molecular profiling of ocular surface squamous neoplasia identifies multiple dna copy number...
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1
Identification of Multiple DNA Copy Number Alterations Including Frequent 8p11.22
Amplification in Conjunctival Squamous Cell Carcinoma
Laura Asnaghi1*, Hind Alkatan
4*, Alka Mahale
4, Maha Othman
4, Saeed Alwadani
1,2,5,
Hailah Al-Hussain4, Sabah Jastaneiah
4, Wayne Yu
6, Azza Maktabi
4, Deepak P. Edward
2,4,
Charles G. Eberhart1,2,3
*Equal contribution
1Department of Pathology,
2Ophthalmology, and
3Oncology, Johns Hopkins University, School
of Medicine, Baltimore, MD, USA 4
King Khaled Eye Specialist Hospital, Riyadh, Saudi Arabia
5 Department of Ophthalmology, King Saud University, Riyadh, Saudi Arabia
6Microarray Core Facility, Sidney Kimmel Cancer Center, Johns Hopkins University, School of
Medicine, Baltimore, MD, USA.
Corresponding author: Charles G. Eberhart, Department of Pathology, Ophthalmology and
Oncology, Johns Hopkins University, School of Medicine, Smith Bldg, 400 N. Broadway Ave,
Baltimore, MD, 21287.
Phone: 410-502-5185; fax: 410-955-9777; e-mail: [email protected]
Running title: Comprehensive molecular profiling of cSCC
Manuscript information: 3850 words
Keywords: Conjunctiva Squamous Cell carcinoma (cSCC); array CGH; NanoString assay
Financial support: King Khaled Eye Specialist Hospital (KKESH) – Wilmer Eye Institute (WEI)
Collaborative Research Grant, Research to Prevent Blindness (Wilmer)
IOVS Papers in Press. Published on December 9, 2014 as Manuscript iovs.14-14920
Copyright 2014 by The Association for Research in Vision and Ophthalmology, Inc.
2
ABSTRACT 1
Purpose: Little is known about the molecular alterations which drive formation and growth of 2
conjunctival squamous cell carcinoma (cSCC). We therefore sought to identify genetic changes 3
which could be used as diagnostic markers or therapeutic targets. 4
Methods: DNA extracted from 10 snap frozen cSCC tumor specimens and 2 in situ tumors 5
carcinomas was analyzed using array-based Comparative Genomic Hybridization (aCGH), and 6
further examined with NanoString and quantitative polymerase chain reaction (PCR). 7
Results: The number of regions of DNA loss ranged from 1 to 23 per tumor, while gains and 8
amplifications ranged from 1 to 15 per tumor. Most large regions of chromosomal gain and loss 9
were confirmed by NanoString karyotype analysis. The commonest alteration was amplification 10
of 8p11.22 in 9 tumors (75%), and quantitative PCR analysis revealed 100-fold or greater 11
overexpression of ADAM3A mRNA from 8p11.22 locus. In addition, recurring losses were 12
observed at 14q13.2 and 22q11.23, both lost in 5 of the 12 tumors (42%), and at 12p13.31, lost in 13
4 of the 12 samples (33%). Of the 8 loci associated with the DNA damage repair syndrome 14
Xeroderma Pigmentosum, 3 showed loss of at least one allele in our aCGH analysis, including 15
XPA (9q22.33, 1 tumor), XPE/DDB2 (11p11.2, 1 tumor) and XPG/ERCC5 (13q33.1, 3 tumors). 16
Conclusions: cSCC contain a range of chromosomal alteration potentially important in tumor 17
formation and growth. Amplification of 8p11.22 and overexpression of ADAM3A suggests a 18
potential role for this protease. Our findings also suggest that defects in DNA repair loci are 19
important in sporadic cSCC. 20
21
22
3
INTRODUCTION 23
A range of precancerous and cancerous epithelial lesions can arise in the cornea and conjunctiva, 24
including actinic keratosis, conjunctival intraepithelial neoplasia (CIN), conjunctival carcinoma 25
in situ (cCIS, sometimes grouped with severe CIN), and conjunctival squamous cell carcinoma 26
(cSCC). Carcinomas arise most commonly in the interpalpebral area, and involve the bulbar 27
conjunctiva and the limbus.1,2
Although these tumors have a low metastatic potential, they can be 28
locally invasive.3
Most of the lesions grow slowly and are unilateral. The main reported risk 29
factors for this group of ocular surface squamous neoplasms (OSSN) are high ultraviolet (UV-B) 30
exposure, infection with high risk subtypes of human papilloma virus (HPV), and suppression of 31
the immune response due to infection with human immunodeficiency virus (HIV) or 32
medication.4-6
33
The role of HPV in the pathogenesis of cSCC is somewhat controversial. Early 34
investigations detected HPV, but several recent studies have not identified the virus in CIN or 35
cSCC lesions, calling its role in the initiation and growth of these tumors into question.7-9
The 36
strong association between OSSN and exposure to sunlight, however, is clear, with a striking 37
increase in their incidence in geographic areas that are close to the equator.4,10,11
In addition, 38
patients with Xeroderma Pigmentosum, who are more susceptible to the effects of solar 39
ultraviolet light due to mutation or loss of DNA repair factors, are especially prone to develop 40
the disease.12
Despite this evidence supporting a role for mutations and other DNA alterations in 41
OSSN pathogenesis, the genetic drivers responsible for their initiation and progression are 42
largely unknown. We therefore sought to identify chromosomal abnormalities in a series of 43
cSCC and in situ lesions resected at a single center in Saudi Arabia in order to find molecular 44
alterations potentially useful for diagnosis and serve as targets for therapy. 45
4
MATERIALS AND METHODS 46
Clinical information 47
In situ and invasive conjunctival squamous cell carcinoma cases were identified through review 48
of pathology and tumor bank records at King Khaled Eye Specialist Hospital (KKESH), Riyadh 49
Saudi Arabia. Only cases with tissue snap-frozen at the time of surgery were used in this study. 50
The diagnostic slides in all cases were reviewed by ophthalmic pathologists (HA, DE and AM) 51
to confirm the presence of carcinoma. After Internal Review Board (IRB) approval, relevant 52
clinical data were abstracted from the clinical record and linked to the frozen research cases 53
using anonymized sample identification numbers. The clinical characteristics in these cases are 54
summarized in Table 1. 55
Genomic DNA and total RNA extraction 56
Genomic DNA was extracted from 14 snap frozen OSSN tumor tissues using DNeasy Blood and 57
Tissue Kit (Qiagen, Germantown, MD) following manufacturer’s protocol, and the DNA 58
concentration and quality were determined using a spectrophotometer (NanoDrop Technologies, 59
Wilmington, DE). The DNA in two of the 14 samples was not sufficient to perform array 60
comparative genomic hybridization (CGH), although one of these was used for NanoString 61
analysis. Total RNA was also isolated from the tumor tissues and additionally from 2 normal 62
bulbar conjunctiva specimens taken at autopsy using RNeasy Mini Kit (Qiagen) according to 63
manufacturer’s protocol and used for quantitative RT-PCR. 64
Array CGH 65
The Agilent SurePrint G3 Human 4x 180K Microarray (G4449A) was used in the study (Agilent 66
Technologies, Santa Clara, CA). This array contains 170,334 distinct biological probes chosen 67
from human genome sequences. The integrity of genomic DNA was confirmed by low voltage 68
5
0.6% agarose gel electrophoresis with a mean band size of about 50Kb. Sample labeling was 69
performed following Agilent’s recommendation for array CGH. Briefly, 1.5 g of genomic 70
tumor DNA and normal reference DNA were digested with 50 units of Alu I and Rsa I 71
(Promega, Madison, WI) for 2 hours at 37°C. Samples were then purified by using QIAQuick 72
PCR clean-up kit (Qiagen). Labeling reactions were carried out with restricted and purified DNA 73
for 3 hours at 37°C using a BioPrime Array CGH Genomic Labeling Module (Invitrogen, 74
Carlsbad, CA) with 3 mol Cy5-dUTP or Cy3-dUTP (Perkin Elmer, Waltham, MA). Labeled 75
samples were purified, concentrated on a Centricon YM-30 column (Millipore, Billerica, MA), 76
and then mixed with 10x blocking agent and 2x hybridization buffer (Agilent Technologies). 77
Hybridization mixtures were first denatured at 95°C for 3 min and then immediately transferred 78
to 37°C for 30 min. To remove any precipitates, the mixtures were centrifuged at 14,000g for 5 79
min. These mixtures were then hybridized to the microarrays for 40 hours at 65°C in a rotating 80
oven (Robbins Scientific, Mountain View, CA) at 10 rpm. Hybridized microarrays were washed 81
and dried according to manufacturer’s protocols, and imaged with an Agilent G2565BA 82
microarray scanner using default settings. Data were extracted using Feature Extraction 83
Software v9.1 (Agilent Technologies) and analyzed using Agilent’s Genome Workbench 84
software (ver 7.0). Aberrant regions (gains or losses) were then identified using a build-in 85
Hidden Markov Models (HMM) algorithm with default settings. Dye swap experiments were 86
performed for each sample and only aberrations present in both sets were considered for further 87
analysis. The Log2 [Tumor/Normal] for each probe in a chromosome region was determined and 88
averaged for the region by the algorithm. Values between 0.75 and 1.0 were considered genomic 89
gain, and >1.0 amplification, while those between -0.75 and -1.0 were classified as hemizygous 90
loss, and < -1.0 as homozygous deletion. 91
6
Quantification of chromosomal aberrations by NanoString Analysis 92
For NanoString analysis, 600 ng of purified genomic DNA were used from each sample and 93
hybridized overnight in a thermocycler at 65°C with the human karyotype panel probes 94
(NanoString Technologies, Inc. Seattle, WA). Hybridization mixtures were then loaded into the 95
nCounter Prep Station, and color-coded barcodes on the reporter probes were read and quantified 96
by the nCounter Digital Analyzer (NanoString Technologies). Copy number estimate for each 97
probe was normalized using a normal DNA reference. Quality control, normalization and data 98
analysis were performed using nSolver software (NanoString Technologies). After 99
normalization, copy number estimate values between 2.5 and 3 were scored as genomic gains, 100
greater than 3 high-level amplifications, between 1 and 1.5 hemizygous deletions, and lower than 101
1 homozygous deletions. Technical variability was reported as being 15%, therefore we used a 102
conservative threshold of 25%.13
103
Real time quantitative PCR 104
Expression levels of ADAM3A and ADAM5P transcripts were determined using TaqMan® one-105
step quantitative RT-PCR (Invitrogen), following manufacturer’s protocol. Primers specific for 106
ADAM3A (ID: Hs03297297_m1), ADAM5P (ID: Hs01386884_m1) and 18S, used for 107
normalization (ID: Hs99999901_s1), were purchased from Invitrogen. All reactions were 108
performed in triplicate, using 50 ng of RNA, in an iQ5 Multicolor real-time PCR detection 109
system (Bio-Rad, Hercules, CA), using EXPRESS One-Step Superscript® qRT-PCR universal 110
mix (Invitrogen). The cycling program included the cDNA synthesis at 50°C for 15 minutes, 111
followed by incubation at 95°C for 20 seconds and 40 cycles at 95°C for 3 seconds, and 60°C for 112
30 seconds, according to manufacturer’s protocol. 113
114
7
RESULTS 115
Clinical Characteristics 116
The cases with frozen tissue available included 1 primary and 1 recurrent cCIS, as well as 10 117
primary and 2 recurrent invasive cSCC resected at KKESH between 2005 and 2010. In some of 118
the recurrent cases, cryotherapy had been used in addition to surgery, but no prior chemotherapy 119
exposure was documented. Five of the patients were between 45 and 65 years of age, with the 120
remainder over 65 years old; all but one patient were males. The majority of tumors were well 121
vascularized, and most had invaded into the cornea, sclera or orbit. None of the patients were 122
known to be immunosuppressed or have a history of HPV infection. Clinical features are 123
summarized in Table 1. 124
125
Recurrent DNA alterations detected by array CGH 126
Array CGH analysis was successful in 12 cases, while in 2 tumors DNA extracted was 127
insufficient for this testing, as indicated in Table 1. All of the tumors had DNA copy number 128
abnormalities, and a graphical representation of the regions of gain (red) and loss (green) among 129
the entire cohort is shown in Figure 1. In Figure 2, representative data from selected single cases 130
are shown. The changes illustrated include gains in chromosome 2 (p arm, case #8) and 131
chromosome 3 (q arm, case #6). Representative losses in chromosome 2 (terminal part of the q 132
arm, case #8), chromosome 3 (p arm, case #6), chromosome 11 (part of the p-q arms, case #12), 133
chromosome 13 (q arm, case #6), are also shown. Each tumor sample was analyzed twice using a 134
dye swap protocol, and only alterations identified in both experiments were included in the 135
Tables 2, 3, and S1-S4. 136
137
8
The number of hemizygous or homozygous DNA losses ranged from 1 to 23 per tumor, 138
while gains/amplifications ranged from 1 to 15 (Table 2, 3). These ranged from small changes to 139
entire chromosomal arms. The combined gains and losses varied from 3 to 34 per tumor (mean 140
15). Case #10, which only had 2 gains and 1 loss, was distinct microscopically, with adnexal 141
features. The precise extent of the regions of DNA gain and loss, as well as the amplitude of the 142
changes identified, is summarized in Tables S1 and S2. 143
Large regions of DNA gain or amplification including all of chromosomal arm 3q were 144
identified in 3 cases, including two cCIS and one cSCC, while 5p was gained or amplified in 2 145
tumors. Additional large regions of gain or amplification involving either one or both 146
chromosomal arms were noted in only single tumors at chromosome 1q, 8q, 9q, and 20 (Table 147
S1). The locus 3q22.3-3q28 was found to be amplified in 4 out of 12 cases (33%), and 2p14-148
2p25.2 gain was detected in 3 out of 12 cases (25%). Gains in 2 of the 12 tumors were noted at 149
6p22.1-6p25.2, 6p12.1 or 13q12.12-13q12.3. Finally, the locus 1p31.1 was found to be amplified 150
in three cases and lost in two. Among the regions smaller than 1Mb of increased DNA copy 151
number, the commonest recurring alteration was identified at 8p11.22, which was amplified in 9 152
of 12 (75%) cases. We also examined two frozen normal bulbar conjunctival specimens from 153
adult autopsy patients, and did not identify any large regions of gain or loss. No amplification of 154
8p11.22 was noted in either of these normal controls, but low level DNA gains were found at this 155
locus. 156
Large recurring regions of loss, encompassing one entire chromosome arm, included 3p, 157
4p, 13q, 14q and 17p, with additional large regions lost in only single tumors (Table S2). More 158
focal regions of DNA loss greater than 1Mb in size were present in more than one case at 159
4q34.3-4q35.2, 5q11.1-5q14.3, 8p21.2-8p23.2, 9p13.2-9p24.2, 11p15.2-11p15.4, 12p13.2-160
9
12p13.32, 18q12.3-18q22.3. Recurring small losses (less than 1Mb) were located at 14q13.2 and 161
22q11.23, which were both lost in 5 out of the 12 samples (42%), 12p13.31, lost in 4 of the 12 162
samples (33%), and 8p11.22 lost in 3 of the 12 samples (25%). None of these focal DNA losses 163
were found in the normal bulbar conjunctival specimens. The most common recurrent 164
homozygous deletions greater than 1 Mb included the loci 11p15.4, most of the short arm of 165
chromosome 17 and most of the long arm of chromosome 13, all of which were found to be 166
deleted in 3 out of 12 samples (25%) as shown in Table S2. 167
Interestingly, one of the DNA repair genes associated with Xeroderma Pigmentosum, a 168
condition which predisposes patients to cSCC, is located within the region of loss on 169
chromosome 13q. The 13q33.1 locus encoding XPG/ERCC5 was lost in cases #6, 8, 9. Two 170
other Xeroderma Pigmentosum genes were also found to be lost in single cases: the XPA gene 171
at 9q22.33 (case #8), and the XPE/DDB2 gene at 11p11.2 (case #14). 172
173
Confirmation of gains and losses 174
We used the NanoString platform to confirm chromosomal alterations identified by 175
aCGH (Figure 3). A total of 338 probes were included in the Nanostring karyotype panel, in 176
addition to control probes targeting invariant genomic regions used for normalization. The much 177
lower density of probes as compared to array CGH meant that only very large regions of gain or 178
loss could be identified using this technique. Among larger regions of gain identified by array 179
CGH, 14 out of 26 (54%) were confirmed by all corresponding NanoString probes (complete 180
confirmation), 9 out of 26 (34.5%) were confirmed by some but not all of the corresponding 181
probes (partial confirmation), and 3 were not confirmed (11.5%). For the large deletions, 12 of 182
10
35 were completely confirmed (34.3%), 19 of 35 were partially confirmed (54.3%), and 4 were 183
not confirmed (11.4%), as shown in Figure 3 and Table S3. 184
Representative NanoString analysis for loci on chromosome 3 is shown in Figure 3. We 185
found by array CGH gain of almost the entire 3q arm in samples 1, 3, and 6, while a somewhat 186
smaller region encompassing more than half of the arm (3q22.3-3q28) was amplified in sample 187
12 (Figure 3, central panel). According to the NanoString analysis, all probes on the 3q arm were 188
gained in samples 1 and 6 (complete confirmation) while half of the relevant probes showed 189
significant gains (copy number estimate values greater than 2.5) in sample 3 (partial 190
confirmation). All of the relevant probes within the smaller region of gain identified in case #12 191
were also confirmed using the NanoString platform (Figure 3, lower panel). No NanoString 192
probes were located in proximity to the recurring gain that we identified by array CGH at 193
8p11.22, thus we had to further analyze this locus using other methods. 194
195
196
Analysis of 8p11.22 locus 197
The most frequent genomic alteration was observed on chromosome 8 (Figure 4A), 198
where the locus 8p11.22 was amplified in 9 tumors out of 12 (75%). Interestingly in the other 3 199
cases this locus appeared to be at least partially lost. The region contains a group of genes coding 200
for “a disintegrin and metalloprotease” (ADAM) proteins, which are peptidases involved in the 201
shedding and activation of oncogenic receptors, in tumor formation and cell migration. In 202
particular, the locus includes the ADAM1B, ADAM3A, and ADAM5P genes (Figure 4A, right 203
panel). Upregulation of the ADAM3A locus was confirmed at the RNA level by TaqMan® one-204
step quantitative RT-PCR, normalized to 18S mRNA levels (Figure 4B). Normal bulbar 205
11
conjunctival tissue from 2 eyes was used as an additional control. We observed that the 206
ADAM3A gene was at least 100 times more highly expressed in 5 of the 9 samples (case #2, 3, 207
7, 12, 13) where we detected high-level amplification by aCGH, as compared to case #1 and #5, 208
which showed genomic loss in this locus by aCGH. There was insufficient RNA to analyze 209
samples #6, 8, 9, 10 by real time PCR. These 5 tumors with DNA amplification at the locus 210
8p11.22 also showed mRNA levels of ADAM3A about 100 times higher than non-neoplastic 211
conjunctiva. One tumor (Case #14) was found to have amplification of 8p11.22 by aCGH, but 212
did not show increased expression of ADAM3A. Quantitative RT-PCR was also performed to 213
determine the expression of ADAM5P gene, which maps in a region partially included in the 214
8p11.22 region of gain. However, in contrast to ADAM3A, ADAM5P mRNA was not detected 215
in the tumor samples (data not shown). 216
217
218
219
DISCUSSION 220
OSSN is most common in countries near the equator with increased exposure to sunlight, 221
suggesting a role for DNA damage due to UV exposure.14,15
The incidence in the United States is 222
0.03 cases per 100,000 persons, while in Australia it is 1.9 cases per 100,000 persons.16
223
Conjunctival SCC is also common in Saudi Arabia, with one report noting that most patients 224
spent significant time outdoors, and that advanced cases requiring orbital exenteration were 225
common.17
A more recent study reviewing the KKESH tumor registry over the last three decades 226
found that cSCC incidence has remained steady, and that it still represents the most common 227
ocular malignancy diagnosed in adults at this tertiary eye hospital.18
228
12
Using aCGH, we identified a number of recurring chromosomal aberrations in the 229
analysis of 10 cSCC and 2 cCIS specimens. To our knowledge, no detailed chromosomal 230
analyses of OSSN have been published, thus we are not able to compare these results to prior 231
studies. However, we found that in 4 of the 12 tumor samples there was loss of one or more loci 232
containing three of the eight genes responsible for the repair of the UV-induced DNA defects 233
and altered in Xeroderma Pigmentosum. To the best of our knowledge, the genes XPA, 234
XPE/DDB2, and XPG/ERCC5 have not been previously examined in sporadic cSCC, and their 235
loss suggests DNA repair defects may also contribute to tumor formation or progression in non-236
syndromic cases. 237
Less direct evidence implicating changes to DNA repair were also identified. At 238
chromosome 22q11.23, five tumors showed DNA loss and two DNA gain. Deletions of 239
22q11.23 have previously been reported in cervical squamous cell carcinoma, chronic 240
lymphocytic leukemia, and ovarian adenocarcinoma.24-26
Interestingly this locus contains several 241
genes involved in the glutathione metabolism, including GSTT1 (Glutathione S-Transferase ), 242
whose deletion has been found in ovarian, cervical and endometrial carcinoma cells.27,28
Such 243
deletion has been reported to predispose to mutations in p53 gene in breast cancer.29
Alterations 244
in p53 expression have also been detected in OSSN, and p53 is known to play a crucial role in 245
the nucleotide excision repair (NER), a pathway for repair of UV-induced DNA damage.30,31
246
Thus these changes may relate to the defects in Xeroderma Pigmentosum genes described above. 247
A number of other regions of gain and loss we identified have been implicated in the 248
pathogenesis of other tumor types. For example, chromosome 6p21.32 was lost in 3 out of 12 of 249
our cases. Deletions including this region have also been reported in nasopharyngeal 250
carcinoma.19,20
Chromosome 14q13.2 was lost in 4 of the 12 OSSN samples (33%), and this 251
13
region contains a number of genes, including PAX9 and FOXG1, that play key roles in normal 252
development and carcinogenesis.21
The locus 12p13.31 was deleted in 4 of the 12 samples and 253
gained in 3. Interestingly the region 12p13 has been found to be genetically unstable and fragile 254
in patients with lymphoid and myeloid hematologic malignancies.22
We also found that the locus 255
1p31.1 was lost in two samples and gained in another three. High levels of loss of heterozygosity 256
(LOH) were found in 1p31 locus, along with other regions in the p arm of chromosome 1, in 257
different human solid tumours.23
258
We also examined if any DNA changes in our cases could be linked to cutaneous 259
squamous cell carcinoma (SCC) or HPV-associated cervical and oropharyngeal carcinomas, and 260
a summary of relevant recurring chromosomal abnormalities is shown in supplemental Table 261
4.32-42
Similarities include DNA gains that we observed in 3q22.3-3q28 and in 5p, as well as 262
DNA losses in 9p, 13q, 17p and 18q, which are also found in cutaneous SCC.34
A smaller 263
number of changes, which were similar in our tumors and HPV-associated neoplasms outside the 264
eye are also listed in this supplementary table. 265
Our study was too small to tightly link clinical features with genetic changes. However, 266
one of the four recurrent tumors, case #12, had the highest amount of DNA gains (15) and the 267
second highest amount of DNA losses (19), thus an increased number of DNA alterations may in 268
some cases be associated with more aggressive clinical behavior or tumor progression. Larger 269
studies which include cases drawn from other ethnic groups and geographical regions will need 270
to be performed to address this issue, as well as to confirm our genetic observations in this single 271
institution cohort. 272
The most frequent chromosomal variation was observed on chromosome 8, where 273
8p11.22 was amplified in 9 tumors (75%). This region contains a group of genes coding for 274
14
ADAM proteins known to be involved in the activation of oncogenic receptors and tumor 275
formation and spread.43
ADAMs are transmembrane proteins that contain a disintegrin and a 276
metalloprotease domain, and have both cell adhesion and protease activities. Their functions 277
include activation of membrane receptors (Notch and HER2), cell migration, as well as cytokine 278
and growth factor shedding.44-47
The ADAM gene family includes 29 members, but the function 279
of most of the ADAM gene products is still unknown. 280
The amplicon that we found includes parts of the ADAM1B, ADAM3A, and ADAM5P 281
loci (Figure 4A), and is near to the ADAM9 locus recently found to be commonly amplified in 282
oral mucosal SCC.48
Using Taqman RT PCR, we confirmed an approximately 100-fold or 283
greater increased expression from the ADAM3A locus in amplified cases as compared to deleted 284
ones or normal conjunctiva. We did not detect expression of ADAM5P in the tumors. 285
Interestingly, 8p11.22 was lost in the 3 cases without amplification of the locus. Homozygous 286
loss of ADAM3A locus has been reported in pediatric high-grade glioma, but the biological 287
function of the deletion in this tumor type has not been identified.49
It is not clear why this locus 288
would be either lost or amplified in all cases examined. 289
In summary we found frequent DNA copy number alterations in conjunctival SCC 290
samples, including gains and losses of large DNA regions and more focal changes encompassing 291
a limited number of loci. The most frequent focal homozygous loss was found in 22q11.23, 292
whose deletion predisposes to p53 mutation in breast cancer. Loss of loci encoding 3 genes 293
altered in Xeroderma Pigmentosum was also found in 4 cases. The predominant amplification 294
was observed at the 8p11.22 locus, where ADAM3A is located, and this family of genes has 295
recently been implicated in mucous membrane carcinomas. Our studies thus confirm the 296
importance of DNA repair in cSCC, and suggest that ADAM proteases might also play a role. 297
15
ACKNOWLEDGEMENTS 298
This study was supported by King Khaled Eye Specialist Hospital (KKESH) – Wilmer Eye 299
Institute (WEI) Collaborative Research Grant and by Research to Prevent Blindness (RPB) 300
Wilmer Eye Institute. Samples quality assessment and array CGH analysis were conducted at 301
The Sidney Kimmel Cancer Center Microarray Core Facility at the Johns Hopkins University, 302
supported by NIH grant P30 CA006973 entitled Regional Oncology Research Center. 303
304
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473
19
LEGENDS 474
Table 1. Clinical and pathological features 475
476
Table 2. Chromosomal gains detected in the Ocular Surface Squamous Neoplasia samples. 477
Predicted amplifications are shown in bold. 478
479
Table 3. Chromosomal losses detected in the Ocular Surface Squamous Neoplasia samples. 480
Predicted deletions are shown in bold. 481
482
Figure 1. Graphical representation of all chromosomal changes. The regions of DNA gain 483
(red) and loss (green) among the entire cohort are shown, with the magnitude of the change 484
representing the number of cases with alterations in each region. 485
486
Figure 2. Chromosomal alterations in selected single cases. Each dot corresponds to a single 487
probe, with deflections to the left of the “0” midline representing reduced hybridization ratio of 488
tumor DNA to that probe, and deflections to the right representing an increased hybridization 489
ratio of tumor DNA to that probe. These correspond to loss and gain of DNA at the respective 490
chromosomal loci. Log2 [Tumor/Normal] for each probe in a chromosome region was 491
determined and values between 0.75 and 1.0 were considered genomic gain, >1.0 amplification, 492
between -0.75 and -1.0 hemizygous loss, and < -1.0 homozygous deletion. The thin pairs of lines 493
to the left or right of each alteration indicate that such loss or gain was identified in both of the 494
paired analyses that were performed for each DNA sample according to the dye swap protocol. 495
496
20
Figure 3. Confirmation of chromosomal changes using Nanostring. Comparison between the 497
CNV alterations identified by array CGH and NanoString platform shows that among the larger 498
regions of DNA gain identified by aCGH, 14 out of 26 (54%) were confirmed by all 499
corresponding NanoString probes (complete confirmation), 9 out of 26 (34.5%) were confirmed 500
by some but not all of the corresponding probes (partial confirmation) and 3 were not confirmed 501
(11.5%). For the large deletions, 12 out of 35 were completely confirmed (34.3%), 19 of 35 were 502
partially confirmed (54.3%), and 4 were not confirmed (11.4%). After normalization, Nanostring 503
copy number estimate values between 2.5 and 3 were scored as genomic gains, greater than 3 as 504
high-level amplifications, between 1 and 1.5 as hemizygous deletions, and lower than 1 as 505
homozygous deletions. 506
507
Figure 4. mRNA expression at the 8p11.22 locus. The most frequent DNA amplification was 508
detected at 8p11.22. (A) Genomic aberration summary of chromosome 8 is shown for all the 509
samples analyzed, with dye swap performed for each of them. Green indicates regions of loss 510
and red regions of gain. The heatmap shows DNA gain in the locus 8p11.22 in 9 of the 12 511
samples. The gene view on the right shows that such amplification involves ADAM3A and part 512
of the ADAM5P genes. (B) Upregulation of ADAM3A as compared to non-neoplastic 513
conjunctiva was confirmed in cases #2, 3, 7, 12, 13 at the RNA level using TaqMan® one-step 514
quantitative RT-PCR normalized to 18S mRNA. Normal bulbar conjunctival tissue was used as 515
internal control. For cases #6, 8, 9, 10 there was not enough RNA to perform the real time PCR. 516
517
Supplementary Table S1. List of all the chromosome regions where DNA gains were detected 518
by aCGH, along with the genomic location (expressed in bp), size (1Mb = 106 bp), case number 519
21
and amplitude, which represents the log2 [Tumor/Normal]. Predicted amplifications (amplitude 520
>1) are shown in bold. 521
522
Supplementary Table S2. List of all the chromosome regions where DNA losses were detected 523
by aCGH, along with the genomic location (expressed in bp), size (1Mb = 106 bp), case number 524
and amplitude, which represents the log2 [Tumor/Normal]. Predicted homozygous deletions 525
(amplitude <1) are shown in bold. 526
527
Supplementary Table S3. Summary of the large regions (> 1Mb) of DNA gains and losses that 528
were found by aCGH and partially confirmed (A,C) or not confirmed (B,D) by NanoString 529
analysis. 530
531
Supplementary Table S4. Recurring genetic abnormalities observed in cutaneous squamous cell 532
carcinoma (SCC), HPV-associated carcinomas and other malignancies compared to 533
chromosomal gains (A) and losses (B) detected in the Ocular Surface Squamous Neoplasia 534
(OSSN) samples. 535
536
537
538
539
540
541
542
22
Table 1. Clinical and pathological features of the Ocular Surface Squamous Neoplasia 543
specimens. 544
545
Case Array CGH
Nano String
Age group Sex
Size group Vascularized Histopathology
Invasion Outside
Conjunctiva
Subsequent Recurrence
Immuno- suppression
1 Yes Yes 3 M 4 Yes cCIS Cornea No Unknown
2 Yes Yes 4 M 5 Yes cSCC Cornea Orbit No No
3 Yes Yes 4 M 4 Yes cSCC Cornea No No
4 No No 4 M 5 No cSCC
Cornea Sclera Orbit No Unknown
5 Yes Yes 3 F 5 Yes cSCC Cornea Orbit No No
6 Yes Yes 4 M 4 Yes Recurrent
cCIS Cornea Yes No
7 Yes No 3 M Unknown Unknown Unknown Unknown No Unknown
8 Yes Yes 4 M 4 Yes cSCC Cornea No No
9 Yes Yes 3 M 5 Unknown cSCC Cornea Orbit No No
10 Yes Yes 3 M 5 Unknown
cSCC with focal adnexal
differentiation Orbit No No
11 No Yes 4 M 5 Yes Recurrent cSCC Sclera Yes No
12 Yes Yes 4 M 5 Yes cSCC Cornea Yes No
13 Yes Yes 4 M 5 Yes
Poorly differentiated recurrent cSCC
Cornea Sclera Orbit No Unknown
14 Yes Yes 4 M 5 Yes Recurent cSCC Sclera Orbit Yes No
Age groups: 3 (45-65 years), 4 (>65 years); Sex: M – male, F – Female; Size group: 4 (5-10mm), 5 546
(>10mm); cSCC – conjunctival squamous cell carcinoma, cCIS - conjunctival carcinoma in situ 547
548
549
550
23
Table 2. Chromosomal gains detected in the Ocular Surface Squamous Neoplasia samples. 551
Case # Genomic gains N. alterations
1 5p; 7q11.22; 22q11.23 3q
4
2 1p31.1; 12p13.31; 20q13.33; 8p11.22 4
3 3q; 6p12.1 8p11.22
3
5 16p11.2 1
6 9q; 16p12.3-16p13.2; 18p11.31 1q; 3q; 8p11.22; 9p21.3 7
7 7p14.1-7p22.2; 12p13.31 8p11.22 3
8 2p14-2p25.2; 6p22.1-6p25.2; 8p21.3; 19q13.41-19q13.43; 20 6q22.31-6q27; 13q12.12–13q12.3; 13q14.11 8
9 2p14-2p25.2; 16p11.2; 19q13.2-19q13.31 8p11.22 4
10 1p31.1 8p11.22 2
12 1p12-1p35.1; 2p14-2p25.2; 3q22.3-3q28; 7p22.2; 9p24.2; 10p12.33-10p15.2; 12p13.31; 16q22.2-16q23.3; 20q13.13-20q13.33; 22q11.23;
6p12.1-6p21.2; 6p24.3-6p25.2; 8p11.22; 13q12.12 -13 q12.3; 17q12 15
13 8q; 14q21.3 5p; 8p11.22 4
14 6p22.1-6p25.2; 11p12 -11p15.1; 17q22-17q25.2 8p11.22; 11p11.2-11p12 5
552
553
554
24
Table 3. Chromosomal losses detected in the Ocular Surface Squamous Neoplasia samples. 555
Case # Genomic losses N. alterations
1 1q21.2; 2p23.1; 2q22.1; 9p23-9p24.1; 11q11; 14q13.2-14q21.3;
14q23.3; 15q11.2-15q13.1; 18q12.2-18q12.3; 21q21.1; 1p31.1; 8p11.22; 11p15.4; 20p12.1; 22q12.3
15
2 1q21.2; 10q11.22;
3p22.3; 7p12.1; 11q11 5
3 2p22.1; 22q11.23 2
5 3p21.2; 5q13.2; 5q23.2; 7q11.23; 7q22.3; 10q21.3; 10q24.32; 12q13.3; 12q24.31;
12q23.3-12q24.1; 14q13.2; 15q21.2; 15q22.31; 16p13.12; 16q22.2; 17q21.31; 17q22; 22q12.2; 3q21.2; 3q26.1; 6p22.1; 8p11.22; 22q11.23
23
6 4p; 4q31.21-4q34.3; 6p11.2; 17p;
3p; 6p21.32; 8p21.2-8p23.2; 13q; 22q11.23 9
7 14q; 18q12.3-18q22.3; 1q31.3; 7p12.1; 11q11
5
8 2q22.1; 2q24.2; 3p24.3; 9p13.2-9p24.2; 18q12.3-18q22.3;
2q34-2q37.2; 3q26.1; 7p; 7q31.1-7q36.2; 8p11.22; 9q; 10p; 11p15.2-11p15.4; 12p13.2-12p13.32; 13q14.12-13q33.3; 14q12-14q21.1; 17p12; 22q
18
9 1q21.2; 4p; 6p22.1; 9p13.2-9p24.2; 13q; 14q; 17p; 19q13.33;
1p31.1; 3p; 6p21.32 11
10 12p13.31 1
12 4q34.3-4q35.2; 4q22.1; 5q11.1- 5q14.3; 6p22.3; 7p12.1-7p15.2; 9p21.3; 10q11.22; 10q22.2; 10q23.31-10q26.2; 11p13-11p15.4; 11q23.2-11q24.3; 16p12.1-16q12.3;
16q12.2; 18; 20p12.1-20p12.3; 21q22.2; 21q21.2-21q22.11; 8p21.2-8p23.2; 14q21.1 19
13 9p21.3; 17q21.31; 19p13.11-19p12; 12p13.31; 22q12.3 5
14 5q; 9p13.2-9p24.2; 11p11.12-11p12; 12p; 6p21.32; 11p15.2-11p15.4; 22q11.23 7
556
557
558
25
Supplementary Table S1. Regions of gain. Predicted amplifications are shown in bold. 559
Chromosome region Genomic location Size (Mb) Case # Amplitude
1p12 – 1p35.1 1p31.1
1q
33099302 - 121322377 72768855 - 72795480
145264630 - 249212668
88.22
0.026
103.94
#12
#2,10 #6
0.75 – 1
1 – 1.5 1
2p14 – 2p25.2 30341 - 65502509 65.47 #8,9,12 0.75 - 1
3q 3q22.3 – 3q28
93575285 - 197837049 135247918 - 197837049
104.26
62.58 #1,3,6
#12 0.75 - 1.5 0.75 - 1
5p 26142 - 46100367 46.07 #1,13 0.75 - 1
6p12.1 – 6p21.2
6p12.1
6p22.1 – 6p25.2
6p24.3 – 6p25.2
6q22.31 – 6q27
38455507 - 58686125
55673790 - 57062142
184779 - 29502309 402016 - 9720048
123246923 - 170890049
20.23 1.38
29.31
9.32
47.64
#12
#3
#8,14
#12
#8
1 – 1.5 0.5 – 1
0.75 – 1
1
1
7p14.1 – 7p22.2
7p22.2
7q11.22
65558 - 44253840 65558 - 7288958
63641029 - 69563017
44.18
7.22
5.92
#7
#12
#1
0.5
0.5
0.5
8p11.22
8p21.3
8q
39249352 - 39362887
19203294 - 20072511
46953844 - 146294098
0.11
0.87
99.34
#2,3,6,7,9, 10,12,13,14
#8
#13
1 – 1.5
0.5
0.75 - 1
9p21.3
9p24.2
9q
21188940 - 21208560
1615613 - 8718674 71048619 - 141018984
0.02
7.10
69.97
#6
#12
#6
1.5
0.5
0.75 – 1
10p12.33 – 10p15.2 136361 - 17735233 17.59 #12 0.5
11p12 – 11p15.1
11p11.2 – 11p12
21516459 - 36842348
43213408 - 43850127
15.32
0.64
#14
#14
0.5 – 1 1
12p13.31 9637323 - 9713425 0.07 #2,7,12 0.75 - 1
13q12.12 – 13q12.3
13q14.11
25373508 - 29753481
40371457 - 42118802
4.38
1.75
#8,12
#8
0.75 – 1.5
1 – 1.5
14q21.3 46116942 - 49629363 3.51 #13 0.75
16p12.3 – 16p13.2
16p11.2
16q22.2 – 16q23.3
215724 - 20851995 32573808 - 33487672
74147464 - 83297838
20.63
0.91
9.15
#6
#5,9
#12
0.75 – 1
0.5
0.75
17q12 17q22 – 17q25.2
31852012 - 33552682 49790717 - 75222975
1.70 25.43
#12 #14
1 0.75 – 1
18p11.31 131700 - 7906550 7.77 #6 0.75 - 1
26
Table S1 cont’d. Regions of gain. Predicted amplifications are shown in bold. 560
561
Chromosome region Genomic location Size (Mb) Case # Amplitude
19q13.2 – 19q13.31
19q13.41 – 19q13.43 43173815 - 43690041
48941148 - 59092570 0.52
10.15 #9
#8 0.5
0.75
20
20q13.13 – 20q13.33
20q13.33
67778 - 62904501
48903042 - 62186836
60430656 - 60563664
62.84
13.28
0.13
#8
#12
#2
0.5 – 1
0.5
0.5
22q11.23 24347959 - 24390254 0.04 #1,12 0.75
562
563
564
565
566
567
568
569
570
571
572
573
574
575
27
Supplementary Table S2. Regions of loss. Predicted homozygous deletions are shown in bold. 576
Chromosome region Genomic location Size (Mb) Case # Amplitude
1p31.1
1q21.2
1q31.3
72768855 - 72795480
149041933 - 149243967 196742735 - 196799302
0.026
0.20
0.06
#1,9
#1,2,9
#7
-2; -3
-0.5
-2
2p23.1
2p22.1
2q22.1
2q24.2
2q34 – 2q37.2
32019286 - 32153941
38907085 - 38932067
141882067 - 142017750
154906204 - 154988848
211687462 - 243017884
0.13
0.025
0.13
0.08
31.33
#1
#3
#1,8
#8
#8
-0.5
-1
-0.5
-0.5
-1
3p
3p24.3
3p22.3
3p21.2 3q21.2
3q26.1
73914 - 90004020 22567818 - 22605208
36271361 - 36337759
51474959 - 51645105 122687754 - 124238386
162514534 - 162619141
89.93
0.04
0.07 0.17 1.55
0.10
#6,9
#8
#2
#5
#5
#5,8
-1
-1
-0.75
-0.5
-1
-2
4p
4q22.1
4q31.21 – 4q34.3 4q34.3 – 4q35.2
71552 - 45038836
91321265 - 91813218
141527109 - 190874516
179266646 - 190874516
44.96
0.49
49.35
11.60
#6,9
#12
#6
#12
-0.75
-0.5
-0.5 -0.5
5q 5q11.1 – 5q14.3
5q13.2
5q23.2
49584189 - 180684501 49584189 - 88348206
68369526 - 70369959
125882578 - 126119218
131.10
38.76
2.00
0.23
#14
#12
#5 #5
-0.5 -0.5
-0.3
-0.3
6p22.3
6p21.32
6p22.1
6p11.2
19102216 - 26642819
32480027 - 32521929
29854870 - 29873992
57270000 - 57503312
7.54
0.04
0.02
0.23
#12
#6,9,14
#5,9
#6
-0.5
-2
-0.75
-0.5
7p
7p12.1 – 7p15.2 7p12.1
7q31.1 – 7q36.2 7q11.23
7q22.3
42976 - 55022636
23554100 - 55022636 53461023 - 53583184
110372588 - 159075079 73492241 - 75546088
104928616 - 105216153
54.97
31.46
0.12 48.7
2.05
0.29
#8
#12
#2,7
#8
#5
#5
-1
-0.75
-1
-1
-0.5
-0.3
8p21.2 – 8p23.2
8p11.22 193250 - 25314730 39237438 - 39322744
25.12 0.08
#6,12
#1,5,8
-1
-1; -1.5
9p13.2 – 9p24.2
9p23 – 9p24.1
9p21.3 9q
204193 - 39140270
7809142 - 9505954
21957735 - 21968099 69194069 - 140994780
38.93
1.69
0.01 71.80
#8,9,14
#1
#12,13
#8
-0.75
-0.35
-0.75
-1
577
28
Table S2 cont’d. Regions of loss. Predicted homozygous deletions are shown in bold. 578
Chromosome region Genomic location Size (Mb) Case # Amplitude
10p 10q23.31 – 10q26.2
10q11.22
10q22.2
10q21.3
10q24.32
136361 - 37468455 92748093 - 135372492
46984913 - 47138325
75533236 - 75641567
70110227 - 70726973
104687140 - 105169607
37.33
42.62
0.15
0.11
0.62
0.48
#8 #12
#2,12
#12
#5
#5
-1 -0.5
-0.5
-0.5
-0.3
-0.3
11p13 – 11p15.4 11p15.1 – 11p15.4 11p15.2 – 11p15.4 11p11.12 - 11p12
11p15.4 11q11
11q23.2 – 11q24.3
210300 - 32589746 210300 - 21495396
210300 - 13768924
37442379 - 48645948 7817491 - 7826827
55377910 - 55450788 110895304 - 134927114
32.38
21.28
13.56
11.2 0.009
0.07 24.03
#12
#14
#8
#14 #1
#1,2,7
#12
-0.75
-0.5
-0.75
-0.5
-1
-0.5; -2 -0.5
12p 12p13.2 – 12p13.32
12p13.31 12q23.3 – 12q24.1
12q24.31
12q13.3
163593 - 33507921 163593 - 12356288
9637323 - 9672658 105322025 - 109832355
121919949 - 123321611
57704197 - 57823931
33.34
12.19
0.03 4.5
1.4
0.12
#14 #8
#10,13
#5
#5
#5
-0.75 -1
-0.75
-0.75
-0.3
-0.3
13q
13q14.12 – 13q33.3 19504053 - 115077940 19535444 - 115077940
95.57
95.54 #6,9
#8 -1; -0.75
-1
14q
14q12 – 14q21.1
14q13.2 – 14q21.3
14q13.2
14q21.1 14q23.3
19376762 - 107230635 29538004 - 43470527 36473822 - 43059336
35565871 - 35724752
37810624 - 38191313 67069527 - 67746168
87.85
13.93
6.58
0.16
0.38 0.67
#7,9
#8
#1 #5,12
#12
#1
-0.75
-1
-0.5
-0.5
-1
-0.5
15q21.1 – 15q22.31
15q11.2 – 15q13.1
15q21.2
15q22.31
50659717 - 66828846
24085574 - 30001259
50659717 - 51207771
66580165 - 66828846
16.16
5.91
0.55
0.25
#5
#1
#5
#5
-0.3
-0.3
-0.3
-0.3
16p12.1 – 16p12.3
16p13.12
16q12.2
16q22.2
19112966 - 31693787
14649516 - 16171621
46500741 - 62907202 68599611 - 69936199
12.58
1.52
16.40
1.33
#12
#5
#12
#5
-0.5
-0.3
-0.5
-0.3
17p
17p12
17q21.31
17q22
76263 - 21255056 8434728 - 20219464
44188441 - 44297143
56694946 - 58078230
21.18
11.78
0.11
1.38
#6,9
#8 #3,5,13
#5
-0.5
-1 -0.5
-0.3
579
29
Table S2 cont’d – Regions of loss. Predicted homozygous deletions are shown in bold. 580
Chromosome region Genomic location Size (Mb) Case # Amplitude
18
18q12.3 – 18q22.3
18q12.2 – 18q12.3
118760 - 78010032
40402263 - 77982126 36925871 - 37339977
77.89
37.57
0.41
#12
#7,8
#1
-0.5
-0.75
-0.5
19p13.11 – 19p12
19q13.33
19486091 - 21902958
49272728 - 50126589 2.42
0.85 #13
#9 -0.5
-0.3
20p12.1 – 20p12.3
20p12.1 129916 - 18262941
14416991 - 15259324 18.33
0.84 #12
#1
-0.5
-0.5
21q21.2 – 21q22.11
21q22.2
21q21.1
20610978 - 35068667
41354418 - 42222204
22756905 - 23415306
14.46
0.87
0.66
#12
#12
#1
-0.3
-0.5
-0.5
22q
22q11.23
22q12.3 22q12.2
16133474 - 51186249
24371205 - 24390254
39359112 - 39385485
31756218 - 32202127
35.05
0.02
0.03
0.44
#8
#3,5,6,14
#1,13
#5
-1
-1; -1.5
-1
-0.3
581
582
583
584
585
586
587
588
589
590
30
Supplementary Table S3. Summary of the large regions (> 1Mb) of DNA gains (A,B) and losses 591
(C,D) that were partially or not confirmed by NanoString analysis. 592
593
31
Supplementary Table S4. Recurring genetic abnormalities observed in cutaneous squamous 594
cell carcinoma (SCC), HPV-associated carcinomas and other malignancies compared to 595
chromosomal gains (A) and losses (B) detected in the Ocular Surface Squamous Neoplasia 596
(OSSN) samples. 597
598
599
Table S4A 600
Chromosomal gains detected in the OSSN samples (frequency)
Chromosomal abnormalities in cutaneous SCC and other malignancies
Chromosomal abnormalities in HPV-associated carcinomas
1p31.1 (3/12) Lung SCC, Ref. 32 Solid tumours, Ref. 23
3q22.3 – 3q28 (4/12) Lung SCC, Ref. 32 Head and neck squamous cell carcinoma, Ref. 33 Skin SCC, Ref. 34
Cervical cancer, Ref. 37 Cervical cancer , Ref. 38 Cervical cancer , Ref. 39
3q12.2 – 3q22.3 (3/12) Cutaneous SCC, Ref. 35 Cutaneous SCC, Ref. 34
5p (2/12) Cutaneous SCC, Ref. 34
6p22.1 – 6p25.2 (2/12) Cervical cancer, Ref. 37
12p13.31 (3/12) Hematologic tumors, Ref. 22
13q12.12 – 13q12.3 (2/12) Lung SCC, Ref. 36
20q13.13 – 20q13.33 (2/12) Head and neck squamous cell carcinoma, Ref. 33
601
602
603
604
605
606
607
32
Table S4B 608
Chromosomal losses detected in the OSSN samples
(frequency)
Chromosomal abnormalities in cutaneous SCC and other malignancies
Chromosomal abnormalities in HPV-associated carcinomas
1p31.1 (2/12) Solid tumours, Ref. 23 Cutaneous SCC, Ref. 35
3p (2/12) Cutaneous SCC, Ref. 35 Head and neck squamous cell carcinoma, Ref. 40 Cutaneous SCC, Ref. 34
Cervical cancer , Ref. 39
6p21.32 (3/12) Nasopharyngeal carcinoma, Ref. 19,20
7p12.1 – 7p15.2 (2/12) Cervical cancer , Ref. 39
8p11.22 (3/12) Pediatric high-grade glioma, Ref. 38
9p13.2 – 9p24.2 (3/12) Head and neck squamous cell carcinoma, Ref. 40 Cutaneous SCC, Ref. 34
12p13.31 (4/12) Hematologic tumors, Ref. 22
13q14.12 – 13q33.3 (3/12) Cutaneous SCC, Ref. 35 Cutaneous SCC, Ref. 34 13q33.1 is NER DNA mismatch repair gene XPG/ERCC5, Ref. 41
14q13.2 (4/12) Holoprosencephaly, Ref. 21
16q22.2 (2/12) Oropharynx SCC, Ref. 42
17p12 (3/12) Cutaneous SCC, Ref. 34
18q12.3 – 18q22.3 (3/12) Cutaneous SCC, Ref. 34
22q11.23 (5/12) Cervical squamous cell carcinoma, chronic lymphocytic leukemia, and ovarian adenocarcinoma, Ref. 24-26
609
Figure 1
Figure 2
Chr. 2, Case #8 Chr. 3, Case #6
Chr. 11, Case #12 Chr. 13, Case #6
AMPLIFICATIONS >1Mb
Total = 26
14 confirmed (54%)
9 partially confirmed (34.5%)
3 not confirmed (11.5%)
DELETIONS >1Mb
Total = 35
12 confirmed (34.3%)
19 partially confirmed (54.3%)
4 not confirmed (11.4%)
Figure 3
Chr. 3q
Case # 1 3 6 12
: gain
Copy
Number
Es!mate 1 2 3 5 6 8 9 10 11 12 13 14
3q13.11 3.3 2.05 2.54 1.28 3.92 2.16 2.24 2.24 1.84 2.15 2.33 2.05
3q13.32 3.09 2.2 2.28 2.5 3.91 2.35 2.02 2.14 2.07 2.07 2.32 2.12
3q21.3 2.87 1.69 2.09 1.65 3.61 1.45 1.83 1.83 1.67 2.03 1.95 1.7
3q23 3.76 2.2 2.65 2.32 4.11 2.55 2.42 2.42 2.11 3.14 2.47 2.44
3q25.2 3.24 2.44 2.77 2.22 5.15 2.47 2.39 2.58 2.3 2.98 2.44 2.14
3q26.1 2.59 2.23 2.55 2.44 4.5 2.14 2.42 2.33 1.82 2.84 2.43 2.33
3q26.32 2.66 1.96 2.24 3.02 4.11 1.04 1.95 1.91 1.76 2.65 2.18 1.94
3q28 2.62 2.01 2.23 2.05 3.91 1.85 2.19 1.93 1.81 2.75 2.08 2.03
Copy Number Estimate = 2.5 - 3.0 genomic gain
> 3.0 high-level amplification
= 1.0 - 1.5 hemizygous deletion
< 1.0 homozygous deletion
B
Figure 4
A
Case #: 12 14 14 13 13 12 10 10 9 9 8 8 7 7 6 6 5 5 3 3 2 2 1 1
Log2 [Tumor/Normal]
-4 -3 -2 -1 0 1 2 3 4
mR
NA
AD
AM
3A
/18
S
8p11.22 Amplification - + + - + + + + -
Case # 1 2 3 5 7 12 13 14 Norm.
Conj.
mRNA levels of ADAM3A