Whole-Exome Sequencing of Syndromic Adrenocortical Carcinoma Reveals Distinct Mutational Profile From Sporadic ACC
Norman G. Nicolson,1 James M. Healy,1,2 Reju Korah,1 and Tobias Carling1
1Yale Endocrine Neoplasia Laboratory, Department of Surgery, Yale School of Medicine, New Haven, Connecticut 06520; and 2Connecticut Children’s Medical Center, Hartford, Connecticut 06106
ORCID numbers: 0000-0002-9986-6327 (N. G. Nicolson); 0000-0002-1941-4452 (J. M. Healy).
Next-generation sequencing has provided genetic profiles of a large number of sporadic adrenocortical carcinomas (ACCs), but the applicability of these results to ACC cases associated with tumor predisposition syndromes is unclear. Although the germline features of these syndromes have been well described, the somatic mutational landscape of the tumors they give rise to is less clear. Our group obtained germline and tumor tissue from a pediatric patient who developed ACC during her first year of life, which was treated successfully. She was subsequently diagnosed with additional tumors later in childhood. Whole exome sequencing analysis was performed followed by in silico protein function prediction, revealing a probably deleterious germline TP53 L265P mutation. The somatic mutational burden was comparable between the index case and a previously published cohort of 40 sporadic cases, but the mutational spectrum was distinct in terms of raw base-change frequency as well as in a trinucleotide context-specific analysis. No canonical somatic genetic drivers of ACC were identified in the reported case, suggesting that syndromic adreno- cortical tumors may represent a genetically distinct entity from sporadic tumors.
Copyright @ 2019 Endocrine Society
This article has been published under the terms of the Creative Commons Attribution Non- Commercial, No-Derivatives License (CC BY-NC-ND; https://creativecommons.org/licenses/by-nc- nd/4.0/).
Freeform/Key Words: adrenocortical carcinoma, Beckwith-Wiedemann, Li-Fraumeni, p53, mutational spectrum
Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with a poor prognosis [1]. Although these tumors are rare overall, they are somewhat more common in several known predisposition syndromes, including Beckwith-Wiedemann syndrome and Li-Fraumeni syn- drome [2]. The somatic genomic landscapes of sporadic adrenocortical carcinoma have been well described, but these previous studies have typically excluded syndromic cases [3-5]. As a result, although the germline events leading to tumor predisposition disorders have been well de- scribed, the somatic genetic profiles of these syndromic tumors are not well understood, which calls into question their tumor biology, and potential response to available targeted therapies. A previous series has examined the landscape of pediatric ACC in a cohort with a high prevalence of a founder Brazilian TP53 R337H germline variant using whole-genome, whole-exome, and transcriptome profiling techniques, but did not examine mutational signature of syndromic cases specifically [6]. We therefore performed whole exome sequencing (WES) of tumor and germline tissue from a pediatric patient with ACC with suspected tumorigenic syndrome.
The patient initially presented at five months of age when she developed progressive hemiparesis and was found to have a large left adrenal mass with apparent spinal metastasis.
Abbreviations: ACC, adrenocortical carcinoma; SNV, single nucleotide variants; WES, whole exome sequencing.
She had been noted previously to have some atypical developmental features at birth, in- cluding macroglossia and hemihypertrophy. There was no family history in either parent or any other relatives of childhood cancers or other apparent tumor syndromes. The child underwent resection of the adrenal and spinal masses. Pathology revealed a 4.5 cm, 19.5 g adrenocortical tumor with increased mitotic rate, atypical mitoses, and nuclear pleomor- phism without necrosis, vascular, or capsular invasion or lymph node metastases; in light of the spinal metastasis, the tumor was noted to be an European Network for the Study of Adrenal Tumors stage IV adrenocortical carcinoma. The patient was treated postoperatively with adjuvant chemotherapy including ifosfamide, carboplatin, etoposide, and intrathecal methotrexate, after which she was apparently cured of her ACC, which never recurred. Unfortunately, she subsequently developed a pelvic osteosarcoma at the age of 12 years, which was refractory to multiple resections and local and systemic therapies, and she ul- timately died of that tumor four years later.
1. Materials and Methods
The patient’s family signed informed consent for research and the project was approved by the Yale institutional review board. DNA was extracted from archival formalin-fixed paraffin-embedded tissue samples representing both the ACC tumor and uninvolved normal adrenal, using a proprietary enzymatic deparaffinization and extraction protocol. Sequencing was performed on the Illumina (Illumina, Inc., San Diego, CA) platform using the same approaches described previously for our group’s sporadic ACC cohort [4]. Somatic and germline single nucleotide variants (SNVs) were called using an in-house pipeline, and variants with an allele fraction of 5% or more were retained. Functional protein consequences of mutations were predicted by MutationTaster [7]. Somatic and germline nonsynonymous mutational burdens were compared with a previously published cohort of sporadic ACCs. Mutational signatures taking into account the tribase context of each SNV (including silent, splice-site, and nonsynonymous) were tabulated for each tumor using the deconstructSigs algorithm normalized to the whole exome [8]. Cases with five or fewer SNVs were excluded from signature analysis.
2. Results
WES revealed a comparable mutational burden in the syndromic case relative to the pre- viously investigated cohort (n = 40) of sporadic cases (Fig. 1). However, the base-change spectrum of the syndromic case’s mutations relative to the sporadic cases was distinct (P < 0.001 by x2 analysis of syndromic vs pooled sporadic variants). The syndromic case had fewer
A.
40
B.
250
35
Mean variants per tumor
Non-synonymous variants [mean +/- SEM]
30
200
25
20
Silent
150
15
Nonsense
Missense
100
10
Splice site
5
50
0
Syndromic
Sporadic (mean)
0
Syndromic Sporadic
C>T variants and relatively more T>A variants (Fig. 2A). These corresponded to a relative lack of Signature 1 and an abundance of Signature 25 in the syndromic case (Fig. 2B) [9]. Nonsynonymous somatic SNVs were identified in 25 genes, including several annotated in the Catalogue of Somatic Mutations in Cancer Cancer Gene Census, although none are known to be drivers in sporadic ACC (Table 1) [10]. Many of these were predicted to be damaging in silico by the MutationTaster algorithm [7]. Nonsynonymous germline variants were identified in 8 genes, including a probably damaging heterozygous TP53 variant not identified in the previous pediatric ACC cohort (Table 2) [6].
3. Discussion
In this study, we performed WES of an ACC occurring in a pediatric patient initially sus- pected to have Beckwith-Wiedemann syndrome. The genetic findings in our study (and indeed the patient’s ultimate clinical course) would suggest that the more appropriate di- agnosis was likely Li-Fraumeni syndrome. In any case, it is clear that the somatic genetic events in syndromic ACC are likely somewhat distinct from those events that occur in sporadic cases.
We identified a similar overall burden of somatic mutations, but a distinct mutational signature, in the syndromic case. In particular, the tumor carried relatively few Signature 1 mutations (associated with aging) and an unusually high number of Signature 25 mutations (a signature of unknown etiology that has been previously identified in Hodgkin lymphoma cells). Admittedly, the lack of Signature 1 mutations is possibly a consequence of the patient’s young age, rather than the tumor predisposition syndrome per se. Of course, many pediatric cases of ACC are associated with germline predisposition syndromes, so pediatric and syndromic ACC are overlapping categories.
A.
100%
80%
.C>T
.C>A
60%
WC>G
40%
IT>C
IT>A
20%
IT>G
0%
Syndromic
Sporadic
B.
100%
Signature 1
Signature 2
Signature 3
Signature 4
80%
Signature 5
Signature 6
Signature 7
.Signature 8
Signature 9
Signature 10
60%
Signature 11
Signature 12
· Signature 13
Signature 14
.Signature 15
. Signature 16
40%
Signature 17
Signature 18
· Signature 19
Signature 20
. Signature 21
· Signature 22
20%
Signature 23
Signature 24
Signature 25
Signature 26
Signature 27
Signature 28
0%
1
7
Z
”
”
2
/
=Signature 29
Signature 30
Syndromic
Sporadic
« Unassigned
| Gene | Base Change | AA Change | LOH | MutationTaster | COSMIC |
|---|---|---|---|---|---|
| AFF3 | A>T | L1088I | * | D | * |
| BTF3 | A>T | K158M | D | ||
| CACNA2D3 | G>T | L571F | D | ||
| CCDC14 | C>T | E347K | B | ||
| CLASP1 | C>G | E489Q | D | ||
| CWC22 | A>T | S106R | * | B | |
| DIAPH2 | A>T | K293I | * | D | |
| DMD | A>C | Y425D | D | ||
| DYSF | A>T | R293W | D | ||
| FGF13 | A>T | C52S | * | D | |
| FLT3 | A>C | S762A | D | * | |
| HPRT1 | A>T | D18V | D | ||
| KLHDC2 | A>G | Y163C | D | ||
| LARS | G>T | A820D | * | D | |
| LUC7L3 | T>C | I61T | D | ||
| PDHA2 | T>G | N245K | * | B | |
| PHLDA1 | C>G | Q383H | * | B | |
| PLEK2 | A>T | F159Y | D | ||
| PLEKHD1 | A>T | K150X | * | D | |
| PSMA8 | G>T | G46V | * | D | |
| PSMD1 | A>T | T755S | * | D | |
| SLC24A1 | T>C | L513P | * | D | |
| SMS | A>C | K230T | * | D | |
| SYNRG | G>T | P518H | D | ||
| ZMPSTE24 | A>T | T174S | D |
Asterisk indicates LOH at this locus. Asterisks in the final column indicate that the mutation is listed in the COSMIC Cancer Gene Census as of February 2019 [10].
Abbreviations: AA, amino acid; B, benign or D, damaging by the Mutation Taster algorithm [7]; COSMIC, Catalogue of Somatic Mutations in Cancer; LOH, loss of heterozygosity.
Furthermore, despite the patient’s pathologically advanced disease, she experienced an apparent cure after aggressive treatment, with more than a decade ACC-free, which would be highly unusual in sporadic adult ACC. Indeed, prior reports of pediatric ACC have dem- onstrated superior survival in tumors presenting in the first few years of life, compared with those presenting in older children or in adults [6]. These molecular (and clinical) findings may call into question the appropriateness of taking a “one size fits all” approach for the use of newer therapeutic agents in syndromic compared with sporadic ACC.
It may therefore be particularly important to separate these patients from sporadic ACC in future studies in the field, as the carcinogenesis and progression of these tumors may likely
| Gene | Base Change | AA Change | LOH | MutationTaster |
|---|---|---|---|---|
| ATP1A4 | G>C | D685H | D | |
| FAM114A2 | T>C | K433E | D | |
| GBP5 | C>T | R221H | D | |
| MMACHC | T>C | F158L | D | |
| MUC12 | G>C | R4631T | B | |
| RAPGEF4 | T>C | L204P | * | D |
| TP53 | A>G | L265P | D | |
| TPH2 | G>T | M432I | D |
Asterisk indicates LOH.
Abbreviations: AA, amino acid; B, benign or D, damaging by the MutationTaster algorithm [7]; LOH, loss of heterozygosity.
proceed via different mechanisms than those found in sporadic ACC. Furthermore, an im- proved understanding of the somatic events leading to tumors in the setting of germline predisposition disorders may provide insight for early detection or even prevention of these life-limiting cancers in patients and families suffering with these diseases.
Acknowledgments
The authors would like to acknowledge the patient and her family, without whom this study would not be possible. Additionally, the authors acknowledge the contribution of Gerald Goh, who per- formed the initial variant calling in this data set. Some data was presented at 15th Academic Surgical Congress, 5 February 2019, Houston, Texas.
Financial Support: The work was funded by institutional support without extramural grant funding.
Additional Information
Correspondence: Tobias Carling, MD, PHD, Section of Endocrine Surgery, Department of Sur- gery, Yale School of Medicine, 333 Cedar Street, PO Box 208062, New Haven, Connecticut 06520. E-mail: tobias.carling@yale.edu
Disclosure Summary: The authors report no conflicts of interest or commercial relationships relevant to the present work.
Data Availability: The datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.
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