1 Copy Number Aberrations in Benign Serous Ovarian Tumors: a Case for Reclassification?

Author Manuscript Published OnlineFirst on October 5, 2011; DOI: 10.1158/1078-0432.CCR-11-2080 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Copy number aberrations in benign serous ovarian tumors: a case for reclassification?

Sally M. Hunter1, Michael S. Anglesio2, Raghwa Sharma3, C. Blake Gilks2,5, Nataliya Melnyk2, Yoke-Eng Chiew4,7, Anna deFazio for the Australian Ovarian Cancer Study Group1, Teri A. Longacre6, Anna deFazio4,7, David G. Huntsman2,5, *Kylie L. Gorringe1, *Ian G. Campbell1.

1Centre for Cancer Genomics and Predictive Medicine, Peter MacCallum Cancer Centre, Melbourne, Australia. 2The Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada. 3Anatomical Pathology, University of Sydney and University of Western Sydney at Westmead Hospital, Australia. 4Department of Gynaecological Oncology, Westmead Hospital, Westmead, Australia. 5Genetic Pathology Evaluation Centre of the Prostate Research Centre and Department of Pathology, Vancouver General Hospital and University of British Columbia, Vancouver BC, Canada. 6Stanford University School of Medicine, Stanford, CA 94305, United States. 7Westmead Institute for Cancer Research, University of Sydney at Westmead Millennium Institute, Westmead Hospital, Westmead, Australia.

*Co-senior authors

Running title: Copy number aberrations in benign serous ovarian tumors

Keywords: ovarian, fibroma, serous, benign, borderline.

Financial support: This work was supported by a grant (ID 628630) from the National Health and Medical Research Council of Australia (NHMRC). The AOCS was supported by the U.S. Army Medical Research and Materiel Command under DAMD17-01-1-0729, The Cancer Council Tasmania and The Cancer Foundation of Western Australia and the National Health and Medical Research Council of Australia (NHMRC).

Corresponding author: Ian Campbell, VBCRC Cancer Genetics Research Laboratory, Peter MacCallum Cancer Centre, Locked Bag 1, A’Beckett Street, Melbourne, Victoria 8006, Australia. Phone: 613-9656-1803; Fax: 613-9656-1411; E- mail: [email protected]. Conflicts of interest: None.

Manuscript notes: Word count: 3390 Figures/tables: 6 Supplementary figures/tables: 11

Abbreviations: BL, borderline; CN, copy number; CNA, copy number aberrations; CN LOH, copy neutral; FISH, fluorescence in situ hybridisation; H&E, haematoxylin and eosin; LGSC, low grade serous carcinoma; LOH, loss of heterozygosity; NOS, not otherwise specified; SBT, serous borderline tumor; SNP, single nucleotide polymorphism; WT, wildtype.

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Statement of translational relevance Ovarian cancer is a very significant health burden and the seventh leading cause of cancer death in women. At the time of diagnosis, women often have advanced disease and as a consequence their prognosis is extremely poor. Our understanding of the progression of ovarian cancer through precursor stages and the molecular genetic events underlying these changes is very limited. Although a number of candidate precursor lesions have been proposed, the true contribution of these precursor lesions to the onset of ovarian cancer is unresolved. Identifying genuine ovarian cancer precursors and defining key molecular genetic events initiating and promoting tumorigenesis has important implications for early detection and treatment of ovarian cancers.

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Abstract Purpose: Serous ovarian carcinomas are the predominant epithelial ovarian cancer subtype and it has been widely believed that some or all of these may arise from precursors derived from the ovarian surface epithelium or fimbriae, although direct molecular evidence for this is limited. This study aimed to perform copy number analysis using a series of benign and borderline serous ovarian tumors to identify underlying genomic changes that may be indicative of early events in tumorigenesis.

Experimental Design: High resolution copy number (CN) analysis was performed on DNA from the epithelial and fibroblast components of a cohort of benign (N=39) and borderline (N=24) serous tumors using the Affymetrix OncoScan assay and SNP6.0 arrays.

Results: CN aberrations were detected in the epithelium of only 2.9% (1/35) of serous cystadenomas and cystadenofibromas. In contrast, CN aberrations were detected in the epithelium of 67% (16/24) of the serous borderline tumors (SBTs). Unexpectedly, CN aberrations were detected in the fibroblasts of 33% (13/39) of the benign serous tumors and in 15% (3/20) of the SBTs. Of the 16 cases with CN aberrations in the fibroblasts, 12 of these carried a gain of chromosome 12.

Conclusions: Chromosome 12 trisomy has been previously identified in pure fibromas, supporting the concept that a significant proportion of benign serous tumors are in fact primary fibromas with an associated cystic mass. This is the first high resolution genomic analysis of benign serous ovarian tumors and has demonstrated not only that the majority of benign serous tumors have no genetic evidence of epithelial neoplasia but that a significant proportion may be more accurately classified as primary fibromas.

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Ovarian cancer is a very significant health burden and the 7th leading cause of cancer

death in women worldwide1. At the time of diagnosis, women with epithelial ovarian

cancer usually have advanced disease and as a consequence their prognosis is

extremely poor (5 year survival for stage III & IV disease is only 25-30%)2, 3. For

such a clinically significant disease, remarkably little is known about the molecular

events that initiate the disease. While the paradigm that malignancies arise through a

stepwise progression from benign precursors has been established for many

malignancies, the archetypical example being colorectal carcinogenesis4, it remains

unclear if this holds true for ovarian cancer. There is still considerable controversy as

to what constitutes a true ovarian cancer precursor; an important definition that needs

to be made in order to understand the origins and identify new clinical interventions

for this lethal disease.

Serous ovarian carcinomas are the predominant clinically important subtype but at

present there is little experimental evidence from which to draw convincing

conclusions about what constitutes the precursor(s) to this sub-type. It has been

widely believed that some or all of these arise from precursors originating from the

ovarian surface epithelium, via inclusion cysts or serous benign and borderline

tumors5-8. Obvious candidate precursors are serous ovarian cystadenomas and

cystadenofibromas, which are benign lesions with a cystic mass ≥1cm in diameter,

lined with a single layer of cuboidal to columnar epithelium and commonly associated

with a fibromatous stromal mass (Supplementary Figure S1). The serous epithelial

layer of these tumors typically displays minimal cellular proliferation and no nuclear

atypia. These are relatively common tumors, accounting for 60% of all serous ovarian

tumors, while serous borderline tumors (SBTs) and low grade serous carcinomas

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(LGSCs) account for 10-15% and 2-9% of all serous ovarian tumors, respectively9-11.

Benign serous tumors are presumed by many to be precursors to SBTs based on

similarities in the cystic structure of some SBTs and the frequent detection of cases

with a benign cyst co-existing with a SBT or cases comprising predominantly benign

cysts with regions of atypical proliferation13. Despite the co-occurrence of benign,

borderline and low grade carcinoma epithelial components, direct molecular evidence

supporting benign lesions as precursors is limited. While some studies have shown the

existence of KRAS and BRAF mutations in ovarian serous cystadenomas and

cystadenofibromas co-existing with a region of atypical proliferation or adjacent

SBT14, mutations in these genes have not been detectable in solitary benign tumors.

SBTs have been firmly established as the likely precursor lesions to LGSCs, sharing

similar rates of KRAS and BRAF mutation and low levels of genomic instability15-18.

This is in contrast to the high rates of TP53 mutation and high levels of genomic

alteration observed in high grade serous carcinomas18, 19. Traditionally, these have

been believed to arise from ovarian surface epithelium and epithelial inclusion cysts

formed from invaginated surface epithelium5. More recently, circumstantial evidence

has been published suggesting that serous ovarian carcinomas may not arise from the

ovary at all and may in fact originate from fallopian tube epithelium20, 21, 22, 23.

With the aim of identifying somatic genomic changes that may be indicative of early

events in tumorigenesis and which could assist in determining if these represent

precursors to some invasive serous ovarian carcinomas we have performed high

resolution copy number analysis on a series of benign and borderline serous ovarian

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tumors. This is the first ultra-high resolution copy number analysis of benign serous

tumors of the ovary.

Methods

Tissue Samples

Only fresh frozen tissue samples were used in this for copy number and mutation

analyses. All samples were collected with the patient's informed consent and the study

was approved by the Human Research Ethics Committees at the Peter MacCallum

Cancer Centre, Queensland Institute of Medical Research, University of Melbourne

and all participating hospitals. Patients with ovarian tumors were identified through

two primary sources: a) 9 at hospitals in Southampton24, UK, b) 54 through the

Australian Ovarian Cancer Study25. Pathology reviews were performed independently

by two gynaecological pathologists (RS and CBG). Pathology review was conducted

on cryosections adjacent to the tissue from which DNA was extracted (n = 63).

Microdissection and DNA Extraction

A representative haematoxylin and eosin stained section was assessed and needle

microdissection was performed using 10 µm sections to obtain high percentage tumor

epithelial cell and fibroblast cell components. DNA was extracted using the Qiagen

Blood and Tissue Kit (Qiagen, Valencia, CA, USA). Normal DNA extracted from

blood lymphocytes was available for all 63 patients.

Copy number arrays

A subset of cases were processed by Affymetrix for the OncoScan (Molecular

Inversion Probe) assay, which consists of a 330 k probe set that allows the detection

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of genome-wide, allele-specific copy number. OncoScan data normalisation was

performed by Affymetrix Inc. (Santa Clara, CA) as previously described26, 27. Where

sufficient material (≥ 250 ng DNA) was available, the Affymetrix SNP6.0 (1.8M

probe set) array were utilised for ultra-high resolution allele-specific copy number

analysis, although prior to its release the Affymetrix 500K array was used. For the

SNP6.0 array the input was reduced from the recommended 500 ng to 250 ng with no

detectable difference in the quality of the data. Reaction volumes were halved

accordingly prior to the SNP6.0 PCR step. MAPD scores (pass ≤ 0.4) for samples run

on OnsoScan and SNP6 platforms are available in Supplementary Tables 5 and 6.

Data analysis

The SNP and OncoScan data were analysed using Partek® Genomics Suite 6.5,

employing paired and unpaired copy number generation, allele-specific copy number

analysis and circular binary segmentation (CBS) to identify regions of copy number

aberration and LOH. Regions of CN aberration and LOH were confirmed through

examination of allele-specific copy number ratios.

Mutation Screening

DNA sequencing was performed by Sanger sequencing using BDT v3.1 reagents

(Applied Biosystems) and an ABI3130 sequencer. Sequencing was used to identify

the most common serous ovarian tumor mutations: BRAF codon 600, KRAS codons

12 and 13, TP53 exons 5-8 and ERBB2 exon 20 (Supplementary Figure S3). Primer

sequences are detailed in Supplementary Table S1 and specific mutations identified

are detailed in Supplementary Table S2.

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FISH and Aneusomy

Fluorescence in situ hybridisation was performed on 6 μm FFPE sections using the

following commercial and in-house BAC probes: Cep12 (orange, 12p11.1-q11,

Abbottt Molecular 30-160012), RP11-1K3 Green d-UTP (chr12q23.3-q24.11), RP11-

13G14 Green d-UTP (chr12q23.3-q24.11) and RP11-209C18 Alexa Fluor 647 d-UTP

(blue, chr15q25.3). BAC probes were labeled using nick translation kit (Abbott

Molecular; #32-801300). Aneusomy analysis was performed on 6 μm FFPE sections

using a commercial assay from Abbott Molecular (formerly Vysis), Breast Cancer

Aneusomy Multi-Color Probe Kit: Vysis LSI 1 - 1p12 Spectrum Gold (yellow), Vysis

CEP 8 -8p11.1 Alpha Satellite DNA Spectrum Red, Vysis CEP 11 - 11p11.11-q11

Spectrum Green and Vysis CEP 17 - 17p11.1-q11.1 Spectrum Aqua. All

hybridizations were performed as previously described28. Imaging was performed on

a Zeiss Axioplan epifluorescent microscope.

Results

Benign serous tumor copy number analysis

Haematoxylin and eosin (H&E) stained sections from a cohort of benign serous

tumors (serous cystadenomas and cystadenofibromas) were reviewed to identify cases

where sufficient epithelial and stromal tissue could be microdissected to perform high

resolution, genome-wide, allele-specific copy number analysis. A total of 14 serous

cystadenomas and 21 serous cystadenofibromas were identified where copy number

could be analysed in matching epithelium, fibroblasts and germline (lymphocyte)

DNA.

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All tissues were microdisssected to achieve >80% pure epithelia or fibroblast cell

populations (Supplementary Figure S2), which we have shown previously is sufficient

purity to reliably identify copy number (CN) aberrations and loss of heterozygosity

(LOH)29. CN aberrations and/or LOH were detected in the epithelial component in

one of the 35 cases (2.9%) but surprisingly, 12 of the 35 cases (34.3%) harboured CN

aberrations and/or LOH in the fibroblast component (Table 1). In one of these cases

unique copy number changes were detected in both the epithelium and fibroblasts,

consistent with this case comprising two distinct and independent tumors (Figure 1).

Overall, fibroblast components from 14.3% of the serous cystadenomas and 47.6% of

the cystadenofibromas harbored CN/LOH alterations (Supplementary Table S3).

Among the 12 cases with CN/LOH changes, 9 (75%) showed gain of chromosome 12

and three (25%) showed LOH of chromosome 22.

In a further attempt to identify evidence of somatic genetic events in the epithelial

component, each sample was analysed for mutations in KRAS codons 12 and 13,

BRAF codon 600, TP53 exons 5-8 and ERBB2 exon 20 but no mutations were

detected in the either the fibroblast or epithelial component of any case.

The observed rate of CN/LOH alterations in the fibroblasts was nearly three times

greater in cystadenofibromas compared to cystadenomas. However, as these cases

were selected on the basis of the presence of micro-dissectible epithelium we

considered that it was possible they may not have been representative of the typical

spectrum of benign serous tumors. Therefore, the fibroblast component from 10

consecutive benign serous tumors (five cystadenomas and five cystadenofibromas; six

of these are also reported in Table 1) were analysed for CN/LOH alterations using

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SNP6.0 arrays (Supplementary Table S4). The frequency of CN/LOH alterations

present in the fibroblasts from these cystadenofibromas was similar to the original set

of selected cases (40.0% versus 42.9%) but was much higher among the

cystadenomas (40% versus 14.3%) although this was not statistically significant

(p=0.2722, Fisher’s exact test). All four cases with CN/LOH alterations included gain

of chromosome 12. Among the entire cohort, 13/39 cases (33.3%) showed CN/LOH

alterations in the fibroblast component.

Serous borderline tumor copy number analysis

Given that many of the benign serous tumors appeared to be neoplastic fibromatous

masses rather than genuine epithelial tumors it was plausible that a similar situation

might also exist for SBTs. Consequently we performed high-resolution, genome-wide

copy number analysis on epithelial and fibroblast components from a series of SBTs.

Among the 24 SBTs analysed, CN/LOH aberrations were detected in 16/24 epithelial

components (67%) and in 3/20 fibroblast components (15%) (Table 2). BRAF or

KRAS mutations were detected in the epithelial component in 57.9% of cases (Table

2); however, no TP53 or ERRB2 mutations were detected. Overall, CN/LOH and/or

BRAF/KRAS mutations were detected in the epithelial component of all but one of the

borderline tumors. In all three cases where CN aberrations were detected in the

fibroblasts component, unique CN aberrations were also detected in the adjacent

epithelial component demonstrating they were not clonal variants of a common

initiating tumor (Table 2, Supplementary Figure S4).

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Fluorescence in situ hybridization

As noted above, gain of chromosome 12 was the most common copy number

aberration amongst stromal components of benign tumors. To confirm the presence of

the chromosome 12 gain in the fibroblasts from the benign serous tumors and to

determine absolute copy number, FISH was performed on two cases where suitable

tissue was available (cases A2 and A6). Both A2 and A6 were confirmed to have

trisomy of chromosome 12 in the majority of fibroblasts. Interestingly, A2 appeared

to have four copies of chromosomes 12 and 15 (a chromosome 15 probe was used as a

control) in some of the epithelial cells (Figure 2). Balanced tetrasomy in clusters of

epithelial cells was confirmed using an aneusomy detection kit and centromeric

probes for chromosomes 1, 8, 11, and 17 (Supplementary Figure S5). Completely

balanced tetrasomy is not readily detectable by SNP array as copy number is

calculated after a median-centred normalisation procedure. Further examination of the

SNP allelic ratio was also unable to detect imbalance, suggesting either this

duplication is nearly perfectly balanced or an insufficient proportion of affected cells

were present to make the distinction. FISH was also performed on a tumor microarray

with tissues from normal ovary (6), benign cord-stromal tumors (3), benign

endometriosis (4) and serous borderline tumors (42). Only a single SBT was

identified with chromosome 12 trisomy in approximately 45% of fibroblast cells,

although this case also had features of low grade serous

carcinoma/psammocarcinoma.

Discussion

This study has demonstrated that CN/LOH aberrations in the epithelium of benign

serous tumors are rare, occurring in just one (2.9%) of cases studied, whereas

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aberrations in the fibromatous components were more frequent, occurring in 33.3% of

cases. Where CN/LOH aberrations were observed in the epithelial components these

were similar to those identified in previous studies of benign serous tumors (Table

3)30, 31. Although the rate of epithelial CN/LOH events in this study is significantly

lower than previously reported, artefactual LOH events may have been introduced

when performing microsatellite analyses on low quantities of DNA in these previous

studies32. Our findings are consistent with the findings of Cheng et al. (2004)33 who

showed in a cohort of 29 ovarian serous cystadenomas that only 14% of these tumors

had detectable monoclonal expansion in the epithelium.

The absence of detectable KRAS, BRAF, ERBB2 or TP53 mutations in the epithelial

component of any benign serous lesion in this study is consistent with the low rate of

such mutations reported from previous studies17, 31. Reports of KRAS and BRAF

mutations in ovarian serous cystadenomas and cystadenofibromas have been limited

to a small number of cases with a co-existing region of atypical proliferation or

adjacent SBT14. Despite the small number of CN/LOH aberrations present in the

epithelium of benign serous tumors, combining our data with published studies

demonstrates that they have similar profiles to a proportion of SBTs, such as gain of

6p and 7q. If the benign serous tumors with CN/LOH aberrations are indeed

precursors to SBT, the absence of either KRAS or BRAF mutations, characteristic of

SBTs, might indicate that acquisition of these mutations accompanies progression to a

SBT. This possibility is consistent with the observations of Singer et al. (2002)6 who

showed that bilateral SBTs frequently shared a subset of CN aberrations but rarely

shared identical KRAS mutations, indicating that the CN aberrations are likely

occurring earlier than the acquisition of KRAS mutations and precede dissemination to

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the contralateral ovary. CN aberrations as early events in ovarian serous neoplasia is

further supported by studies that have identified aneuploidy, but not mutations, in

ovarian inclusion cysts and even at low levels in ovarian surface epithelium7, 8.

Alternatively, the absence of identifiable KRAS or BRAF mutations in benign serous

tumors with clear clonal expansion events leaves open the possibility that they may be

precursors for other serous ovarian cancers. The tetrasomy identified in the epithelial

component in case A2, adjacent to fibroblasts with chromosome 12 trisomy, is a

particularly intriguing finding in light of ploidy studies by Pradhan et al. (2009) that

identified tetraploidy in 7/40 high grade serous carcinomas, 0/22 LGSCs and 0/245

SBTs34.

In contrast to the benign serous tumors, CN/LOH events were identified in the

epithelial component in 16/24 (67%) of SBTs; similar aberrations have been detected

in previous studies (Table 3). Although limited CN/LOH data is available that

distinguishes low grade serous from high grade serous and other histological types of

ovarian cancer, overlapping CN/LOH events support the model of SBTs as precursors

to LGSCs (Table 3). Among the borderline tumors 57.9% harboured KRAS or BRAF

mutations, consistent with previous reports for both SBTs and LGSCs18, 35.

An important finding of this study is that the fibromatous components of benign

tumors frequently harbour identifiable clonal CN/LOH alterations, strongly

suggesting that many tumors originally classified as benign epithelial tumors are in

fact primary fibroblastic tumors with an associated epithelial cyst. This supports the

work of Seidman and Mehrota (2005)38, who published a review of 113 unselected

benign serous ovarian tumors to assess the rate of epithelial “neoplasia” based on the

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presence of a minimum of 1 mm2 area of epithelial proliferation, finding that only 7%

of cases passed this criterion. Seidman and Mehrotra predicted that 81% of serous

cystadenofibromas are in reality fibromas with epithelial inclusions and that 99% of

serous cystadenomas are cystically dilated glandular inclusions. Consequently, they

concluded that the majority of these tumors should not be considered serous

neoplasms. Consistent with Seidman and Mehrota’s finding of greater evidence of

“true” neoplasia in cystadenofibromas compared to cystadenomas, we only observed

CN aberrations in the epithelium of cystadenofibromas and none in the epithelium of

cystadenomas.

Pathologist reviews of the cases in this study failed to identify any distinguishing

histological features associated with detectable CN aberrations in the fibromatous

components compared to those tumors without detectable fibromatous CN

aberrations. One explanation may be that in fact the majority of these benign tumors

do contain clonal fibromatous neoplasms that this molecular study has not detected

due to the focus on copy number analysis and mutations most commonly present in

the epithelium of serous ovarian tumors. To date no highly recurrent mutated genes

have been identified in fibromas that can be used to assess the true frequency of

benign serous tumors that are fibromas.

Trisomy 12 has been previously identified as a characteristic copy number aberration

of the fibroma-thecoma group of sex cord-stromal tumors39-42. Persons et al. (1994)43

found chromosome 12 gain in 40% of pure fibromas, very similar to the rate detected

in the fibroblasts of this study. Although chromosome 12 gain has previously been

identified in benign ovarian tumors these studies were performed on whole tumors

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and therefore failed to identify the cell type of origin42. Our data clearly demonstrates

for the first time that at least 40% of benign serous tumors are likely primary

fibromas. It has been previously speculated that recurrent gain of chromosome 12

may be driven by a requirement for an extra copy of the KRAS oncogene for neoplasia

in this milieu44. A correlation between trisomy 12 and increased KRAS expression

has been demonstrated in an adenosquamous carcinoma of the lung45, however, this

has not been demonstrated experimentally in ovarian tumors. Other known oncogenes

are located on chromosome 12, including CDK4, CCND2, MDM, WNT1, and ERBB3,

along with a number of other genes of potential relevance in neoplasia.

An intriguing finding of this study was the identification of one benign tumor and

three borderline tumors with co-existing but unique CN aberrations in the epithelial

and fibroblast components. Interestingly, loss of chromosome 22, the second most

frequent CN aberration in fibromas detected in this study, was also detected by Qiu et

al. (2005)29 in the fibroblast component of a high grade endometrioid tumor.

Although this was a single case in 25 cases analysed, the presence of an underlying

fibroma in another histological subtype suggests this phenomenon may not be limited

to serous tumors. This finding raises some interesting questions about the potential

biological relationship between these tumor populations: are these biphasic tumors,

collision tumors, or is it possible that the fibromatous component promotes

tumorigenesis in the adjacent epithelium? The absence of shared point mutations and

CN aberrations (within the limits of our assays) between the adjacent epithelial and

fibroblast components would appear to rule out a mesenchymal-epithelial or

epithelial-mesenchymal transition event underlying a biphasic tumor. The likelihood

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of these being collision tumors also seems highly unlikely given that the frequency of

fibromas and genuine benign serous tumors is extremely low46.

Speculatively, our data points to a unique ovarian tumorigenesis pathway whereby a

pre-existing fibroma promotes proliferation and occasional transformation of the

adjacent ovarian epithelium. Fibroblasts have long been recognised as an integral part

of the tumor microenvironment influencing tumorigenesis, capable of both inhibitory

and stimulatory influences on neoplasia in neighbouring epithelia47. In model system,

artificially induced genetically altered fibroblasts have been previously demonstrated

to promote both initiation and progression of tumorigenesis in adjacent epithelia48-50.

It has been proposed that in primary human cancers, adjacent fibroblasts (so called

cancer associated fibroblasts) almost universally acquired clonal somatic genetic

mutations in genes that promoted the progression of the adjacent cancer epithelium.

While that theory has subsequently been disproved29, our data for the first time

suggests that in rare instances a genuine stromal tumor can promote tumorigenesis in

adjacent epithelium. A possible mechanism for this to occur could be entrapment of

epithelial cells within proliferating stroma that subsequently drives the formation of

cystic masses. Under these circumstances we propose that growth factors secreted by

the fibroma, or inhibitory factors no longer secreted by the fibroma, promote

proliferation of the epithelium, which subsequently increases the likelihood of the

epithelium acquiring or selecting for genetic alterations leading to neoplasia (Figure

3). The capacity for genetically altered fibroblasts to induce neoplasia in epithelial

cells has been demonstrated in a number of mouse models51-52. This alternative

pathway may exist independently of the classic pathways of low grade serous tumors

characterized by the presence of activating mutations in the oncogenes KRAS and

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BRAF in the epithelium and may even explain some high grade serous and

endometrioid tumors.

This is the largest high-resolution study of benign serous tumors performed to date

and provides evidence that the majority of benign ovarian serous tumors are not

epithelial neoplasms, but are in fact primary fibromas. This finding significantly

deflates the prevalence of serous histological subtype of ovarian neoplasms, of which

benign tumors accounted for 60%. The findings of this study also provide preliminary

evidence for a possible novel pathway of tumorigenesis dependent on a promoting

primary fibroma.

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Acknowledgements

We gratefully acknowledge the cooperation of the participating institutions in

Australia. We also acknowledge the contribution of the study nurses, research

assistants and all clinical and scientific collaborators and would like to thank all of the

women who participated in the study. Members of the Australian Ovarian Cancer

Study Group, collaborators and hospitals involved in AOCS can be found at

http://www.aocstudy.org

Grant Support

This work was supported by a grant (ID 628630) from the National Health and

Medical Research Council of Australia (NHMRC). The AOCS was supported by the

U.S. Army Medical Research and Materiel Command under DAMD17-01-1-0729,

The Cancer Council Tasmania and The Cancer Foundation of Western Australia and

the National Health and Medical Research Council of Australia (NHMRC; IDs 40028

and 400413).

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Figure legends

Figure 1: Whole genome copy number plot. Benign serous cystadenofibroma (case 5) identified with unique copy number aberrations in the epithelium (+6p, +7q, -6q, - 7p) and adjacent fibroblasts (+12, -22).

Figure 2: Triploidy and tetrasomy. Case A2 was found to have clusters of epithelial cells with balanced tetrasomy (arrow) alongside fibroblast cells triploid for chromosome 12 (arrow heads). Probes: orange, 12p11.1-q11; green, 12q23.3-q24.11; blue, 15q25.3.

Figure 3: Primary fibroblastic neoplasia. (A) Genetically altered fibroblast secretes growth/proliferation signals to influence surrounding cells. (B) Clonal expansion of genetically altered fibroblasts with secretion of growth/proliferation signals that influence surrounding cells and adjacent epithelium to form benign glandular structures. (C) Genetic alteration of epithelial cells resulting in neoplasia in response to adjacent fibroma.

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Table 1: Copy number analysis of benign serous tumors Affymetrix Sample ID Morphology Epithelium Copy Number Aberrations Fibroblast Copy Number Aberrations Platform 5 Serous cystadenofibroma Gain: 6p, 7q; LOH: 6q, 7p, 13q12.11-12.12 Gain: 12; LOH: 22 OncoScan 467 Serous cystadenofibroma None Gain: 9q, 16q12.1-12.2; LOH: 16q13-24.3 OncoScan A1 Serous cystadenofibroma LOH: X a LOH: X (low level)a OncoScan A2 Serous cystadenofibroma None Gain: 12 OncoScan A3 Serous cystadenofibromac None Gain: 12 OncoScan A4 Serous cystadenofibromac None Gain: 12; LOH: 17q, 22 OncoScan A5 Serous cystadenofibroma None Gain: 12 OncoScan 158 Serous cystadenofibroma None Gain: 12 SNP6.0 450 Serous cystadenofibroma None LOH: 22 SNP6.0 A6 Serous cystadenofibroma None Gain: 12 SNP6.0 A7 Serous cystadenofibroma None Gain: 12 SNP6.0 A8 Serous cystadenofibroma None None OncoScan A9 Serous cystadenofibroma None None OncoScan A10 Serous cystadenofibromac None None OncoScan A11 Serous cystadenofibroma None None OncoScan A12 Serous cystadenofibroma None None OncoScan A13 Serous cystadenofibroma None None OncoScan A14 Serous cystadenofibroma None None OncoScan 103 Serous cystadenofibroma None None 500K/SNP6.0 164 Serous cystadenofibroma None None SNP6.0 A15 Serous cystadenofibroma None None SNP6.0

A16 Serous cystadenoma None LOH: 3p21.33-14.3, 7q11.21-11.23, 7q22.1 OncoScan

A17 Serous cystadenomab None Gain: 8, 10, 12, 13, 15, 18, 19 OncoScan A18 Serous cystadenoma None None OncoScan A19 Serous cystadenoma None None OncoScan A20 Serous cystadenoma None None OncoScan A21 Serous cystadenoma None None OncoScan A22 Serous cystadenomab None None OncoScan 7 Serous cystadenomab None None 500K/SNP6.0 148 Serous cystadenoma None None 500K/SNP6.0 A23 Serous cystadenoma None None SNP6.0 A24 Serous cystadenomab None None SNP6.0 A25 Serous cystadenoma, papillary None None OncoScan A26 Serous cystadenoma, papillary None None SNP6.0 A27 Serous cystadenoma, papillaryb None None OncoScan LOH = loss of heterozygosity (copy number loss). aApparent germline mosaic loss of X with loss of same allele in all tissues, therefore not considered a tumor-specific loss. bFibrous stroma noted on review. cFibrous stroma not noted on review.

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Table 2: Copy number aberrations borderline serous tumors Sample ID Morphology Epithelium Copy Number Aberrations Fibroblast Copy Number Aberrations Affymetrix Platform Mutations Gain: 8q21.3-q22.1, 8q22.2-qter; A32 SBT, NOS None SNP6.0 BRAF V600E CN LOH: 7q Gain: 7, X A33 SBT, NOS None SNP6.0 BRAF V600E CN LOH: 7q22.1-qter A34 SBT, NOS CN LOH: 7q11.22-qter Untested SNP6.0 BRAF V600E A35 SBT, NOSa LOH: X None SNP6.0 KRAS G12V A36 SBT, NOS Gain: 2, 7, 8, 12, 18 None SNP6.0 KRAS G12D A37 SBT, NOSa LOH: 1p35.1-36.32, 19 Untested SNP6.0 KRAS G12V A38 SBT, adenofibromaa Gain: 8q, 12p None SNP6.0 KRAS G12V A39 SBT, NOSa Gain: 2, 7, 8, 12, 18, 20 None SNP6.0 WT A40 SBT, NOS CN LOH: 12p None SNP6.0 WT Gain: 8q; A41 SBT, NOSa None SNP6.0 WT LOH: 1p35.3-pter A42 SBT, papillary cystica CN LOH: 17q None SNP6.0 WT A43 SBT, papillary CN LOH: 19q13.32-13.43 None SNP6.0 WT A44 SBT, papillarya Gain: 1q; LOH: 16q None SNP6.0 WT Gain: 7q32.2; A45 SBT, NOS Gain: 12 SNP6.0 WT CN LOH: 11q13.5-qter Gain: 12, 14, 22q11.21; A46 SBT, NOSa CN LOH: 17q SNP6.0 WT LOH: 21 Gain: 8q22.1-qter; A47 SBT, cystica LOH: X SNP6.0 WT LOH: 6q23.2-qter 558 SBT, NOS None None SNP6.0 BRAF V600E A48 SBT, NOS None None SNP6.0 BRAF V600E A49 SBT, adenofibromaa None None SNP6.0 BRAF V600E A50 SBT, papillary None None SNP6.0 BRAF V600E A51 SBT, papillary cystica None None SNP6.0 BRAF V600E A52 SBT, papillary cystic None Untested SNP6.0 BRAF V600E A53 SBT, papillary cystica None Untested SNP6.0 BRAF V600E A54 SBT, papillary cystic None None SNP6.0 WT SBT = serous borderline tumor, NOS = not otherwise specified, CN LOH = copy neutral loss of heterozygosity, WT = wildtype. aFibrous stroma noted on review.

Table 3: Summary of serous CN/LOH studies Benign CN aberrations SBT CN aberrations Reported benign CN aberrations Reported SBT CN aberrations Reported LGSC CN aberrations (this study) (this study)

Gain: Gain: Gain : Gain: 6p, 7q 1q, 2, 7, 8, 8q, 8q21.3-q22.1, 1q, 6p 1q, 2, 2q, 6p, 6q, 7, 8, 8q, 9p, 12, 8q22.1-qter, 10, 12, 12p, 18, 20 13q, 16, 16p

LOH: LOH: LOH: LOH: LOH: 6q, 7p, 13q12.11-12, X. 1p35.1-pter, 6q23.3, 7q, 11q13.5-1p32-p11, 4q13-34, 5q11-q23, 1p, 1p36,4, 9, 9p21.3, 12q, 14q, 1p36, 9p21.3 qter, 12p, 16q, 17q, 19q13.32- 6q12-q23, 6q16.3, 6q22.2, 15q, 16p, 17, 17p, 17q, 19p, 19q, 13.43, 19, X 7p22.2, 7p15.3, 7p12.3, 7q22.1, 22q 7q31.1, 7q36.1, 9p21.3, 11q23.3

AI: AI: 1p, 5q, 8p, 18q, 22q, Xp 1p, 5q, 8p, 18q, 22q, Xp

Benign20, 25, 31; SBTs20, 31, 32; LGSCs6, 20, 31. Bold indicates overlapping CN aberrations between current study and previous reports.

Copy number aberrations in benign serous ovarian tumors: a case for reclassification?

Sally M Hunter, Michael S Anglesio, Raghwa Sharma, et al.

Clin Cancer Res Published OnlineFirst October 5, 2011.

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