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Translational Cancer Mechanisms and Therapy

Distinct Biological Types of Ocular Adnexal Sebaceous Carcinoma: HPV-Driven and Virus-Negative Tumors Arise through Nonoverlapping Molecular-Genetic Alterations

Michael T. Tetzlaff, Jonathan L. Curry, Jing Ning, Oded Sagiv, Thomas L. Kandl, Bo Peng, Diana Bell, Mark Routbort, Courtney W. Hudgens, Doina Ivan, Tae-Boom Kim, Ken Chen, Agda Karina Eterovic, Kenna Shaw, Victor G. Prieto, Anna Yemelyanova and Bita Esmaeli
DOI: 10.1158/1078-0432.CCR-18-1688 Published February 2019

Abstract

Purpose: Ocular adnexal (OA) sebaceous carcinoma is an aggressive malignancy of the eyelid and ocular adnexa that frequently recurs and metastasizes, and effective therapies beyond surgical excision are lacking. There remains a critical need to define the molecular-genetic drivers of the disease to understand carcinomagenesis and progression and to devise novel treatment strategies.

Experimental Design: We present next-generation sequencing of a targeted panel of cancer-associated genes in 42 and whole transcriptome RNA sequencing from eight OA sebaceous carcinomas from 29 patients.

Results: We delineate two potentially distinct molecular-genetic subtypes of OA sebaceous carcinoma. The first is defined by somatic mutations impacting TP53 and/or RB1 [20/29 (70%) patients, including 10 patients whose primary tumors contained coexisting TP53 and RB1 mutations] with frequent concomitant mutations affecting NOTCH genes. These tumors arise in older patients and show frequent local recurrence. The second subtype [9/29 (31%) patients] lacks mutations affecting TP53, RB1, or NOTCH family members, but in 44% (4/9) of these tumors, RNA sequencing and in situ hybridization studies confirm transcriptionally active high-risk human papillomavirus. These tumors arise in younger patients and have not shown local recurrence.

Conclusions: Together, our findings establish a potential molecular-genetic framework by which to understand the development and progression of OA sebaceous carcinoma and provide key molecular-genetic insights to direct the design of novel therapeutic interventions.

Translational Relevance

Ocular adnexal (OA) sebaceous carcinoma is an aggressive malignancy that frequently recurs and/or metastasizes. Its delicate anatomic location on the surface of the eye or eyelid and high propensity for multifocal and infiltrative growth make OA sebaceous carcinoma a challenging tumor to treat. Surgical excision may produce functional and aesthetic morbidity, and effective systemic therapies are lacking. Thus, there is a critical need to define molecular-genetic drivers of OA sebaceous carcinoma to devise rational and efficacious therapies. DNA/RNA sequencing on OA sebaceous carcinomas delineated two potentially distinct molecular-genetic subtypes. The first harbors mutations in TP53 and/or RB1 with concomitant NOTCH family member mutations, shows higher nuclear grade features, arises in older patients and more commonly recurs locally. The second arises in younger patients, shows low-to-intermediate grade nuclear features, lacks mutations in TP53, RB1, or NOTCH, but instead harbors transcriptionally active high-risk human papillomavirus (HPV). These findings provide a potential molecular-genetic framework to understand the pathogenesis of OA sebaceous carcinoma.

Introduction

Ocular adnexal (OA) sebaceous carcinoma is an aggressive cancer that accounts for ∼5% of malignant epithelial eyelid tumors (1–5). Surgical excision remains the principal treatment modality. However, OA sebaceous carcinoma has a high propensity for multifocal intraepithelial and locally infiltrative growth that each contribute to frequent local recurrence. Given the tumor's delicate anatomic location on the surface of the eye or eyelid, these characteristics make this a challenging tumor to treat (1, 6, 7). Aggressive surgery is often required but may produce appreciable functional and aesthetic morbidity, and orbital exenteration is necessary to achieve local control of disease in 13% to 23% of patients (5, 7, 8). Further, regional nodal or distant metastasis occurs in 8% to 22% of patients, and up to 22% of patients diagnosed with OA sebaceous carcinoma die of the disease (7–9). Nevertheless, systemic therapies for OA sebaceous carcinoma remain largely ineffective (10). Collectively, these properties underscore a critical need to define the complete set of molecular-genetic alterations driving the development and progression of OA sebaceous carcinoma to possibly improve patient outcomes through the application of rationally designed therapeutic strategies in patients with metastatic sebaceous carcinoma for whom effective drug treatments are currently unavailable.

We and others previously reported that a subset of OA sebaceous carcinomas contain somatic mutations (11–13) and differentially expressed miRNAs (14) that culminate in common activation of the PI3K pathway, suggesting that PI3K inhibitors may be effective in the management of advanced disease (13). However, further mechanistic studies are needed to delineate additional pathways/mechanisms driving OA sebaceous carcinoma and identify additional candidates to exploit in targeted or immune-based therapies to reduce the morbidity and mortality related to this aggressive cancer.

One important risk factor for OA sebaceous carcinoma is long-standing immunosuppression, including that related to solid organ transplant (15–17) and HIV infection or AIDS (18). These associations not only emphasize the importance of a competent immune system in the pathogenesis of OA sebaceous carcinoma, but also suggest a potential etiologic relationship to an infectious agent. The clinical evolution of OA sebaceous carcinoma in the setting of long-standing conjunctivitis or blepharitis further implicates an infectious origin in some cases.

In this study, we report next-generation sequencing of a targeted panel of cancer-associated genes in specimens from 42 OA sebaceous carcinomas (31 primary or locally recurrent tumors from 29 patients and 11 matched metastases lesions from 8 patients) and whole transcriptome RNA sequencing on eight of these tumors. We demonstrate nonoverlapping, potentially distinct molecular-genetic subtypes of primary OA sebaceous carcinoma. The first subtype (20/29; 69% patients) harbors mutations in TP53 and/or RB1 with concomitant NOTCH family member mutations, arises in older patients, shows higher nuclear grade and has a greater tendency for local recurrence [all locally recurrent tumors (n = 5) harbored mutations in both TP53 and RB1]. The second subtype (9/29; 31% patients) arises in younger patients, shows lower/intermediate nuclear grade, and lacks mutations in TP53, RB1, or NOTCH. Instead, these tumors harbor transcriptionally active high-risk human papillomavirus (HPV) in 44% of cases (four of nine patient tumors). Together, our findings provide a potential molecular-genetic framework by which to understand the pathogenesis of OA sebaceous carcinoma and further suggest novel opportunities for therapy and/or prevention.

Materials and Methods

Tissue samples

The study was performed with an approved protocol from the Institutional Review Board (IRB) of The University of Texas MD Anderson Cancer Center (UTMDACC). All aspects of our research were performed in accordance with recognized ethical guidelines (e.g., Declaration of Helsinki, Belmont Report). Only left-over archival formalin-fixed, paraffin-embedded (FFPE) tissue (beyond that required for routine patient-care) was utilized for this study. We identified patients with OA sebaceous carcinoma diagnosed at our institution during 2007 to 2017 for whom sufficient tissue was available for molecular studies. FFPE tissue blocks and hematoxylin and eosin–stained sections were retrieved and reviewed. For each tissue sample, the diagnosis of OA sebaceous carcinoma and adequacy of the specimen for molecular testing were confirmed by at least three pathologists (MTT, DB, DI, VGP, and/or JLC). The diagnosis of OA sebaceous carcinoma relied on a combination of histopathologic assessment for features typical of OA sebaceous carcinoma (i.e., intracytoplasmic vacuoles that impinge nuclear contours and intraepithelial extension) together with immunohistochemical confirmation of sebaceous differentiation (i.e., Adipophilin positivity) in the tumor cells in most cases (19, 20). We obtained samples from 31 primary and/or locally recurrent OA sebaceous carcinomas from 29 patients and 11 matched metastatic tumors from eight of those patients. For each patient, normal tissue DNA from an uninvolved lymph node or adjacent skin/conjunctiva was also selected and sequenced to control for germline polymorphisms. Histopathologic features (including nuclear grade/differentiation, necrosis (presence/absence) and intraepithelial spread (presence/absence) were also recorded for each tumor. Histopathologic grade was defined according to the nuclear morphology and cytoplasmic evidence of sebaceous differentiation and determined according to the predominance (>50%) of the tumor cell population. High-grade tumors exhibited enlarged irregular nuclei with high nuclear:cytoplasmic ratio and only focal evidence sebaceous differentiation (intracytoplasmic vacuoles indenting the nucleus). Intermediate grade tumors exhibited similar enlarged nuclei but with lower nuclear:cytoplasmic ratio and more obvious evidence of sebaceous differentiation. Finally, well-differentiated tumors showed only slightly enlarged nuclei and frank evidence of sebaceous differentiation.

DNA extraction

For each primary, locally recurrent, or metastatic tumor included in this analysis, 10 to 20 unstained 5-μm FFPE tissue sections were manually microdissected to enrich for tumor cell DNA using the hematoxylin and eosin–stained slide as a guide. Samples from 23 of the 42 tumors were processed as described in our prior study (13). Briefly, we extracted DNA using the PicoPure DNA Extraction Kit (Arcturus) followed by DNA purification using the AMPureXP Kit (Agentcourt Biosciences), respectively. The resulting genomic DNA was quantified by Qubit dsDNA high-sensitivity assay (Life Technologies). For samples from the remaining 19 tumors, DNA was extracted using the Qiagen FFPE Extraction Kit (Qiagen), the resulting genomic DNA was quantified by Picogreen (Invitrogen), and quality was evaluated using the TapeStation 2200 instrument with Genomic DNA ScreenTape assay (Agilent).

Next-generation sequencing of a targeted panel of cancer-associated genes

For 23 of the tumors, somatic mutations and copy number variations in 409 cancer-related genes (and the methodologies for identifying those) have been previously described (13, 21). Briefly, we used the Ion Torrent Comprehensive Cancer Panel (Life Technologies) for target enrichment and sequencing using Ion Proton sequencer. Sequencing, data analysis, and identification of somatic genomic aberrations was performed by excluding variants identified in germline DNA from normal tissue in paired tumor samples as described previously (13, 21).

For the remaining 19 tumors, somatic mutations were identified using a different sequencing platform (T200.2) based on lack of commercial availability of the previous platform. T200.2 was initially developed by the institute for personalized cancer therapy (IPCT) genomic laboratory and currently consists of 323 cancer-related genes (22). Libraries were made using KAPA Hyper [KAPA library prep kit (KAPA)] using the “with beads” manufacturer protocol. Briefly, we performed enzymatic reactions for end repair, A-tailing, and adaptor ligation and then inserted the barcode by PCR using KAPA HiFi polymerase (six cycles). The resulting libraries were purified using the Agencourt AMPure PCR Purification Kit (Agentcourt Biosciences). Following library preparation, sample size was analyzed using the TapeStation 2200 (Agilent) or Fragment Analyzer (AATI). The KAPA qPCR Quantification Kit was used to quantify samples. Biotin-labeled probes (Roche Nimblegen) were designed to capture all exons in 323 cancer-related genes, and captures were performed following the manufacture's protocol. Briefly, equimolar amounts of DNA were pooled (8–16 samples), capture reagents and probes were added, and samples were incubated at 47°C on a thermocycler with heated lid (57°C) for 64 to 74 hours. We recovered targeted regions using streptavidin beads, and the streptavidin-biotin-probe-target complex was washed and then subjected to PCR amplification according to the manufacturer's protocol. The quality of each capture pool was validated using the TapeStation DNA High-Sensitivity Kit (Agilent), and enrichment (minimum cutoff 50×) was assessed with qPCR using specific primers (Roche Nimblegen). The captured libraries were sequenced on a HiSeq 4000 (Illumina Inc.) for 2 × 100 base paired end reads according to the manufacturer's instructions. All regions were covered by more than 20 reads. For data analysis, we aligned the T200.2 target-capture deep-sequencing data to human hg19 using BWA (23) and removed duplicate reads using Picard (24). We called single nucleotide variants and small in-dels using a proprietary custom analysis pipeline (25), which classified variants as somatic, germline, or loss of heterozygosity on the basis of variant allele frequencies in the tumor and the matched normal tissues. To assess the potential functional consequences of detected somatic variants, we compared them with dbSNP, COSMIC (26), and TCGA databases and annotated them using VEP (27), Annovar (28), CanDrA (29), and other programs. A detailed summary of the mutations in each primary, locally recurrent and metastatic tumor can be found in Supplementary Table S1, and mean sample coverage is provided in Supplementary Table S2. The raw data files of the targeted sequencing reported in our manuscript will be deposited in dbGAP in accordance with the consenting rules and guidelines from dbGAP. All other data reported herein will be made available from the corresponding author(s) upon reasonable request.

RNA isolation, cDNA creation, library preparation, and library capture

Eight distinct patient tumors, including two primary tumors with TP53 mutations, two primary tumors with both TP53 and RB1 mutations, and four tumors wild type for TP53 and RB1 (including three primary and one metastatic tumor) were subjected to whole transcriptome RNA sequencing. Total RNA was isolated using the FFPE RNA Purification Kit (Norgen). Extracted RNA was quantified by Picogreen (Invitrogen), and quality was assessed using the TapeStation 2200 (Agilent). cDNA was created from 10 to 100 ng of total RNA using the Ovation RNA Seq system V2 Kit (Nugen). cDNA was sheared by sonication using the E220 instrument (Covaris). To ensure the proper fragment size, samples were checked on the TapeStation 2200 using the TapeStation DNA High-Sensitivity Kit (Agilent). Libraries were prepared from the cDNA using the KAPA Library Prep Hyper Kit following the “with beads” manufacturer protocol, and quality control was performed as described above. Samples were then quantified using the KAPA qPCR Quantification Kit. Library capture was performed as described above (pooling two to six samples per pool).

RNA sequencing

The captured libraries were sequenced on a HiSeq 2500 (Illumina) on a version 3 TruSeq paired end flowcell according to the manufacturer's instructions at a cluster density of 700,000 clusters/mm2 to 1,000,000 clusters/mm2. The resulting BCL files containing the sequence data were converted into “.fastq.gz” files, and individual libraries within the samples were de-multiplexed using CASAVA version 1.8.2 with no mismatches. All regions were covered by more than 20 reads.

RNA sequencing data analysis

Raw RNA sequence data were processed by an in-house RNA seq data analysis pipeline, which, among other tools, uses the STAR aligner to align raw reads to the hg19 version of the human reference genome, featureCounts to quantify aligned reads to produce raw counts, Oncofuse to filter and prioritize fusion candidate generated by the STAR aligner, and FastQC and QualiMap to evaluate the quality of raw reads and feature counts.

We used VirusFinder, version 2.0, to align reads that did not map to the human reference genome to a viral database that contains viruses of all known classes (32,102 in total; ref. 30), including all classes of human papillomavirus and human polyomavirus. Relative levels of RNA expression was quantified in “reads per kilobase million (RPKM)” as follows:

RPKM = Number of reads/([gene length/1,000] × total number of reads in million)

In situ hybridization for high-risk HPV subtypes

In situ hybridization to detect expression of RNA from high-risk HPV subtypes (HPV 16, 18, 31, 33, 35, 45, 52, and 58) was performed using RNAscope 2.5 LS Probe-New Target (HPV 8; ACD Biotech; ready to use) according to the manufacturer's instructions. Cases with punctate nuclear and cytoplasmic signals within the tumor cells on the HPV HR preparations were considered positive for a transcriptionally active high-risk HPV.

Immunohistochemistry

IHC was performed using the polymeric biotin-free horseradish peroxide method on the Leica Microsystems Bond stainer using antibodies to RB (Calbiochem; clone OP66; 1:30), TP53 (Dako; clone DO-7; 1:100), and Adipophilin (Fitzgerald; clone AP125; prediluted). The Bond Refine Polymer Detection Kit (Leica) was used for detection.

Analysis of relationship between clinicopathologic variables and somatic mutations in primary tumors

We analyzed whether various clinical and pathologic variables (including sex, age at primary diagnosis, anatomic location, clinical size, clinical stage (AJCC 7th and 8th editions) correlated with the presence or absence of mutations in primary tumors. For continuous variables, t test was used to compare the means between two groups. For categorical variables, Fisher exact test was applied to test whether the distributions of the variables differ across the groups. Statistical analyses were performed using R version 3.4.3. Fisher exact test determined whether the differences in the frequency of various mutations (TP53, RB1) were statistically significant between primary tumors and local recurrences (at a significance level of 0.05).

Results

Next-generation DNA sequencing of a targeted panel of cancer-associated genes identifies distinct subgroups of OA sebaceous carcinoma

Our cohort included OA sebaceous carcinoma specimens from 29 patients, including 16 women and 13 men, with a median age of 68 years (range, 44–93 years) at primary diagnosis. From these 29 patients, we sequenced (i) 26 primary OA sebaceous carcinomas from 26 patients; (ii) five locally recurrent OA sebaceous carcinoma from five patients (two with and three without matched primary tumor tissue); and (iii) 11 matched metastatic OA sebaceous carcinomas from eight patients whose primary tumor we also sequenced (Table 1).

View this table:
Table 1.

Patient demographics and anatomic distribution of OA sebaceous carcinomas

Results of next-generation sequencing of a targeted panel of cancer-associated genes in the primary and locally recurrent OA sebaceous carcinomas are summarized in Fig. 1 (detailed information regarding the specific mutation in each tumor is summarized in Supplementary Table S1). The most common somatic mutations in these tumors were mutations affecting TP53 (66%; 19/29 patients) and RB1 (48%; 14/29 patients). In 13 of the 29 patients (45%), TP53 and RB1 mutations coexisted in the same tumor, and Fisher exact test confirmed the significance of the frequency of TP53 and RB1 comutation (P = 0.0051). Fifteen of the 19 TP53-mutated tumors with sufficient tissue for testing showed variably increased (aberrant) TP53 protein expression, whereas 8 of the 10 TP53 wild-type tumors showed either absent or variably weak nuclear TP53 protein expression by immunohistochemical studies (Figs. 1 and 2; Supplementary Table S3). IHC studies demonstrated absence of significant nuclear RB protein expression in 12/14 RB1-mutant tumors with sufficient tissue for testing, whereas 12/15 RB1 wild-type tumors showed preserved, strong (albeit variable) nuclear RB expression (Figs. 1 and 2; Supplementary Table S3). Fisher exact test confirmed that aberrant TP53 protein expression in TP53 mutant tumors (P = 0.001) and loss of RB protein expression in RB1 mutant tumors (P < 0.001) were statistically significantly associated (Supplementary Table S3). A correlation between loss of RB protein expression and the type or position of RB1 gene mutations was not identified. Two of the three TP53 mutant tumors lacking aberrant TP53 protein expression were nonsense mutations occurring at amino acid position 196 (patient 7) and 146 (patient 19), respectively.

Figure 1.
Figure 1.

Mutational signatures of primary and locally recurrent OA sebaceous carcinomas. A, Heat map shows genes mutated more than once across the cohort of primary and locally recurrent OA sebaceous carcinomas. Genes are listed according to relative frequency of mutations. Each OA sebaceous carcinoma (primary or locally recurrent tumor) is represented in a column, and primary tumors are designated by a number whereas locally recurrent tumors are designated by a number plus “LR.” Tumors are separated according to the presence of TP53 and RB1 mutations. A colored box indicates the presence of a somatically acquired mutation or transcriptionally active high-risk HPV infection by in situ hybridization; gray boxes indicate the absence of a somatically acquired mutation or transcriptionally active high-risk HPV infection by in situ hybridization; asterisks indicate that HPV RNA was also detected by RNA sequencing in one of the samples from that patient. B, Immunohistochemical studies for TP53 and RB1 confirm reciprocal relationships between TP53 and RB1 mutations and aberrant protein expression of TP3 and RB1, respectively (all images shown as 200× magnification) for patient 11, 17, 20, 25, and 27.

Figure 2.
Figure 2.

Molecular-genetic subtypes of OA sebaceous carcinoma. Left panel shows a tumor carrying both a TP53 and RB1 mutation (A; hematoxylin and eosin; 400×). Right panel shows a tumor wild type for TP53 and RB1 (B; hematoxylin and eosin; 400×). IHC studies demonstrate positivity in both tumors for adipophilin (C, D; 400×). However, the tumor on the left panel shows aberrant nuclear accumulation of TP53 protein (E; 400×) compared with no aberrant nuclear accumulation of TP53 protein in the other tumor (F; 400×). There is also loss of RB protein expression (G; 400×) on the left, but mostly preserved RB protein expression on the right (H; 400×). Finally, whereas the tumor on the left shows no detectable transcriptionally active high-risk HPV (for the subtypes studied) by in situ hybridization (I; 400×), in situ hybridization confirms presence of transcriptionally active high-risk HPV in the tumor cells (nuclear and cytoplasmic signal distribution; J; 400×).

Finally, somatic mutations affecting NOTCH family genes were present in six tumors from five patients (NOTCH1 mutations in five tumors from four patients, NOTCH2 mutation in one tumor), and all tumors with NOTCH family mutations also carried coexisting TP53 and RB1 mutations. (NOTCH2 mutations are not included in Fig. 1 because the figure only includes genes mutated in more than 1 tumor.)

Analysis of relationships between clinical variables and the presence or absence of TP53 and/or RB1 mutations in primary tumors revealed that the 17 patients whose primary tumors contained somatic mutations affecting TP53 and/or RB1 were significantly older than the nine patients whose primary tumors did not contain mutations affecting these genes (mean age at primary diagnosis, 70.3 years vs. 58.1 years; P = 0.02; Table 2). Patients with and without mutations affecting TP53 and/or RB1 did not differ with respect to sex, anatomic location of the tumor, clinical tumor size, stage according to the 7th edition of the American Joint Committee on Cancer staging manual or mutational load.

View this table:
Table 2.

Comparison of clinical and pathologic variables between patients with primary OA sebaceous carcinomas with TP53 and/or RB1 mutations and patients with primary OA sebaceous carcinomas lacking TP53 or RB1 mutations

Somatic mutations affecting TP53 and RB1 are enriched in locally recurrent tumors

All five (100%) locally recurrent tumors included in our study harbored mutations in both TP53 and RB1 (Fig. 1). For two of these locally recurrent tumors, the primary tumor was also sequenced; in both cases, the original primary tumor also harbored mutations in both TP53 and RB1. In our experience, most paired primary and recurrent or metastatic OA sebaceous carcinomas do not differ with respect to their TP53 and RB1 mutation status. Therefore, under the assumption that the primary tumors corresponding to the other three locally recurrent tumors in our study shared similar mutations in TP53 and RB1, TP53 and RB1 mutations coexist in 13 primary tumors from 13 patients; TP53 and RB1 mutations coexist in five recurrent tumors from five patients; TP53 and RB mutations did not coexist in tumors from 16 patients. Fisher exact test showed that the enrichment of somatically acquired mutations affecting both TP53 and RB1 in recurrent OA sebaceous carcinoma compared with their frequency in primary OA sebaceous carcinoma overall was statistically significant (P = 0.046), suggesting that abrogation of both TP53 and RB1 tumor suppressor function together may confer a more locally aggressive clinical phenotype in OA sebaceous carcinoma with a greater propensity for local recurrence.

OA sebaceous carcinomas with somatic mutations affecting RB1 exhibit poorly differentiated histopathologic features that may contribute to local recurrence

To determine if TP3 and/or RB1 mutant tumors exhibit distinctive histopathologic features (that might account for their unique clinical tendency for local recurrence), we classified each primary tumor according to grade/differentiation of the tumor cells (high, intermediate, or well; see Materials and Methods and Supplementary Fig. S1), necrosis and pagetoid/intraepithelial spread (presence vs. absence for each; see Materials and Methods and Supplementary Table S3). Although TP53 mutant tumors showed more frequent necrosis compared with TP53 wild-type lesions (P = 0.05), RB1-mutated OA sebaceous carcinomas more commonly showed a predominance of high-grade nuclear morphology compared with RB1 wild-type tumors (P = 0.03; Supplementary Table S4). Further, TP53/RB1 comutant tumors (patients 1–13) were enriched for high-grade tumor cells compared with non-comutated tumors (patients 14–29; P = 0.02). Not surprisingly, there was a similar nonsignificant trend for high-grade nuclear features among the primary tumors that recurred compared to those that did not (P = 0.06). No additional statistically significant correlations were observed between histopathologic features and the mutational background were observed in this cohort of tumors (Supplementary Tables S3 and S4).

Whole transcriptome sequencing and in situ hybridization confirm high-risk HPV RNA expression in TP53/RB1 wild-type OA sebaceous carcinoma

Given the dichotomous distribution of TP53 and RB1 mutations in our cohort of primary OA sebaceous carcinomas and the distinct age at primary tumor diagnosis, histopathologic and local recurrence profiles of TP53/RB1 mutant versus TP53/RB1 wild-type tumors, we hypothesized that a viral or bacterial infection that directly or indirectly abrogates TP53 and RB (or dependent pathways) might drive or promote the development of TP53/RB1 wild-type OA sebaceous carcinoma—similar to what has been described in Merkel cell carcinoma, a cancer with a similar binary distribution of TP53/RB1 mutations and polyomavirus infection (31, 32). Thus, we submitted eight OA sebaceous carcinomas from eight patients for whole transcriptome RNA sequencing: four primary tumors with TP53 mutations (including two tumors with coexisting TP53 and RB1 mutations), and four tumors (three primary and one lymph node metastasis) wild type for TP53 and RB1. RNA sequencing identified variable levels of high-risk HPV RNA (type 16 and type 18) in two of the four TP53/RB1 wild-type tumors but none of the four TP53/RB1 mutant tumors. For patient 21, HPV type 18 RNA sequences were detected at an average of 141 RPKM (see Materials and Methods), whereas HPV type 16 RNA sequences were detected at an average of 7 RPKM for patient 22. No additional unique viral or bacterial RNA sequences were identified in any of the 8 tumors tested.

To confirm and expand on these findings, we performed in situ hybridization to detect RNA expression of high-risk HPV RNA in the entire cohort of patient tumors. We identified transcriptionally active high-risk HPV RNA in four of the nine patients (44%) with TP53/RB1 wild-type OA sebaceous carcinoma (including the two patients in whom high-risk HPV was previously identified in the primary tumor by RNA sequencing), but in none of the 20 patients whose tumors harbored somatic TP53 and/or RB1 mutations (including the four tumors originally subjected to RNA sequencing; Figs. 1–3). Fisher exact test confirmed the statistical significance of the dichotomous relationship between either TP53 and/or RB1 mutation versus the presence of transcriptionally active HPV (P = 0.0053). Furthermore, patients with HPV-positive OA sebaceous carcinoma were significantly younger (mean age at primary diagnosis, 54 years) than patients with HPV-negative OA sebaceous carcinoma (mean age at primary diagnosis, 68.3 years; P = 0.01) and patients whose primary OA sebaceous carcinoma contained somatic mutations affecting TP53 and/or RB1 (mean, 70.3 years; P = 0.006).

Figure 3.
Figure 3.

Mutational signatures of metastatic OA sebaceous carcinomas. Genes mutated more than once are listed according to relative frequency of mutations. Each metastatic OA sebaceous carcinoma is represented in a column, and tumors are grouped according to the frequency of TP53 and RB1 mutations. Red boxes indicate the presence of a somatically acquired mutation or transcriptionally active high-risk HPV infection by in situ hybridization; gray boxes indicate the absence of a somatically acquired mutation or transcriptionally active high-risk HPV infection by in situ hybridization; asterisks indicate that HPV RNA was also detected by RNA sequencing in one of the samples from that patient.

Metastatic OA sebaceous carcinomas harbor somatic mutations in TP53 and/or RB1 or infection by transcriptionally active high-risk HPV

Results of next-generation sequencing of cancer-associated genes in the metastatic OA sebaceous carcinomas (11 metastatic tumors from 8 patients) are summarized in Fig. 3, and a pairwise comparison of somatically acquired mutations in primary tumors and paired metastases is provided in Supplementary Table S1. The somatically acquired mutation profile in metastatic OA sebaceous carcinoma largely reflected that seen among primary OA sebaceous carcinoma: six of eight patients (75%) had TP53 mutations, and four of eight (50%) had RB1 mutations (all of those coexisted with TP53 mutations). Finally, somatic mutations affecting NOTCH genes were identified in four metastatic tumors from four patients; mutations in NOTCH1 were identified in two TP53/RB1 mutant tumors, and mutations in NOTCH3 were identified in two TP53 mutant tumors.

Two metastatic OA sebaceous carcinomas from two patients lacked mutations in TP53, RB1, or NOTCH family members but harbored transcriptionally active high-risk HPV subtypes. However, neither the relative frequency of TP53 and/or RB1 mutations nor the relative frequency of HPV infection differed significantly between metastatic and primary/locally recurrent OA sebaceous carcinomas (P > 0.05 for all comparisons using Fisher exact test).

Discussion

Our findings in this study provide a potential molecular-genetic framework by which to understand the development and progression of primary and locally recurrent OA sebaceous carcinomas and have important clinical implications. We demonstrated a bimodal, potentially distinct distribution of somatically acquired mutations and transcriptionally active HPV infection that define at least two distinct molecular-genetic subtypes of primary OA sebaceous carcinoma. Seventy-one percent (22/31) of the primary and locally recurrent tumors from 69% (20/29) of the patients in this series had OA sebaceous carcinomas of the first subtype, harboring mutations in TP53 and/or RB1. Fifteen of the 16 tumors (94%) harboring RB1 mutations also harbored TP53 mutations (P = 0.0051), and six TP53/RB1 mutant OA sebaceous carcinomas (from five patients) also carried concomitant NOTCH family member mutations: NOTCH1 mutation in five tumors and NOTCH2 mutation in one tumor. In contrast, 29% (9/31) of the primary and locally recurrent tumors and 31% (9/29) from the patients in this series had OA sebaceous carcinomas of the second subtype. These lacked mutations in TP53, RB1, or NOTCH and, in four of the nine tumors, harbored infection by transcriptionally active high-risk HPV subtypes. The clinical significance of this dichotomization is underscored by our findings that (i) age at primary tumor diagnosis was significantly higher for primary tumors with mutations in TP53 and/or RB1 than for primary tumors lacking these mutations or primary tumors with transcriptionally active HPV infection; (ii) tumors with RB1 mutations and TP53/RB1 comutant tumors exhibited higher grade features histopathologically compared with the RB1 wild-type and TP53/RB1 non-comutant tumors, respectively; and (iii) all five locally recurrent tumors harbored mutations in both TP53 and RB1, a statistically significant enrichment of this genotype among locally recurrent compared to primary OA sebaceous carcinomas (P = 0.046), indicating a more locally aggressive phenotype among tumors with this genotype that might possibly reflect their enrichment for higher nuclear grades. Our demonstration of potentially distinct molecular-genetic subtypes of OA sebaceous carcinoma with apparently distinct clinical phenotypes represents a pivotal development in our understanding of the disease.

Our finding that somatic mutations affecting TP53 were the most common alteration in OA sebaceous carcinoma is consistent with prior studies demonstrating TP53 mutations occurred in approximately 63% (37/59) of tumors (11–13, 33). Prior studies have shown that TP53 alterations are largely restricted to OA sebaceous carcinomas of the eyelid and further occur in a mutually exclusively fashion with defects in mismatch repair (13, 34), as the latter are predominantly seen in sebaceous carcinomas away from the eyelid. Together, abrogation of TP53 or of mismatch repair represent distinct pathways in the development of sebaceous carcinoma, but as previously described, these largely occur in accordance with their distinctive anatomic distributions (34).

Our finding that somatic RB1 mutations were the second most common somatically acquired mutation in OA sebaceous carcinoma, occurring in 48% (14/29) of our patients, is consistent with the long-established propensity for patients with hereditary retinoblastoma to also develop OA sebaceous carcinoma (35–41). More strikingly, 93% (13/14) of the patients in our series who had RB1 mutation in their primary and/or locally recurrent OA sebaceous carcinoma also harbored TP53 mutations in the same tumor, and this coalescence of somatic mutations in TP53 and RB1 was enriched in older patients and in locally recurrent tumors, which not only supports a pivotal role for concomitant TP53 and RB1 abrogation in the development of OA sebaceous carcinoma, but also suggests that this molecular-genetic subtype confers a more locally aggressive clinical phenotype, possibly related to their higher nuclear grade features.

The apparent dichotomous distribution of TP53 and RB1 mutations across primary or recurrent OA sebaceous carcinomas in this series was similarly striking: 69% (20/29) patients with primary or recurrent OA sebaceous carcinoma harbored mutations in one and/or the other gene [including 45% (13/29) with mutations in both] and occurred in older patients (mean 70.3 years) and exhibited higher nuclear grade, whereas 31% (9/29) of patients with OA sebaceous carcinoma lacked mutations in either gene, and these tumors occurred in younger patients (mean 58.1 years; P = 0.02) and more commonly showed intermediate and well-differentiated nuclear grade features. The mutual exclusivity of TP53/RB1 comutant tumors from HPV-positive tumors was statistically significant (P = 0.0053). Similar dichotomous distributions have been observed in other tumors, including Merkel cell carcinoma (31, 32) and squamous cell carcinoma of the head and neck (42). In each of these cancers, significant proportions of cases are caused by infection with an oncogenic virus: Merkel cell carcinoma polyomavirus (MCPyV) in Merkel cell carcinomas and HPV in many different squamous cell carcinomas. Furthermore, MCPyV-positive Merkel cell carcinomas contain significantly fewer somatic mutations than their MCPyV-negative counterparts, which harbor a high frequency of ultraviolet light–induced mutations and are specifically enriched for mutations affecting TP53 and RB1 (31, 32). Based on these observations, we hypothesized that OA sebaceous carcinoma lacking TP53 or RB1 mutations might also contain oncogenic viral sequences that interfere directly or indirectly with TP53- and RB-dependent pathways. RNA sequencing and subsequent in situ hybridization studies confirmed this prediction, as we identified transcriptionally active high-risk HPV infection in 44% (4/9) of the TP53/RB1 wild-type OA sebaceous carcinomas but none of the TP53/RB1-mutated tumors. HPV-positive tumors developed in younger patients (mean age at primary diagnosis, 54 years) than HPV-negative tumors (mean age at primary diagnosis, 68.3 years; P = 0.01) and tumors with mutations in TP53 and/or RB1 (mean age at primary diagnosis, 70.3 years; P = 0.006) and exhibit similarly distinctive nuclear grade/differentiation histopathologically. The apparent mutually exclusive relationship observed between TP53/RB1 mutations and HPV infection most likely reflects the targets of HPV infection. Integration of HPV viral DNA into the host genome results in expression of viral proteins E6 and E7, which subsequently bind to and inactivate host cell tumor suppressor proteins TP53 and RB, respectively, interfering with TP53/RB-regulated cellular pathways directing DNA damage repair, apoptosis, and proliferation (43). Our findings suggest that the development of OA sebaceous carcinoma relies exquisitely on the abrogation of TP53 and/or RB function—either mutational or viral protein–mediated. Furthermore, our results at least partially explain the previously reported predisposition for OA sebaceous carcinoma among patients with long-standing immunosuppression, including immunosuppression due to solid organ transplant (15–17) and HIV infection or AIDS (18).

Prior studies have identified HPV sequences in 13.1% (14/107) of OA sebaceous carcinomas (11, 44–47), a frequency similar to the frequency of transcriptionally active high-risk HPV in patients with primary or locally recurrent tumors in our study: 13.8% (4/29). However, no previous study specifically interrogated the relationship between HPV infection and TP53/RB1 mutational status in OA sebaceous carcinomas. As such, prior efforts have produced conflicting results on the relationship between OA sebaceous carcinoma and HPV infection. Some studies (11, 44, 46) failed to confirm any relationship with HPV infection across 38 patient tumors, and a study in which a single HPV-positive case was identified by PCR for HPV DNA failed to confirm transcriptionally active HPV infection by in situ hybridization (48). These differences are partially attributable to the application of differing technologies for HPV detection, including in situ hybridization for HPV DNA or RNA or PCR for HPV DNA. However, to the extent that aberrant expression of p53 measured by IHC correlates with TP53 gene mutation, previous findings partially align with our finding of a mutually exclusive relationship between either TP53/RB1 mutation or HPV infection. Hayashi and colleagues (45) applied immunohistochemistry to detect HPV and TP53 proteins to 21 cases of OA sebaceous carcinoma and found that five cases were positive for HPV only, five were positive for TP53 only, and four were negative for both HPV and TP53, all consistent with our findings, although seven tumors in their series were positive for both HPV and TP53 (45). However, it is unclear whether these latter TP53-positive tumors actually carried a somatic TP53 mutation. Of note, detection of p16 protein expression—typically a surrogate for HPV positivity in HPV-driven tumors—has not been shown to predict HPV infection in prior studies on OA sebaceous carcinoma (48). This is likely because HPV-negative OA sebaceous carcinomas harbor a high frequency inactivating RB1 mutations, and RB1 inactivation itself typically correlates with elevated levels of p16 protein expression (49). Nevertheless, additional mechanisms abrogating TP53 and RB function in HPV-negative TP53/RB1 wild-type OA sebaceous carcinoma remain to be determined and may involve additional epigenetic controls.

The distinct segregation of NOTCH mutations in tumors with concomitant TP53 and RB1 mutations provides additional explanation for the observed dichotomy between OA sebaceous carcinomas with TP53/RB1 mutations and those with high-risk HPV infection as etiopathogenic. NOTCH genes were mutated in six primary or locally recurrent tumors from five patients (NOTCH1 in five tumors and NOTCH2 in one tumor) and in metastatic tumors from four patients (NOTCH1 in two and NOTCH3 in two). NOTCH mutations occurred exclusively in TP53 mutated tumors, and all but two tumors with NOTCH mutations were TP53/RB1 double mutant tumors. In addition to binding and inactivating TP53 protein, HPV E6 protein binds and inactivates MAML1 protein (Mastermind Like Transcriptional Coactivator-1), a transcriptional coactivator and key effector of NOTCH-mediated transcription (50, 51). Previous studies have shown similar mutually exclusive relationships between HPV infection and NOTCH mutations. In a study of head and neck squamous cell carcinomas, HPV-negative tumors more commonly carried NOTCH1 mutations whereas HPV-positive tumors more commonly carried a wild-type NOTCH1 gene (P = 0.031; ref. 52).

Important limitations to this study include the relatively small sample size (including only four patients with transcriptionally active high-risk HPV OA sebaceous carcinoma), the reliance on FFPE tissue resources, and the targeted nature of the DNA-sequencing studies. Five of our patients did not fit into either “type” of OA sebaceous carcinoma. Additional studies (ideally with fresh tissue) on a larger cohort of patients (including expanded whole exome DNA sequencing and whole transcriptome RNA-sequencing studies) are needed to fully understand the molecular landscopae of this rare cancer type.

In summary, we have identified two potentially distinct subgroups of OA sebaceous carcinoma (Fig. 4). Tumors in the first subtype are more common, tend to occur in older patients (mean age at primary diagnosis, 70.3 years), and have a high frequency of mutations in TP53 and/or RB1, which occasionally coexist with mutations in NOTCH genes. In the vast majority of cases with RB1 mutation, TP53 is also mutated (P = 0.0051). To the extent that locally recurrent tumors in our series consist exclusively of this genotype, TP53/RB1 mutated tumors appear to exhibit a more locally aggressive clinical phenotype including higher grade nuclear features and may warrant more aggressive postoperative surveillance. Tumors comprising the second subtype of OA sebaceous carcinoma occur in younger patients (mean age at primary diagnosis, 58.1 years) and lack mutations in TP53, RB1, or NOTCH family members. Instead, almost half of the tumors in this subtype harbor infection by transcriptionally active high-risk HPV subtypes. The mean age of patients with these HPV-positive tumors at primary diagnosis is only 54 years. Our findings position clinicians treating this aggressive malignancy to deploy next-generation sequencing strategies to identify either genome-matched targeted therapies directed at the clinically actionable mutations or to identify immune-based therapies (possibly incorporating HPV vaccination strategies) to further improve patient outcomes.

Figure 4.
Figure 4.

A model for the development of OA sebaceous carcinoma. Type I tumors defined by the presence of somatic TP53 and/or RB1 mutations. They occur in younger patients and exhibit increased frequency of local recurrence. Type II tumors lack mutations in TP53 or RB1 and instead show high frequency infection by HPV high risk subtypes, and viral E6 and E7 proteins abrogate TP53 and RB protein in these tumors. This subtype of tumor occurs in younger patients and exhibits less frequent local recurrence.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors' Contributions

Conception and design: M.T. Tetzlaff, V.G. Prieto, B. Esmaeli

Development of methodology: M.T. Tetzlaff, C.W. Hudgens, A. Yemelyanova, B. Esmaeli

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M.T. Tetzlaff, O. Sagiv, D. Ivan, A.K. Eterovic, K. Shaw, B. Esmaeli

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M.T. Tetzlaff, J. Ning, B. Peng, M. Routbort, C.W. Hudgens, T.-B. Kim, K. Chen, A.K. Eterovic, K. Shaw, B. Esmaeli

Writing, review, and/or revision of the manuscript: M.T. Tetzlaff, J.L. Curry, J. Ning, O. Sagiv, B. Peng, D. Bell, M. Routbort, D. Ivan, K. Shaw, V.G. Prieto, A. Yemelyanova, B. Esmaeli

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M.T. Tetzlaff, T.L. Kandl, A.K. Eterovic, K. Shaw, B. Esmaeli

Study supervision: M.T. Tetzlaff, O. Sagiv, B. Esmaeli

Acknowledgments

This study was funded by the Avery Orbital Oncology Fund for Research and Education. We thank Ms. Kim-Anh Vu for her outstanding help with figure generation and graphic design. We thank Ms. Stephanie Deming for magnificent medical editing.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

  • Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

  • Received May 31, 2018.
  • Revision received July 25, 2018.
  • Accepted November 2, 2018.
  • Published first November 12, 2018.

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Distinct Biological Types of Ocular Adnexal Sebaceous Carcinoma: HPV-Driven and Virus-Negative Tumors Arise through Nonoverlapping Molecular-Genetic Alterations
Michael T. Tetzlaff, Jonathan L. Curry, Jing Ning, Oded Sagiv, Thomas L. Kandl, Bo Peng, Diana Bell, Mark Routbort, Courtney W. Hudgens, Doina Ivan, Tae-Boom Kim, Ken Chen, Agda Karina Eterovic, Kenna Shaw, Victor G. Prieto, Anna Yemelyanova and Bita Esmaeli
Clin Cancer Res February 15 2019 (25) (4) 1280-1290; DOI: 10.1158/1078-0432.CCR-18-1688
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