC patients.
2. Evaluate whether LINE-1 methylation represents a field defect for CRC using the
same cohort of healthy individuals, adenoma and CRC patients.
3. Analyze progressed adenomas in order to assess chromosomal heterogeneity and define
the specific populations and chromosomal alterations involved in the adenoma-to-
carcinoma transition.
4. Characterize tetraploidy in vitro as it is considered an intermediate stage in the progression
of many cancers, including CRC, and a way to tolerate CIN.
Objectives

22. 22
67 healthy
individuals
40 adenoma
patients
41 colon cancer
patients
Bisulfite conversion
and Pyrosequencing
A. LINE-1 methylation levels of
ACF vs. normal mucosaNormal mucosa:
- Rectum (NRM)
- Descending colon (NDM)
ACF (1-3)
PPi
ATP
Sulfurylase
Luciferase
Pyrosequencing
B. LINE-1 methylation levels of
normal mucosa from the different
CRC risk groups (field defect)
ACF, aberrant crypt foci
Experimental design
Materials and Methods II
LINE-1 methylation
Bisulfite conversion

23. NDM ACF
60
70
80
90
100
LINE-1methylation(%)
***
A B
NDM ACF NDM ACF NDM ACF
60
70
80
90
100
LINE-1methylation(%)
Controls Adenoma CC
* ** n.sP < 0.001P < 0.05
NRM NDM
60
70
80
90
100
LINE-1methylation(%)
***
P < 0.0001
23
Evaluation of LINE-1 hypomethylation in ACF and normal colonic mucosa
ACF, aberrant crypt foci; NRM, normal rectal mucosa; NDM, normal descending mucosa; and CC, colon cancer.
LINE-1 methylation levels in normal mucosa differ between colonic segments
B
NRM ACF NRM ACF NRM ACF
60
70
80
90
100
LINE-1methylation(%)
Control Adenoma CC
*** *** ***
P < 0.0001 P < 0.0001 P < 0.0001
Results II
ACF do not display LINE-1 hypomethylation
A. LINE-1 methylation levels of ACF vs. normal mucosa:

24. 24
Evaluation of LINE-1 hypomethylation in ACF and normal colonic mucosa
Control Adenoma CC
60
70
80
90
100
LINE-1methylationofNRM(%)
n.s
Control Adenoma CC
60
70
80
90
100
LINE-1methylationofNDM(%)
n.s
LINE-1 methylation levels do not represent a field defect for CRC
ACF, aberrant crypt foci; NRM, normal rectal mucosa; NDM, normal descending mucosa; and CC, colon cancer.
Results II
B. LINE-1 methylation levels of normal mucosa from the different CRC risk groups (field defect):

25. 25
1. Determine whether ACF are predictive biomarkers for CRC by analyzing morphological and
molecular features described for colorectal carcinogenesis in a wide cohort of ACF samples
from different risk groups, including healthy individuals, adenoma and CRC patients.
2. Evaluate whether LINE-1 methylation represents a field defect for CRC using the same
cohort of healthy individuals, adenoma and CRC patients.
3. Analyze progressed adenomas in order to assess chromosomal heterogeneity and
define the specific populations and chromosomal alterations involved in the
adenoma-to-carcinoma transition.
4. Characterize tetraploidy in vitro as it is considered an intermediate stage in the progression
of many cancers, including CRC, and a way to tolerate CIN.
Objectives

26. 26
B) Non-advanced adenomas (N=6)
- Low-grade dysplasia
- Tubular architecture
- Size <10mm
Experimental design
Tissue disaggregation and
single cell suspension
Fluorescence in situ hybridization (FISH)
ADADK
Materials and Methods III
A) Progressed adenomas (N=23)
ADENOMA (AD)
ADENOCARCINOMA (ADK)

27. 27
FISH analysis
Panel of six gene-based homemade FISH probes Raw FISH data: Signal and imbalance patterns
1 2 3 4 5 6
MYC (8q) 2 2 3 4 2 2
EGFR (7p) 2 3 3 3 2 2
SMAD4 (18q) 1 2 2 2 2 1
CDX2 (13q) 4 4 4 5 2 2
ZINF217 (20q) 3 4 3 4 2 2
APC (5q) 1 2 2 2 2 1
Normal (2)
Gained (>2)
Lost (<2)
Sequential FISH analysis of approximately 200 nuclei per sample
2-3
2-2
2-4
1-4
4-2
3-1
MYC (8q)
EGFR (7p)
SMAD4 (18q)
CDX2 (13q)
ZINF217 (20q)
APC (5q)
Materials and Methods III
2,2,1,4,3,1
APC
EGFR
MYC
CDX2
SMAD7
ZNF217

28. 28
Patterns of chromosome alterations during CRC development
Normal (2) Gained (>2) Lost (<2)
Non-advanced adenoma (N_AD)
Progressed adenoma (AD)
Progressed adenoma (ADK)
Results III
Case2N_ADCase5ADCase5ADK
8q MYC 1.65% 2.2%
7p EGFR 2.2% 2.2%
18q SMAD7 2.2% 2.2%
13q CDX2 2.75% 1.1%
20q ZNF217 7.14% 14.84%
5q APC 6.04% 1.65%
Loss
65.38% 14.29% 5.49% 2.75 12.09%
Case2N_AD
Locus
182 nuclei
Marker Gain
1 3
3
1
1
3
3
8q MYC 92.62% 0.00%
7p EGFR 8.05% 0.67%
18q SMAD4 0.00% 87.92%
13q CDX2 3.36% 1.34%
20q ZNF217 32.21% 1.34%
5q APC 12.08% 9.4%
Case5AD
Locus
149 nuclei
Marker Gain
42.95% 19.46% 4.7% 4.03% 3.36 2.68 22.82%
Loss
5 3 6 5
3
1 1 1
4
1 1
6
3
1
3
1
3
4
1
3
5
1
3
5
1
1 3
3
5
31
5
1
8q MYC 97.63% 0.00%
7p EGFR 17.75% 1.18%
18q SMAD4 0,00% 86.98%
13q CDX2 11.83% 1.18%
20q ZNF217 95.27% 0,00%
5q APC 1.78% 74.56%
Marker Gain Loss
5.33% 9.47%2.37%62.72% 9.47% 8.28%
Case5ADK
Locus
169 nuclei
8
5
8
4
7
5
8
3
8
5
1
1
1
1
1
1
1
1
5
5
1
1
7
4
4
5
1
1
1
1
1
5
5
3
7
3
5
5
6
0
111
11
1
7
1
8
1
6 5
1
5
4
1
7
3
1
5
8
3
1
5
1 1
5
1
1

29. N_AD AD ADK
0
20
40
60
80
100
Instabilityindex(%)
***
*
**
A B
N_AD AD ADK
0
20
40
60
80
100
Chromosomeimbalances(%)
***
***
***
P < 0.001
P < 0.0001
P < 0.05
29
Patterns of chromosome alterations during CRC development
N_AD, non-advanced adenoma; AD, adenoma region of progressed adenoma; and ADK, adenocarcinoma region of progressed adenoma.
Aneuploidy and chromosomal instability increase during CRC development
Results III
N_AD AD ADK
0
20
40
60
80
100
Chromosomeimbalances(%)
P < 0.0001
P < 0.0001
P < 0.0001
5q 7p 8q 13q 18q 20q
0
20
40
60
80
Chromosomeimbalances(%)
AD
ADK
N_AD
P < 0.0001
P < 0.0001 P < 0.0001
P < 0.0001
P < 0.0001
P < 0.0001
P < 0.001
P < 0.001
P < 0.05
P < 0.05

30. 30
Phylogenetic evolution of CRC
Clone
Nodedepth
ADK exhibit greater potential for progression
than AD and N_AD
N_AD AD ADK
0
5
10
15
Nodedepth
***
P < 0.0001
2,2,2,2,2,2
2,4,2,2,2,22,2,2,2,3,22,2,2,2,2,1 2,2,2,2,2,3
2,2,2,2,3,3 2,2,2,3,2,3 2,3,2,2,2,3
2,2,1,2,3,3 3,2,2,3,2,3
3,4,2,3,2,3
119
2 5 10 1
3 2 5
5 1
2
FISH signal pattern: 8q, 7p, 18q, 13q, 20q, 5q
Number of cells forming the clone
N_AD, non-advanced adenoma; AD, adenoma region of progressed adenoma; and ADK, adenocarcinoma region of progressed adenoma.
Results III
Chowdhury, et al. 2013.
FISHtree software

31. 31
A. Single chromosome copy number alterations drive the AD-to-ADK transition (70%):
AD ADK
Phylogenetic evolution of CRC
Results III
+20q

32. B. A whole genome duplication (WGD) event leads the AD-to-ADK transition (30%):
32
AD ADK
Phylogenetic evolution of CRC
Results III
WGD

33. 33
5q 7p 8q 13q 18q 20q
0
10
20
30
Chromosome
Fitnessvalue
mono
di
tri
tetra
poly
Drivers of progression
Phylogenetic evolution of CRC
Results III
Drivers of AD-to-ADK transition
AD, adenoma region of progressed adenoma; and ADK, adenocarcinoma region of progressed adenoma.
Low-frequency clones define both AD and ADK populations
Chromosome alterations can define the AD-to-ADK transition and the evolution of CRC
Loss
5qG
ain
7pG
ain
8qG
ain
13qLoss
18qG
ain
20q
W
G
D
0
10
20
30
40
Cases(%)
Chromosome alteration
Loss
5qG
ain
7pG
ain
8qG
ain
13qLoss
18qG
ain
20q
W
G
D
0
10
20
30
40
Cases(%)
Chromosome alteration
WGD during evolution:
20% AD
35% ADK
Common ancestor exhibit
trisomy in driver chromosome

34. 34
1. Determine whether ACF are predictive biomarkers for CRC by analyzing morphological and
molecular features described for colorectal carcinogenesis in a wide cohort of ACF samples
from different risk groups, including healthy individuals, adenoma and CRC patients.
2. Evaluate whether LINE-1 methylation represents a field defect for CRC using the same
cohort of healthy individuals, adenoma and CRC patients.
3. Analyze progressed adenomas in order to assess chromosomal heterogeneity and define
the specific populations and chromosomal alterations involved in the adenoma-to-
carcinoma transition.
4. Characterize tetraploidy in vitro as it is considered an intermediate stage in the
progression of many cancers, including CRC, and a way to tolerate CIN.
Objectives

35. 35
Experimental design
DLD1, CRC cell line: spontaneously generated tetraploid
cells, MSI, CIN-, TP53 mutated
RKO, CRC cell line: induced cytokinesis failure to generate tetraploid cells, MSI, CIN-, TP53 wild-type
Materials and Methods III
24h 24h 24h
DCB treatment:
Cytokinesis failure Release
Modelling tetraploidy in vitro

36. 36
Modeling tetraploidy in vitro
Diploid (2N) and near-tetraploid (4N) clones isolation
Materials and Methods III
FACS Sorted Cells
Single Cell
2N clone
4N clone
CRC cell lines: DLD1 and RKO

37. 37
Gene expression
Near-tetraploid clones overexpress genes related to DNA replication and mitosis
2N, diploid; and 4N, near-tetraploid.
Results IV

38. 38
Replication stress response
DLD-1 clones:
P < 0.0001
2N, diploid; and 4N, near-tetraploid.
Results IV

39. 39
DLD-1 clones:
RKO clones:
2N 4N 2N 4N
0
2
4
6
Fragilesites(%)
*** ***
- Aph + Aph
P < 0.0001 P < 0.0001
Near-tetraploid clones exhibit replication stress
2N, diploid; and 4N, near-tetraploid.
Replication stress response
Results IV

40. 40
Progression through cell cycle
B. BrdU pulse assay: C. Mitotic timing:
A. Growth assays:
P < 0.001
P < 0.001
2N, diploid; and 4N, near-tetraploid.
Results IV
P < 0.001
0 24 48 72 96
0
50
100
150
Time (h)
Numberofcells(x10,000)
2N
4N
Near-tetraploid clones display a delay in S-phase
P < 0.0001

41. 41
DNA damage
A. Total DNA damage:
2.7-fold change 2-fold change
P < 0.0001 P < 0.0001
2N, diploid; and 4N, near-tetraploid.
Results IV
20 µm
2N 4N
53BP1gH2AX

42. 42
B. DNA damage in S-phase:
C. DNA damage in M-phase:
10 µm
2N, diploid; and 4N, near-tetraploid.
Near-tetraploid clones show increased DNA damage
DNA damage
Results IV
20 µm
P < 0.0001
20 µm
2N 4N
0
2
4
6
8
γH2AXfocipermitoticcell
2.4-fold change
P < 0.0001

43. 43
Intercellular chromosomal heterogeneity and aneuploidy
A. Numerical chromosome alterations:
B. Structural chromosome alterations:
2N, diploid; and 4N, near-tetraploid.
Results IV
The levels of chromosomal
heterogeneity and aneuploidy
are higher in near-tetraploid
clones compared to their diploid
counterparts

44. 44
A. Micronuclei (MNi) formation:
B. Centrosome number:
Chromosomal instability
2N, diploid; and 4N, near-tetraploid.
P < 0.0001
P < 0.0001
Results IV
1 >1
0
10
20
30
40
50
60
70
80
90
100
Centrosome number
Cells(%)
RKO 2N
RKO 4N_1
RKO 4N_2
DLD-1 2N
DLD-1 4N_1
DLD-1 4N_2
P < 0.05

45. 45
C. Mitotic defects:
DLD-1
RKO
Kinetochores, Microtubules, DNA
2N, diploid; and 4N, near-tetraploid.
Lagging chromosome Chromosome bridge
Near-tetraploid clones exhibit significant chromosomal instability
Chromosomal instability
Results IV
10 µm
2N 4N 4N
0
20
40
60
80
Cellsinanaphase(%)
Tripolar
Chr. Fragments
Lagg chr.
Chromosome bridges
2N 4N
P < 0.0001
1 2 3 4 5 6
0
10
20
30
40
50
Cellsinprometaphase(%)
Monopolar
Multipolar
Unfocused poles
Unaligned chromosomes
2N 4N
P < 0.0001
C1 C3 C28 B4 B6 B8
0
20
40
60
80
Cellsinanaphase(%)
Tripolar
Chr. Fragments
Lagg chr.
Multiple mis-segregating chr.
Chromosome bridges
2N 4N
P < 0.0001

46. 46
Chromosomal instability and replication stress
Replication stress not only triggers
structural chromosomal instability but
also numerical one
Results IV
A. MNi formation after aphidicolin-induced replication stress:
B. Mitotic defects after aphidicolin-induced replication stress:
Normal conditions
2N 4N 4N
0
20
40
60
80
Cellsinanaphase(%)
Tripolar
Chr. Fragments
Lagg chr.
Chromosome bridges
2N 4N
RKO
P < 0.0001
2N 4N 4N
0
20
40
60
80
Cellsinanaphase(%)
Tripolar
Chr. Fragments
Lagg chr.
Chromosome bridges
2N 4N
RKO
P < 0.0001
C1 C3 C28 B4 B6 B8
0
20
40
60
80
Cellsinanaphase(%)
Tripolar
Chr. Fragments
Lagg chr.
Multiple mis-segregating chr.
Chromosome bridges
2N 4N
P < 0.0001
DLD-1
C1 C3 C28 B4 B6 B8
0
20
40
60
80
Cellsinanaphase(%)
Tripolar
Chr. Fragments
Lagg chr.
Multiple mis-segregating chr.
Chromosome bridges
2N 4N
DLD-1
P < 0.0001
Aphidicolin
2N 2N + Aph 4N 4N + Aph
0
5
10
15
20
25
BNcellswithmicronuclei(%)
P < 0.0001
P < 0.0001 P < 0.0001
MNi, micronuclei; 2N, diploid; and 4N, near-tetraploid.

47. 47
A. Migration:
24 h
48 h
Migratory and invasive capabilities
2N, diploid; and 4N, near-tetraploid.
Results IV
1 >1
0
20
40
60
80
100
Migratingcells(%)
DLD-1 2N
DLD-1 4N-1
DLD-1 4N-2
Number of centrosomes

48. 48
B. Invasion:
2N 4N
Migratory and invasive capabilities
2N, diploid; and 4N, near-tetraploid.
P < 0.05
Near-tetraploid clones show phenotypic advantages in comparison with diploid clones
Results IV

49. 49
Migratory and invasive capabilities
2N, diploid; and 4N, near-tetraploid.
Tumor
Invasive
front
The invasive front of colorectal tumors
has more aneuploidy and near-
tetraploid cells than the main tumor
mass
Results IV
Tumor Invasive front
0
10
20
30
Cellswithextracentrosomes(%)
P < 0.05
10 µm
N = 9
FISH signals
Percentage(%)
P < 0.0001

50. 50
Conclusions I
Aberrant crypt foci and LINE-1 methylation as predictive biomarkers for CRC development
1. The inability to reliably classify ACF based on their histology hinders their use as surrogate markers for CRC.
2. Endoscopic and morphologic characteristics do not indicate ACF are suitable biomarkers for CRC risk.
3. Only a few of the genetic and epigenetic CRC alterations are detected in ACF and they do not correlate with the CRC
risk group.
4. ACF might merely be preneoplastic and not an intermediate endpoint for colorectal carcinogenesis.
5. ACF do not exhibit global DNA hypomethylation and our results do not support LINE-1 methylation field defect for
sporadic CRC.
Chromosome copy number alterations and whole genome duplication events during CRC evolution
1. Chromosome imbalances progressively increase during colorectal carcinogenesis, leading to extensive intratumor
heterogeneity.
2. The amount and patterns of chromosome alterations distinguish non-advanced adenomas from progressed
adenomas.

51. 51
Conclusions II
3. The AD-to-ADK transition and CRC progression are defined by specific chromosome alterations, mainly involving the
sequential gain of either 7p, 13q or 20q, or the combination of them.
4. WGD events are not only implicated in CRC progression but also in tumor initiation, thus demonstrating the crucial
role of genome doubling events in CRC evolution.
5. The first gain in driver chromosomes is acquired during the early stages of CRC formation. Moreover, low-frequency
clones and the absence of strong selection define the landscape of this type of cancer.
6. Increased DNA ploidy influences the level of genomic instability and the existence of heterogeneity in a population.
Furthermore, near-tetraploid cells are able to progress and show tolerance to aneuploidy.
7. Near-tetraploid cells systematically undergo replication stress, which increases the levels of both structural and
numerical CIN.
8. Although near-tetraploid cells exhibit a growth impairment due to replication stress and genomic instability, they
show greater migratory and invasive capabilities than their diploid counterparts, thus supporting their being observed
in the invasive fronts of primary tumors.