Clinical Chemistry - Case Study
https://doi.org/10.1093/clinchem/hvae103
The proband is a now 4-year-old female, who presented at 9 months of age following a 6-week history of virilization including development of body odor, facial acne, fine oily hair and scalp, and pubic hair.
Student Discussion Document (pdf)
Patrick R. Blackburn, Shaohua Lei, Sujuan Jia, Ruth G. Tatevossian, and Selene C. Koo
The proband is a now 4-year-old female, who presented at 9 months of age following a 6-week history of virilization including development of body odor, facial acne, fine oily hair and scalp, and pubic hair. Laboratory findings showed elevated adrenal androgens and hypercortisolism with suppressed adrenocorticotropic hormone. Renal ultrasound showed a well-marginated solid mass measuring 36 × 34 × 29 mm in the right suprarenal space, worrisome for adrenocortical carcinoma.
A laparoscopic right adrenalectomy was performed to remove the tumor with negative margins. Histologically concerning features included elevated mitotic count (20 per 50 high-power fields), multiple atypical mitoses, and tumor necrosis. Her labs normalized postoperatively, consistent with stage 1 adrenocortical carcinoma. The family initially declined germline variant reporting. Comprehensive whole genome sequencing and whole exome sequencing (WES) with germline subtraction to remove false positives, and tumor-only whole transcriptome sequencing (RNA-seq) were performed on frozen tissue from the resected tumor specimen and the patient’s peripheral blood specimen. Only variants that had mutant allele present in >10% of total reads in the germline sequence data were retained; variants at lower levels were filtered out, since they usually represent low-quality calls or tumor-in-normal contamination. The sequencing data underwent analysis using internal bioinformatics software on a secure HIPAA-compliant high-performance computing system. The alignment was performed against the hg19 (GRCh37) human genome reference sequence.
Sequencing analysis identified a TP53 variant (NM_000546.5: c.657_668del; p.Tyr220_Pro223del) in a region of copy-neutral loss of heterozygosity on the p-arm of chromosome 17, with a variant allele frequency (VAF) of approximately 69% in whole genome sequencing, approximately 60% in WES, and approximately 59% in RNA-seq. Additional abnormalities included a focal amplification including RAC1 [7p22.1(5645827_6605937)x∼9] and a complex genome with several whole and partial chromosomal gains and losses and copy-neutral loss of heterozygosity (cnLOH).
Plasma cell-free DNA (cfDNA) testing was performed on a sample collected 5 months after complete suprarenal mass resection. cfDNA was extracted from 1 mL plasma with the NucleoSnap® cfDNA kit and quantified with the Qubit 1xds DNA HS assay kit. The ThruPLEX® Tag-Seq HV kit was used to prepare the library. A clinically validated targeted DNA sequencing capture panel (St. Jude Pediatric Panel) was used to enrich the library, following the standard manufacturer’s protocol. The enriched library was sequenced on the NovaSeq platform and analyzed using in-house developed bioinformatic software. Analysis of the plasma cfDNA sample detected the TP53 c.657_668del variant identified in the primary tumor at approximately 2.5% VAF. No other shared single nucleotide variants, gross or focal copy number alterations, or loss of heterozygosity were detected in the cfDNA sample compared to the original tumor resection.
At approximately 16 months of age (about 2 months after collection of the blood sample for cfDNA testing), the patient again developed signs of virilization with similar associated lab findings and respiratory findings. Computed tomography with positron emission tomography scan showed an approximately 1.2 cm avid lung nodule, for which she underwent a lung wedge resection; pathologic examination confirmed metastatic adrenocortical carcinoma. WES and RNA-seq of the formalin-fixed paraffin-embedded tumor tissue from the resection specimen was performed. The sequencing analysis showed the same TP53 c.657_668del variant at 33% VAF in WES and 36% VAF by RNA-seq. Similar copy number alterations and regions of cnLOH to the primary tumor were noted in the metastatic lesion.
As part of her diagnostic workup, the patient was referred for genetic evaluation with the cancer predisposition team. The patient’s family history was largely noncontributory. Given the patient’s history of adrenocortical carcinoma, germline genetic testing was ordered for Beckwith–Wiedemann syndrome (OMIM #130650), including methylation analysis with high-resolution copy number analysis for chromosome 11p15 (University of Pennsylvania), as well as a targeted custom exome-based sequencing panel with copy number variant detection (Prevention Genetics; genes tested: APC, CDKN1C, EPCAM, MEN1, MLH1, MSH2, MSH6, NF1, PMS2, PRKAR1A, RET, and TP53), all of which were negative.