Chromosomal Alterations in Ulcerative Colitis-Related and Sporadic Colorectal Cancers by Comparative
Genomic Hybridization1
Daniela E. Aust,2 Robert F. Willenbucher, Jonathan P. Terdiman, Linda D. Ferrell, Cornell G.
Chang, Dan H. Moore II, Annette Molinaro-Clark, Gustavo B. Baretton, Udo Loehrs and
Frederic M. Waldman
Cancer Center (DA, CC, AC, FW) and Departments of Laboratory Medicine (FW), Medicine
(RW, JT), and Pathology (LF), University of California San Francisco, San Francisco, CA
94143-0808; Geraldine Brush Cancer Research Institute, California Pacific Medical Center, San
Francisco, CA (DM); and Pathologisches Institut der Ludwig-Maximilians-Universität, 80337
München, Germany (GB, UL).
Running Title: CGH alterations in UC-related cancers
Keywords:
Comparative Genomic Hybridization, Ulcerative Colitis, Chromosomal
Alterations
1
Supported by NIH CA 74826 and Deutsche Forschungsgemeinschaft Au141/1-1.
1
CGH alterations in UC-related cancers
2
To whom requests for reprints should be addressed, at: UCSF Cancer Center, Box 0808, San
Francisco, CA 94143-0808. Phone: 415-476-3822, Fax: 415-476-8218, email:
[email protected]
3
The abbreviations used are: UC, ulcerative colitis; CGH, comparative genomic hybridization;
UICC, Union International Contre le Cancer; LOH, loss of heterozygosity.
2
CGH alterations in UC-related cancers
ABSTRACT
Both ulcerative colitis (UC)-related and sporadic colorectal cancers are thought to evolve
through a multistep process of genomic instability, accumulation of genomic alterations and
clonal expansion. This process may involve different genomic changes in UC-related cancers
than in sporadic cancers due to the origin of UC-related cancers in an inflammatory field. This
study was designed to define the specific genomic events in UC-related cancers.
Comparative genomic hybridization (CGH) was performed on 32 UC-related and 42
stage-matched sporadic colorectal cancers. The mean number of chromosomal alterations per
case was similar in the UC-related and sporadic tumor groups (8.6 in UC, 8.1 in sporadic).
Frequently lost regions in both cancer groups were 18q (78% UC, 69% sporadic), 8p (53% UC,
50% sporadic), and 17p (44% UC, 57% sporadic). The three most frequent gains involved
chromosomes 8q (63% UC, 45% sporadic), 20q (44% UC, 67% sporadic), and 13q (44% UC,
38% sporadic). Chromosome 5q was lost in 56% of UC-related but in only 26% of sporadic
cancers. Losses of 17q and gains of 5p were also more frequent in UC-related cancers, whereas
losses of 14q and gains of Xp were significantly less common in UC-related cancers than in
sporadic tumors. There was a stage-related increase in chromosome 8 alterations in UC-related
but not in sporadic cancers, while only sporadic tumors showed a stage progression for 5p loss,
17q gain, and 18q loss. Thus, differences in the frequency and timing of individual chromosomal
alterations suggest that the genetic pathways in these two tumor groups are distinct from each
other.
3
CGH alterations in UC-related cancers
INTRODUCTION
Ulcerative colitis (UC)3 is a chronic inflammatory disease of the bowel associated with an
increased risk of developing colorectal neoplasia compared to an age-matched control group. The
risk of developing cancer increases with extent and duration of the disease (1, 2). Like sporadic
colorectal cancer, UC-related neoplasia is thought to evolve through a multistep process of
genomic instability, accumulation of mutations and clonal expansion. Unlike sporadic cancer,
which usually develops from an adenomatous polyp, UC-related cancer arises from flat
dysplastic epithelium in a field of chronic inflammation. Therefore UC-related cancer may follow
a different genetic progression pathway than sporadic cancer. Genomic alterations that occur
during the adenoma to carcinoma sequence in sporadic cancers are well characterized (3-7).
However, less is known about the specific genomic events in UC-related cancer initiation and
progression.
The aim of this study was to determine clonal genomic alterations in UC-related
colorectal cancers by comparative genomic hybridization (CGH), and to identify alterations that
are unique to UC-related tumors when compared to sporadic colon cancers.
4
CGH alterations in UC-related cancers
MATERIALS AND METHODS
Clinical Materials. Thirty-two UC-related cancers were identified from the archives of the
University of California San Francisco and Munich University Departments of Pathology. CGH
alterations in 14 of the UC-related cases have been reported previously (8). Forty-two archival
sporadic cancers were selected from a similar time period to match the stage distribution in the
UC-related cancer group. Staging was done according to UICC established criteria (9). Sporadic
tumors were obtained from patients with no family history of colon cancer and no personal
history of inflammatory bowel disease. Normal surgical margins from seven colectomy
specimens removed for diverticular disease served as normal controls. One representative block
of carcinoma was selected for each tumor. DNA for CGH was extracted from thin paraffin
sections as previously described (8, 10). Microdissection of tumor material was followed by 3
day proteinase K treatment prior to CGH analysis.
Comparative Genomic Hybridization. In CGH, total genomic DNA from the tumor sample
and genomic DNA from a normal donor, detected in different colors, are simultaneously
hybridized to a normal metaphase spread (8, 10, 11). The ratio of the colors along the normal
chromosomes provides a quantitative map of the relative copy number of DNA sequences in the
test sample. Thus the entire genome can be surveyed in a single step without the need to first
select which genomic loci to test.
Microdissected DNA was amplified by degenerate oligonucleotide primed (DOP) PCR as
previously described (8, 10). Each PCR run included controls of normal genomic DNA, and the
MPE-600 cell line. Amplification of 1-2 ng microdissected DNA yielded up to 1 µg of PCR
product, averaging 500 base pairs in size (range 200 bp-2 kb). Fifty nanograms of fresh DNA
5
CGH alterations in UC-related cancers
(normal and MPE600 cell line) resulted in approximately 2-3 µg of amplified DNA. In addition
to the 32 successful samples, two UC-related samples (stage I, G1 and stage III, G3) yielded
insufficient PCR product and were not included in the study. PCR amplified DNA was labeled
by nick translation. Normal reference DNA was labeled with Texas Red-5-dUTP or with
Fluorescein-12-dUTP (Dupont), while amplified DNA from paraffin sections was labeled with
Fluorescein-12-dUTP (Dupont) or Digoxigenin-11-dUTP (Boehringer). The optimum size for
CGH was 500 bp to 2000 bp. Nick translated PCR products were close to the original PCR
product size. Each sample was hybridized as previously described, in duplicate, with different
fluorochromes, onto normal male metaphases (8, 10). Successful hybridizations were judged by
the intensity of the tumor and normal signals, by the granularity vs. smoothness of the signals, by
the homogeneity of the signal over the entire metaphase, and by the banding intensity of the 4', 6diamidino-2-phenylindole (DAPI) signals used for chromosome identification. Acquisition was
performed utilizing our QUIPS analysis system (11).
High level amplifications were defined as a peak of the ratio intensity above 2.0, involving
less than a whole chromosome arm. Low level gain or loss was defined as chromosome regions
having a ratio above 1.25 or below 0.8. Inverse CGH, using a second hybridization with reversed
color labels, allowed greater confidence in making these interpretations. The inverse pair was
examined together to allow better discrimination of significant changes. All changes must have
been present in both the forward and inverse hybridizations to be considered real. Interpretation
of changes at 1pter, 19 and 22 (as well as 4 and 13q in the opposite direction) required careful
examination of all chromosome profiles because these loci were likely to show more variability
in their ratios.
6
CGH alterations in UC-related cancers
Statistical Analysis. Comparison of individual changes between groups used the two-sided
Fisher’s exact test, a non-parametric test for independence. In addition, to test the set of gains and
losses by tumor type, an overall chi-square test was formed by summing the individual chi-square
statistics for each comparison. To test whether the observed chi-square statistics could have
occurred by chance, an empirical p-value was determined via a randomization test. The
randomization test was based on repeatedly sampling from the observed data, each time
randomly dividing the sample into two groups. The value of the observed chi-square statistics,
based on the true group assignments, was compared with the distribution of chi-square statistics
formed from the pseudo-groups. The empirical p-value was the fraction of samples with chisquares exceeding the observed chi-square test statistics.
7
CGH alterations in UC-related cancers
RESULTS
CGH was performed on DNA extracted from 32 UC-related and 42 stage matched
sporadic colorectal cancers to identify the specific pattern of chromosomal alterations in UCrelated cancers, and to define differences in the pattern of genomic alterations between the two
groups. Clinical characteristics associated with these samples are listed in Table 1. Note that the
matched set of sporadic tumors shows a relatively high number of right-sided lesions compared
to the UC-related tumors.
The most common chromosomal alterations seen in both groups of tumors are listed in
Table 2. The mean number of changes detected by CGH was similar in both groups (8.6 in UCrelated, 8.1 in sporadic) and did not increase significantly with patient age, tumor stage or tumor
grade in either group. No chromosomal alterations were detected in the seven samples of normal
colonic mucosa from diverticulitis colectomy specimens.
UC-related cancers. Thirty of the thirty-two UC-related cancers (94%) showed
chromosomal alterations detectable by CGH. The mean number of changes was 8.6, with an
average of 3.6 gains and 5.1 losses per case. Two tumors, one located in the ascending colon, the
other located in the descending colon, did not show any CGH abnormalities. These two cases
displayed a high frequency of microsatellite instability (data not shown) (8).
The loss of chromosome arm 18q was the most common alteration in UC-related cancers,
present in 25 of the 32 cases (78%). Other common regions of copy number loss were
chromosome arms 5q, 8p, 17p, and the entire chromosome 4. The most frequent relative gain
involved chromosome arm 8q. Chromosome arms 20q, 13q, and 5p were also frequently gained
in UC-related cancers.
8
CGH alterations in UC-related cancers
The only stage dependent changes in UC-related cancers were gains of chromosome arm 8q
and losses of chromosome arm 8p. Both of these changes were significantly more frequent in
stage III and IV tumors than in stage I and II tumors (Table 3). None of the chromosomal
alterations seen were significantly associated with tumor grade or tumor location.
Sporadic cancers. Thirty-eight of the forty-two sporadic cancers showed CGH detectable
changes. The mean number of changes in the entire group was 8.1, with an average of 4.3 losses
and 3.8 gains per case. Four tumors, all located in the right colon, did not show any CGH
changes. Microsatellite instability analysis showed high frequency instability in two of the four
cases (data not shown).
The most frequent CGH alteration in the sporadic cancers was the loss of chromosome arm
18q, in 29 of the 42 sporadic cases (69%). Losses of chromosome arms 17p, 18p, and 8p also
occurred in more than half of the sporadic cases. The most frequently gained region in sporadic
tumors was chromosome arm 20q (67%). Other frequent gains were seen on chromosome 7, and
chromosome arms 8q and 20p (Table 2).
Three aberrations occurred significantly more often in late stage tumors (III and IV) than in
early stages (I and II): gain of chromosome arm 17q, and loss of chromosome arms 5p and 18q
(Table 3). Tumor grade was not associated with an altered frequency of any specific CGH
aberration. The gain of chromosome arm 20q and the loss of 5q were significantly more frequent
in left-sided tumors than in right-sided tumors (Table 4).
Comparison of UC-related and sporadic cancers. The predominant pattern of genomic
instability in both tumor groups was that of gross chromosomal aberrations. UC-related and
sporadic cancers shared similar frequent changes. Loss of 18q was the most frequent alteration in
9
CGH alterations in UC-related cancers
both tumor groups, followed by loss of 8p and 17p. In both tumor groups the most common gains
seen involved chromosome arms 8q, 20q, and 13q.
However, there were significant differences in the frequency of specific chromosomal
alterations between UC-related and sporadic cancers. Losses of chromosome arms 17q and 5q
were significantly more frequent in UC-related than in sporadic cancers as were gains of
chromosome arm 5p. Losses of chromosome arm 14q and gains of Xp were significantly less
common in UC-related than in sporadic tumors. These findings are summarized in Table 2. In
addition, the loss of chromosome arm 18q was seen more frequently in early stages of UC-related
cancers than in early stages of sporadic cancers. In UC-related cancers there were no differences
in the frequency of individual changes between right and left-sided tumors. In contrast, in leftsided sporadic cancers, losses of 5q and gains of 20q were significantly more frequent than in
right-sided tumors.
To test whether these differences in frequency of individual changes could have arisen by
chance a randomization test was performed. This analysis showed that the differences observed
in the overall distribution of changes between the two tumor groups was highly unlikely to have
resulted from chance (p= 0.0028).
10
CGH alterations in UC-related cancers
DISCUSSION
This study used CGH analysis to compare the chromosomal alterations in UC-related
colorectal cancers to those found in a stage matched set of sporadic tumors. To our knowledge
this represents the largest series reported to date of colorectal cancers analyzed by CGH.
Comparison of CGH alterations between sets of tumors represents a large number of
comparisons in a relatively small group of tumors. One problem arising in studies in which
multiple comparisons are made is that differences detected may occur by chance. To address this
issue we performed a randomization test of the observed data (mixing the two groups together
and randomly repartitioning them to define chance associations), which showed a significant
difference in the overall frequency of changes between the two tumor groups. This analysis
allows us to conclude that the differences and similarities reported for frequencies of individual
chromosome alterations reflect biologically significant relationships.
Sporadic colorectal cancer usually develops from an adenomatous polyp through a
multistep process involving genomic instability and the progressive accumulation of genetic
alterations (4, 6). In our study 90% of the sporadic tumors demonstrated genomic instability
characterized by gross chromosomal alterations, in agreement with previous CGH studies of
sporadic colorectal cancers (12, 13). A similar pattern of genomic instability (gross chromosomal
alterations) was seen in most of the UC-related cancer tumors as well (94%), even though they
generally arise from flat dysplastic epithelium in a chronic inflammatory field.
UC-related and sporadic cancers not only shared the same pattern of genomic instability
but also a similar group of common chromosomal gains and losses. Loss of chromosome arm
18q was the most frequent alteration in both tumor groups, suggesting comparable importance of
11
CGH alterations in UC-related cancers
this alteration in UC-related and sporadic carcinogenesis. These CGH data corroborate the
finding of frequent LOH on 18q in UC-related cancers (14, 15). Chromosome arm 18q is the site
of three tumor suppressor genes known to be frequently lost in sporadic tumors: DCC, SMAD2
and SMAD4 (16-18). The importance of these genes in UC-related carcinogenesis has not yet
been determined. Hoque et al. found SMAD4 to be mutated in a case of UC-related cancer with
LOH on 18q21.1 (19). However, Lei et al. did not find mutations of SMAD4 in their set of 10
UC-related cancers (20).
Loss of chromosome arm 17p, the site of p53, was also frequently seen in both tumor
groups. Loss of heterozygosity (LOH) on chromosome 17p has been described as a frequent
alteration in UC-related dysplasias (67%) and cancers (100%) (21-24) and is a common finding
in sporadic cancers as well (4, 6, 25). P53 mutations may be even more common than 17p LOH,
occurring in 91 – 100% of UC-related cancers and in 40 – 80% of UC-related dysplasias (21-24,
26-31). Further, p53 mutation has been identified in nondysplastic mucosa biopsied from colons
with dysplasia or cancer (23, 32, 33), and it has been identified in colonic mucosa sampled prior
to the occurrence of dysplasia (29, 34). This suggests that p53 mutation is a very early event in
UC-related carcinogenesis. In contrast, p53 mutation is found in 63-76% of sporadic colon
cancers, but in only 3% of sporadic adenomas (35, 36), suggesting that p53 mutation occurs later
in the neoplastic progression pathway in sporadic colorectal cancer.
Loss of chromosome arm 8p was also frequent in both tumor groups, suggesting that this
region may harbor a tumor suppressor gene important in both UC-related and sporadic
carcinogenesis. Loss of 8p in our set of tumors may, to some extent, reflect isochromosome
formation in both tumor groups since 67% (sporadic) and 71% (UC-related) of 8p losses were
accompanied by gains on chromosome arm 8q. The significance of 8p loss in colorectal
12
CGH alterations in UC-related cancers
carcinogenesis is unclear. Ried et al. reported losses on 8p in 0% of colorectal adenoma, but in
38% of sporadic colorectal cancer by CGH (13), while Korn et al. reported losses of 8p in 59% of
the liver metastases of sporadic colorectal cancers (37), indicating an association of 8p loss with
tumor progression. In our set of sporadic tumors frequency of 8p loss did not increase with tumor
stage, whereas the UC-related cancers did show a stage related increase. LOH on 8p has been
described in both sporadic (29%) and UC-related cancers (20%) (15). Chang et al. described
LOH on 8p as a common and early event in UC-related carcinogenesis, present in 44% of
dysplasias and carcinomas (38), whereas Fogt et al. found LOH on 8p in only 14% of UC-related
dysplasias and none of their UC-related cancers (14).
The three most commonly gained regions in both groups were 8q, 13q and 20q. These
gains have been previously reported as frequent CGH alterations in sporadic cancers (12, 13, 37)
suggesting that oncogenes located on these chromosomes (c-myc on 8q24, ZAPC1 and Aurora 2
on 20q13) (39) are important in sporadic carcinogenesis. The role of these oncogenes in UCrelated tumors is not known.
Despite a similarity in the most frequent gains and losses in the two tumor groups there
were also significant differences in the prevalence and timing of individual changes. The frequent
loss of 5q in our set of UC-related samples suggests a crucial role of the APC gene in UC-related
cancer progression. However, the role of APC in UC-related cancer progression has been
controversial. There is evidence of frequent LOH at the site of APC in UC-related cancers (33%
to 50%) and dysplasias (27% to 33%) (14, 15, 24, 27), and the APC protein was found to be
truncated in 40% to 50% of a small series of UC-related cancers (24, 40). Despite this, mutations
within the mutational cluster region of the APC gene (exon15, codons 1125 to 1540) appear to be
infrequent in UC-related neoplasia (0 – 6 %) (41, 42). One possible explanation for these
13
CGH alterations in UC-related cancers
inconsistencies is that APC mutations might occur outside of this mutational cluster region in
UC-related tumors.
APC mutation and LOH on 5q is a frequent and early event in sporadic colorectal
carcinogenesis (4-7, 14, 15, 43, 44). The low frequency of 5q loss detected by CGH in our
sporadic cancers is consistent with other reports of CGH analyses in sporadic colorectal tumors
(12, 13, 37). This low detection rate may be due to the region of loss in sporadic cancers being
too small to be detected by CGH. The difference in frequency of 5q loss between UC-related and
sporadic tumors suggests that the mechanism of alteration is different in the two tumor groups. In
UC-related cancers losses on 5q most often affect the whole chromosome arm, and are
accompanied by a gain of 5p in half the cases, presumably reflecting isochromosome formation
in these tumors. This also may explain the difference in frequency of 5p gains between the two
groups. Only 24% of the sporadic cases with a loss of 5q showed a simultaneous gain of 5p.
A significant difference between sporadic and UC-related tumors was also observed in
the alteration of chromosome arm 17q. Chromosome arm 17q was lost in 34% of the UC-related
tumors, which can be partly explained by the fact that this chromosome was often lost as a whole
(25%). In sporadic tumors, however, loss of all of chromosome 17 was less frequent (7%) and
loss of 17q alone was not observed at all. In contrast, chromosome 17q was gained in 19% of the
sporadic tumors. 89% of these gains occurred together with losses of 17p. These data indicate
that the mechanism of alteration of chromosome 17 is different in the two tumor groups,
preferentially targeting 17p in sporadic tumors and affecting the whole chromosome in UCrelated tumors. In addition, oncogenes located on 17q (erbB2) may be important in sporadic
cancers but may not be influential in UC-related carcinogenesis.
14
CGH alterations in UC-related cancers
Losses of chromosome arm 14q and gains of Xp were detected in over 30% of the
sporadic cancers and were significantly more frequent in sporadic than they were in UC-related
cancers, suggesting that these regions might contain genes relevant to sporadic colorectal
carcinogenesis.
In sporadic colorectal carcinogenesis the loss of chromosome 18q is thought to be a later
event (47), found by CGH in 89% of the hepatic metastases of sporadic colorectal cancers (37).
Our data support this finding in that the loss of 18q was significantly more frequent in late stages
of sporadic tumors (stages III and IV) than in early stages (I and II). In our UC-related cancers the
loss of 18q was equally frequent in early and late stage tumors, suggesting that a tumor
suppressor gene on 18q may play an early role during UC-related carcinogenesis. Our previous
finding that UC-related dysplasia and even nondysplastic UC-involved mucosa show 18q losses
by CGH supports this hypothesis (8, 10).
The differences we observed between UC-related and sporadic cancers are not likely to be
due to differences in tumor stage since our tumors were matched for stage. However, certain
differences may be due to a dissimilarity in the relative distribution of right and left-sided tumors
in our set of cases. While our set of UC-related cancers was comprised of a majority of left sided
tumors due to a high percentage of rectal cancers (31%), the sporadic tumor set contained a
majority of right sided cancers (57%). This distribution may reflect a truly higher prevalence of
left-sided (rectal) cancer in UC or it may have occurred by chance. In either case, the difference
in frequency of 5q losses between the two groups may be partly explained by the finding that 5q
losses in sporadic cancers are less frequent in left-sided tumors than right-sided tumors. The
frequency of 5q alterations may therefore have been low in our set of sporadic cancers since it
consisted of a majority of right-sided tumors (57%). A second chromosomal alteration that was
15
CGH alterations in UC-related cancers
more frequent in left than right-sided sporadic tumors was the gain of 20q. If matched for
location as well as stage 20q gains might have been observed even more frequently in sporadic
cancers than in UC-related cancers. The difference between right and left-sided tumors in the
frequency of these two chromosomal changes also hints at different pathways of carcinogenesis
in right and left-sided sporadic colorectal tumors without microsatellite instability. In contrast to
the sporadic group there were no significant differences between right and left sided UC-related
tumors, suggesting that the underlying chronic inflammation may select for the same genetic
changes in right and left-sided UC-related carcinogenesis.
In summary we have shown considerable overlap in the specific alterations occurring in
UC-related and sporadic colorectal cancers, suggesting that a common set of genes is involved
during development and progression of both groups of tumors. However, differences in the
frequency and timing of individual alterations are evident and indicate that the genomic
progression pathways in these two groups of colorectal cancer are distinct from each other,
possibly reflecting their different fields of origin.
16
CGH alterations in UC-related cancers
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22
CGH alterations in UC-related cancers
Table 1 Clinical characteristics
UC-related Cases
(n=32)
Sporadic Cases
(n=42)
Male
53%
52%
Agea
49 ± 15
68 ± 13
41%
28%
31%
57%
26%
17%
25%
16%
38%
21%
24%
21%
33%
21%
3%
66%
31%
7%
71%
22%
Tumor
Location
right colon
left colonb
rectum
Stage
I
II
III
IV
Grade
G1
G2
G3
a
Mean age in years ± standard deviation
b
at or distal to splenic flexure
23
CGH alterations in UC-related cancers
Table 2 Comparison of common CGH Alterations
Chromosome
arm
UC-relateda
(%)
Sporadicb
(%)
P-valuec
Lossesd
18q
5q
8p
17p
4q
4p
17q
18p
14q
78
56
53
44
38
34
34
31
9
69
26
50
57
29
33
7
52
31
0.44
0.02
0.82
0.35
0.46
1.00
0.01
0.10
0.04
Gains
8q
20q
13q
5p
7p
20p
7q
Xq
Xp
63
44
44
38
28
25
25
19
9
45
67
38
10
48
38
45
36
36
0.25
0.06
1.00
0.02
0.10
0.32
0.09
0.07
0.01
a
n = 32
b
n = 42
c
Two-sided Fisher’s Exact Test comparing UC-related and sporadic cancers
d
All changes ≥ 30% in either UC-related of sporadic groups are listed.
24
CGH alterations in UC-related cancers
Table 3 Stage dependent chromosomal alterations
UC-related cancers
Sporadic cancers
Stage I/IIa
(%)
Stage III/IVb
(%)
P-valuec
Stage I/IId
(%)
Stage III/IVe
(%)
P-valuec
8p loss
8
80
0.00
42
57
0.54
8q gain
33
75
0.03
37
52
0.37
5p loss
8
0
0.38
0
22
0.05
17q gain
0
5
1.00
5
30
0.05
18q loss
75
80
1.00
53
83
0.05
Chromosome
alteration
a
n = 12
b
n = 20
c
Two-sided Fisher’s Exact Test comparing stage I/II with stage III/IV within tumor group
d
n = 19
e
n = 23
25
CGH alterations in UC-related cancers
Table 4 Comparison of right sided vs. left sided tumors
UC-related cancers
Sporadic cancers
right sideda
(%)
left sidedb
(%)
P-valuec
right sidedd
(%)
5q loss
62
53
0.73
13
44
0.03
20q gain
46
42
1.00
50
89
0.01
Chromosome
alteration
left sidede P-valuec
(%)
a
n = 13
b
n = 19
c
Two-sided Fisher’s Exact Test comparing left and right-sided tumors within tumor group
d
n = 24
e
n = 18
26