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Ophthalmology. Author manuscript; available in PMC 2017 January 01. Published in final edited form as: Ophthalmology. 2016 January ; 123(1): 216–217.e1. doi:10.1016/j.ophtha.2015.06.049.

Whole exome profiling of ocular surface squamous neoplasia ANAT GALOR, MD, MSPH1,2, CAROL L. KARP, MD2, DAVID SANT, BS3, MADHURA JOAG, MD2, NABEEL SHALABI, MD2, CHRISTOPHER B. GUSTAFSON, BS3, and GAOFENG WANG, PHD2,3 1Department

of Ophthalmology, Miami Veteran Affairs Medical Center, Miami FL 33136

2Department

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of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami FL 33136 3John

P. Hussman Institute for Human Genomics, University of Miami, Miller School of Medicine, Miami FL 33136

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Ocular surface squamous neoplasia (OSSN) represents a spectrum of diseases ranging from mild dysplasia to invasive squamous cell carcinoma. OSSN can be successfully managed with surgical excision or with medical therapy. Recently, interferon-α-2b (IFNα-2b) treatment has been established as a standard treatment option for OSSN, eliminating the need for surgical excision. However, approximately 15% of tumors do not respond to the IFNα-2b therapy. 1 It remains unclear which tumor-specific factors may affect treatment response and/or course after treatment. This information is important as it can help individualize therapy. For example, physicians may proceed directly to surgery, or use a different agent, in patients in whom IFNα-2b is unlikely to be effective. Understanding the genetic variability of OSSN may provide important information on initial response to a specific therapy and subsequent patient course. Limited data is available, however, on genetic mutations associated with OSSN. The aim of this study was to apply the powerful whole exome sequencing technology to identify mutations in OSSN tumors and correlate these variants with clinical features and treatment response. Seven patients with OSSN undergoing excisional biopsy were prospectively recruited for this study. Approval was obtained from the University of Miami Institutional Review Board

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Correspondence: Gaofeng Wang, PhD, 3John P. Hussman Institute for Human Genomics, University of Miami, Miller School of Medicine, Biomedical Research Building, Rm. 608, 1501 NW 10th Ave, Miami, FL 33136, USA. [email protected]. Conflict of Interests: None Financial Disclosure(s): Supported in part by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Clinical Sciences Research and Development’s Career Development Award CDA-2-024-10S (Dr. Galor), NIH Center Core Grant P30EY014801, Research to Prevent Blindness Unrestricted Grant, Department of Defense (DODGrant#W81XWH-09-1-0675 and Grant# W81XWH-13-1-0048 ONOVA) (institutional), The Dr. Ronald and Alicia Lepke Grant, The Ronald and Alicia Lepke Grant, The Lee and Claire Hager Grant, The Jimmy and Gaye Bryan Grant, The Gordon Charitable Foundation and the Richard Azar Family Grant(institutional grants for Dr. Karp) and by a James and Esther King Biomedical Research Award (3KN08) (Dr. Wang). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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and the methods adhered to the tenets of the Declaration of Helsinki and were HIPAAcompliant. All subjects were white, and 3 self-identified as Hispanic. Three patients had a previous history of OSSN; 4 were initially treated with IFNα-2b for the current tumor and subsequently underwent excisional biopsy due to an incomplete or no response to therapy. On histopathological examination, 1 case was graded as moderate dysplasia, 1 as severe dysplasia and 5 as carcinoma in situ (CIS) (Table 1).

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Whole exome sequencing of these 7 OSSN specimens was conducted in the Sequencing Core facility at the University of Miami. Over 1,000 changes in various genes were presented in each individual sample. To identify mutations that potentially underlie OSSN, we applied an online genomic analysis program Genomes Management Application (https:// genomics.med.miami.edu) to exclude less likely causative genes. First, by excluding synonymous variants or variant located within untranslated regions (UTRs), we narrowed down to 1295 variants in 1003 genes. These variants have frequencies less than 0.5% in the population and the Genomic Evolutionary Rate Profiling (GERP, a measurement of the conservation for each nucleotide in the genome) scores are greater than 2.0. Second, we chose the genes represented at least twice in the dataset and ended up with 192 variants in 76 genes. Third, there were 64 variants in 26 genes which were mutated with the frequency >5% in any types of cancers in COSMIC database (http://cancer.sanger.ac.uk/cosmic). Fourth, after literature review, 32 variants in 10 genes were chosen for verification based on their potential functions in cancer. Finally, only 20 variants in 6 genes were confirmed by capillary sequencing (Figure 1, available at http://aaojournal.org).

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The most frequent mutations were identified in genes Titin (TTN, OMIM188840) and Neuron Navigator 2 (NAV2, OMIM607026). Four OSSN samples (#1, #2, #6 and #7) carry 10 unique mutations in TTN, of which one is novel and the rest have reference single nucleotide polymorphism (RS) numbers but with very low frequencies (< 0.50%) in the population. Three unique mutations in NAV2 were identified in 4 samples (#1, #2, #3 and #5). Mutations in the gene FAT atypical cadherin 2 (FAT2, OMIM604269) were shared by three samples (#5, #6 and #7). Two unique mutations in the gene Hepatocyte Growth Factor (HGF, OMIM142409) were found in the two samples (#6 and #7). Two mutations in the gene dynein axonemal heavy chain 8 (DNAH8, OMIM 603337) were discovered in two samples (#1 and #5) and one mutation in the gene CREB Binding Protein (CREBBP, OMIM600140) was shared by two samples (#3 and #4). Overall, 2 genes (TTN, NAV2) were mutated in 4 different samples; 1 gene (FAT2) was mutated in 3 individual samples; and 3 genes (HGF, DNAH8, CREBBP) with mutations were identified in 2 different samples (Table 2, available at http://aaojournal.org).

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We then attempted to correlate mutations in the six genes to clinical features including recurrence, IFNα-2b treatment outcomes, tumor sizes and locations, or pathological stages. We found that the four samples carrying TTN mutations (100%) had previously failed treatment with IFNα-2b. The other 3 tumors, which were not found to carry TTN mutations, had been excised primarily; thus their potential response to medical therapy is not known. Besides this, no clear correlation was identified between mutations in the five genes and any other clinical features including pathologic grade (Table 1).

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To our knowledge, this study is the first to apply whole exome sequencing technology to examine the genomic mutations profiles in OSSN. Previously, TP53 mutations were reported to be frequent in African population, but rare in European population. 2, 3 Consistent with the latter report, our data showed that no mutation in TP53 was identified in this European descendant’s sample.

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Results from our study, while very preliminary, demonstrate that the samples carrying the mutant TTN had a prior history of non-response to IFNα-2b. A weakness of the study, however, is that the three TTN negative tumors were not ever treated with IFNα-2b and thus, we do not know whether they would have responded to treatment. Additionally, there is a possibility that IFNα-2b treatment itself influenced the mutation profile by differentially affecting certain OSSN cell populations. Further, we cannot comment on mutation profiles in invasive OSSN due to the lack of such samples in this study. TTN encodes a gigantic protein titin, which forms a unique filament network mainly in muscle cells. By whole exome sequencing, TTN was identified as one of the five genes recurrently mutated in hairy cell leukemia.4 By analyzing available data of whole exome sequencing of 11 major cancers, one study concluded that TTN is one of the most frequently mutated genes along with TP53 and few other genes in cancers.5 Little is known about how the mutant TTN contribute to the pathogenesis of cancer. In conclusion, the powerful whole exome sequencing uncovered major genomic mutation profiles in OSSN. The mutant TTN was found in tumors with a prior history of non-response to IFNα-2b. Thus, mutant TTN could be a potential prognostic biomarker for OSSN response to IFNα-2b treatment. Further study is warranted to confirm these findings.

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Supplementary Material Refer to Web version on PubMed Central for supplementary material.

References

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1. Galor A, Karp CL, Chhabra S, et al. Topical interferon alpha 2b eye-drops for treatment of ocular surface squamous neoplasia: a dose comparison study. Br J Ophthalmol. 2010; 94(5):551–4. [PubMed: 19493859] 2. Ateenyi-Agaba C, Dai M, Le Calvez F, et al. TP53 mutations in squamous-cell carcinomas of the conjunctiva: evidence for UV-induced mutagenesis. Mutagenesis. 2004; 19(5):399–401. [PubMed: 15388813] 3. Guthoff R, Marx A, Stroebel P. No evidence for a pathogenic role of human papillomavirus infection in ocular surface squamous neoplasia in Germany. Curr Eye Res. 2009; 34(8):666–71. [PubMed: 19899994] 4. Waterfall JJ, Arons E, Walker RL, et al. High prevalence of MAP2K1 mutations in variant and IGHV4-34-expressing hairy-cell leukemias. Nat Genet. 2014; 46(1):8–10. [PubMed: 24241536] 5. Kim N, Hong Y, Kwon D, et al. Somatic mutaome profile in human cancer tissues. Genomics Inform. 2013; 11(4):239–44. [PubMed: 24465236]

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Figure 1.

Chromatogram depicting mutations in the six genes as determined by capillary sequencing of the OSSN tissues.

Author Manuscript Author Manuscript Ophthalmology. Author manuscript; available in PMC 2017 January 01.

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59

33

#6

#7

M

M

M

M

M

F

M

Sex

W/NH

W/NH

W/H

W/NH

W/H

W/NH

W/H

Race/ethnicity

No

No

No

Yes

No

Yes

Yes

Prior OSSN

Yes

Yes

Untested

Untested

Untested

Yes

Yes

Non-response to interferon

Temporal OD

Nasal OD

Nasal OD

Nasal OD

Temporal OS

Nasal OS

Superior OD

Tumor location

Leukoplakia

Papillomatous

Papillomatous

Leukoplakia

Gelatinous Leukoplakia

Leukoplakia

Corneal opacity

Tumor characteristics 3 1 2

25 mm2

3 3 1

mm2

36 mm2 mm2

4

48

1

9 mm2

9

2 mm2

AJCC stage

mm2

Tumor area

CIN2

CIS

CIS

CIS

CIN3

CIS

CIS

Pathologic grade

Yes

Yes

No

No

No

Yes

Yes

TTN mutation

M = male; F = female; W = white; H = Hispanic; NH = non-Hispanic; OD = right eye; OS = left eye; CIS = carcinoma in situ: CIN = conjunctival intraepithelial; AJCC = American Joint Committee on Cancer, TTN = Titin

51

76

#3

#5

73

#2

64

45

#1

#4

Age

Sample No

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Demographics and clinical features of our study population

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Table 1 GALOR et al. Page 5

Ophthalmology. Author manuscript; available in PMC 2017 January 01.

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Ophthalmology. Author manuscript; available in PMC 2017 January 01. T>C

C>T

ch6:38759442

ch16:3860684

T>C

C>T

ch6:38819392

ch7:81334836

C>T G>T

ch5:150911349

ch7:81334844

T>C

ch5:150922281

A>G

ch2:179458723

ch11:19901481

C>T C>G

ch2:179413186

C>T

G>C

ch2:179550287

G>A

G>A

ch2:179448450

ch11:20089874

C>T

ch2:179588813

ch11:19955640

T>C

ch2:179466171

D = Damaging; N = Non-damaging.

CREBBP

DNAH8

HGF

FAT2

NAV2

T>A G>A

ch2:179410721

T>C

ch2:179612315

ch2:179393898

G>A

ch2:179401724

TTN

Mutation

Position

Gene

149961222

144805357

-

-

373545882

140898888

-

-

142125793

-

201922910

201377736

72650029

56130023

72648964

72646823

142525903

55725279

145581345

-

dbSNP

0.12%

0.00%

-

-

0.00%

0.02%

-

-

0.00%

-

0.00%

0.00%

0.36%

0.12%

0.38%

0.26%

0.12%

0.12%

0.06%

-

Frequency

p.S299G

p.L965F

p.V1803A

p.G627D

p.N624K

p.V3204I

p.K2803E

p.Q129R

p.R1574H

p.A553V

p.G17825A

p.R29415Q

p.L10467V

p.T20179I

p.G6741D

p.K16877R

p.R30107C

p.E33886V

p.M4938V

p.T31730I

Protein change

0.997

0.995

1.000

0.757

0.966

0.197

0.997

1.000

1.000

0.983

1.000

1.000

0.956

1

0.969

1

0.995

0.501

0.902

0.955

Conservation

N

N

D

D

D

D

D

D

D

D

D

D

N

D

D

D

D

N

D

N

Functional prediction

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Recurrently mutated genes identified in Ocular Surface squamous Neoplasia.

#3, #4

#5

#1

#6, #7

#6, #7

#5

#6, #7

#1

#5

#3, #4

#1

#1

#2

#2

#2

#2

#2

#2

#6, #7

#6, #7

Samples

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Table 2 GALOR et al. Page 6