Liquid Biopsy Detection of a TP53 Variant in a “Disease-Free” Pediatric Patient with a History of TP53-Mutant Adrenocortical Carcinoma
Patrick R. Blackburn ID,ª Shaohua Lei,a,b Sujuan Jia,a,c Ruth G. Tatevossian,a,c and Selene C. Kooa
Case Description
The proband is a now 4-year-old female, who presented at 9 months of age following a 6-week history of viriliza- tion including development of body odor, facial acne, fine oily hair and scalp, and pubic hair. Laboratory find- ings showed elevated adrenal androgens and hypercorti- solism with suppressed adrenocorticotropic hormone. Renal ultrasound showed a well-marginated solid mass measuring 36 x 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 transcrip- tome sequencing (RNA-seq) were performed on frozen tissue from the resected tumor specimen and the patient’s peripheral blood specimen (1). Only variants that had mutant allele present in >10% of total reads in the germ- line sequence data were retained; variants at lower levels were filtered out, since they usually represent low-quality
QUESTIONS TO CONSIDER
1. Does the TP53 variant identified by cfDNA testing correspond to tumor minimal residual disease (early evidence of metastatic lesion later detected in the lung)?
2. What might differences in the VAF found in plasma cfDNA vs the tumor specimen at different timepoints indicate?
3. How are we able to determine whether an alteration detected on tumor sequencing is germline or somatic?
4. How might germline mosaicism affect the interpretation of cfDNA results?
calls or tumor-in-normal contamination. The sequen- cing data underwent analysis using internal bioinfor- matics 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 fre- quency (VAF) of approximately 69% in whole genome sequencing, approximately 60% in WES, and approxi- mately 59% in RNA-seq (Fig. 1). Additional abnormal- ities included a focal amplification including RAC1 [7p22.1(5645827_6605937)x~9] and a complex gen- ome with several whole and partial chromosomal gains and losses and copy-neutral loss of heterozygosity (cnLOH) (Fig. 2A).
Plasma cell-free DNA (cfDNA) testing was per- formed 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 sequen- cing capture panel [St. Jude Pediatric Panel (2)] was used to enrich the library, following the standard manufacturer’s protocol. The enriched library was
ªDepartment of Pathology, St. Jude Children’s Research Hospital, Memphis, TN, United States; bCenter of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, TN, United States; “Clinical Biomarkers Laboratory, St. Jude Children’s Research Hospital, Memphis, TN, United States.
Address correspondence to: P.R.B. at Department of Pathology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, United States. E-mail Patrick.blackburn@stjude. org. S.C.K. at Department of Pathology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, United States. E-mail Selene.koo@stjude.org. Received March 19, 2024; accepted June 7, 2024.
https://doi.org/10.1093/clinchem/hvae103
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Clinical Case Study
0
5
Time (months)
9
Primary tumor (adrenal)
Peripheral blood draw
Metastatic tumor (lung)
T
N
N
T
VAF
VAF
Tumor Tissue (T)
60%
33%
I
I
Normal Tissue (N)
5.9%
1.9%
VAF 2.5%
cfDNA
sequenced on the NovaSeq platform and analyzed using in-house developed bioinformatic software (3). Analysis of the plasma cfDNA sample detected the TP53 c.657_668del variant identified in the primary tu- mor at approximately 2.5% VAF. No other shared single nucleotide variants, gross or focal copy number altera- tions, or loss of heterozygosity were detected in the cfDNA sample compared to the original tumor resection (Fig. 2B).
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 tomog- raphy scan showed an approximately 1.2 cm avid lung nodule, for which she underwent a lung wedge resection; pathologic examination confirmed metastatic adrenocorti- cal 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 (Fig. 1). Similar copy number alterations and regions of cnLOH to the primary tumor were noted in the metastatic lesion (Fig. 2C).
As part of her diagnostic workup, the patient was referred for genetic evaluation with the cancer pre- disposition team. The patient’s family history was largely noncontributory. Given the patient’s history of adreno- cortical 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 num- ber variant detection (Prevention Genetics; genes tested: APC, CDKNIC, EPCAM, MEN1, MLH1, MSH2, MSH6, NF1, PMS2, PRKAR1A, RET, and TP53), all of which were negative.
Clinical Case Study
A
Primary tumor (adrenal)
D
Adjacent normal (adrenal)
Coverage
180
Coverage
2700
20
1800
900
-
10
VAR
0.5
VAP
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Copy ratio (og2)
Copy ratio (log2)
6
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4
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6
7
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·
10
11
12
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6
7
8
9
10
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Chromosome
28
19 20 21 22 *
Y
Chromosome
Diploid depth
Gain (with An)
Gain (no Al)
Loss (with All
Loss (no Al)
EnLOH
Diploid depth
Gain (with Al)
Gain (no Al)
Loss (with An)
Loss (no Al)
cnLOH
B
Plasma cfDNA
Coverage
900
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0.5
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Copy ratio (log2)
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1
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3
4
$
6
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9
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11
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24
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Gain (with Al)
Gain (no Al)
Loss (with At)
Loss ino Al)
CnLOH
C
Metastatic tumor (lung)
E
Adjacent normal (lung)
Coverage
210
Coverage
3000
40
1000
TO
1000
1.0
1.0
0.5
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Copy ratio (leg2)
-
Copy ratio (log2)
A
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1
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9
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Loss (with All
Loss (no Al)
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Diploid depth
Gain (with Al)
Gain ino Al)
Loss (with Al)
Loss (no An)
cnLOH
Case Resolution
Initially all germline testing, including Beckwith- Wiedemann syndrome and panel testing, was reported as negative. However, due to the presence of a TP53 c.657_668del variant in this patient’s tumor, a rereview of the germline sequencing panel data was re- quested. The TP53 variant was identified in about 3% of the sequence reads of the patient’s germline blood sample. This putative germline mosaic variant was classified as of uncertain significance at the time. It is
predicted to result in an in-frame deletion within the p53 DNA binding domain and is expected to disrupt protein structure. This variant has been reported in ClinVar with conflicting interpretations of pathogen- icity (one likely pathogenic and one variant of uncertain significance). Given (1) the patient’s history of an adre- nocortical tumor, which can be associated with germline TP53 pathogenic variants, and (2) a second hit (17p copy-neutral loss of heterozygosity) in the tumor, the germline TP53 variant was felt to be likely indicative
Clinical Case Study
of mosaic Li-Fraumeni syndrome (LFS; OMIM #151623).
Tumor-adjacent normal tissue removed at the same time as this patient’s primary and metastatic tumor re- sections were also sequenced using St. Jude Pediatric Panel (2). The same TP53 c.657_668del variant was identified in both normal adrenal gland (VAF 5.9%) and normal lung tissue (VAF 1.9%), supporting the diagnosis of germline mosaicism for this variant (Fig. 1). No other clinically significant single nucleotide variants, insertion-deletion variants, or copy number variants were identified in tumor-adjacent normal tissue (Fig. 2D and 2E).
Case Discussion
LFS is an autosomal dominant tumor predisposition syndrome caused by germline TP53 mutations. (4) Individuals with LFS have an increased risk of develop- ing several types of cancer, including adrenocortical car- cinoma, sometimes at very early ages. (4) Although most patients with LFS have a family history of early-onset cancer, up to 20% of patients have a de novo germline TP53 mutation. (5) With high-depth next-generation sequencing, there is increased sensitivity for detection of mosaic TP53 mutations at low VAF; the frequency of mosaicism in patients with LFS-related cancers and germline TP53 mutation is up to 10.5% (6).
Because this patient was likely mosaic for the TP53 variant, we would expect this patient’s chances of devel- oping a new cancer to be lower than that of a person with classic LFS. However, due to the postzygotic nature of this mosaic germline alteration, we cannot fully assess the distribution of the alteration and allele frequency across all tissues in the patient (7). For that reason, the most conservative approach was to perform LFS surveil- lance according to current recommendations.
The patient completed 10 cycles of her nonprotocol treatment plan as per ARAR0332 (8) and received con- comitant treatment with the chemotherapeutic agent mitotane. She has shown no evidence of tumor recur- rence to date (at the time of publication, >20 months off chemotherapy).
Plasma-derived cfDNA testing (“liquid biopsy”) is swiftly emerging as a minimally invasive complement to traditional tumor biopsies and, in certain scenarios, a potential alternative to invasive tissue biopsy. (9) Liquid biopsy is increasingly recognized as an invaluable tool for molecular testing, aiding in cancer detection and surveillance. cfDNA analysis also has the potential to more comprehensively capture the molecular diversity of multiple distinct clones present in a patient’s tumor compared to tumor needle biopsy. (9) Furthermore, cfDNA analysis holds promise for detecting or monitor- ing tumors in patients without overt clinical symptoms.
POINTS TO REMEMBER
. cfDNA has become a valuable tool in the field of oncology, especially in cases of cancer with limited biopsy options.
. Low-variant frequency alterations in cancer susceptibility genes can be detected through cfDNA testing, presenting both opportunities and challenges for treatment decisions.
. Germline mosaicism can complicate the interpretation of cfDNA results.
· The VAF is a critical parameter in cfDNA analysis, but its interpretation can be challenging due to various factors, including tumor heterogeneity and confounding caused by the presence of germline variants in cancer predisposition genes.
· When faced with potential mosaicism in patients, different testing approaches may be needed to clarify the situation and guide clinical decisions.
Compared to standard tumor testing, cfDNA analysis is limited by the quantity and short half-life of tumor- derived cfDNA, which requires distinct methods for iso- lation, sequencing, and downstream analysis. (9)
The case presented here highlights important con- siderations and limitations of cfDNA testing as its adop- tion continues to expand. Like tumor-only tissue sequencing, in the absence of a germline comparator for cfDNA analysis, variants with a VAF exceeding ap- proximately 35% to 40% in cfDNA may indicate the presence of a germline heterozygous alteration rather than a somatic variant. Unlike tissue-based sequencing where upfront pathologist-assessed tumor cellularity serves as a comparison for sequencing-derived tumor purity, tumor content is not known de novo in cfDNA testing and is in fact determined from the se- quencing data. Reporting tumor content from VAFs in cfDNA thus requires additional caution, as elevated somatic VAFs may also occur with somatic alterations within amplified genomic regions rather than true high tumor content. Additionally, as in our case, germ- line mosaic variants can confound the interpretation of cfDNA results (7, 10). Awareness of potential germline- derived variants is vital, particularly in genes associated with hereditary cancer predisposition, such as TP53. The estimated frequency of mosaicism in cancer suscep- tibility genes is approximately 0.1% of cancer patients. (11) In older adult patients, clonal hematopoiesis of in- determinate potential with TP53 variants may be de- tected incidentally or in conjunction with a primary tumor being monitored by cfDNA testing (9). To ad- dress this challenge, laboratories should thoroughly
Commentary
assess cfDNA assay performance in distinguishing germ- line from somatic variants and are advised to include ap- propriate disclaimers in laboratory reports, acknowledging the potential for incidental germline findings (10). Although this patient’s tumor did not have other alterations that could be used for minimal re- sidual disease monitoring instead of TP53, other pa- tients may have tumors with multiple sequence variants that can be followed over time. There may also be utility in sequencing a germline comparator spe- cimen (e.g., skin biopsy or buccal swab) to distinguish tumor-specific variants from variants of germline origin, particularly in cfDNA collection for sequential tumor burden monitoring.
Author Contributions: The corresponding author takes full responsibility that all authors on this publication have met the following required criteria of eligibility for authorship: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Nobody who qualifies for authorship has been omitted from the list.
Patrick Blackburn (Conceptualization-Equal, Data curation-Equal, Formal analysis-Equal, Investigation-Equal, Methodology-Equal, Project administration-Equal, Supervision-Equal, Writing- original draft-Equal, Writing-review & editing-Equal), Shaohua Lei (Data curation-Equal, Formal analysis-Equal, Investigation-Equal, Methodology-Equal, Software-Equal, Visualization-Equal, Writing-re- view & editing-Equal), Sujuan Jia (Data curation-Equal, Investigation-Equal, Validation-Equal, Visualization-Equal, Writing- review & editing-Equal), Ruth Tatevossian (Conceptualization-Equal, Data curation-Equal, Formal analysis-Equal, Investigation-Equal, Methodology-Equal, Resources-Equal, Supervision-Equal, Writing- original draft-Equal, Writing-review & editing-Equal), and Selene Koo (Conceptualization-Equal, Data curation-Equal, Formal analysis-Equal, Investigation-Equal, Methodology-Equal, Project administration-Equal, Supervision-Equal, Visualization-Equal, Writing -original draft-Equal, Writing-review & editing-Equal).
Authors’ Disclosures or Potential Conflicts of Interest: Upon manu- script submission, all authors completed the author disclosure form.
Research Funding: The authors received funding from the Department of Pathology at St. Jude through Drs. Jeffery M. Klco and Charles G. Mullighan.
Disclosures: None declared.
Acknowledgments: The authors would like to thank Dr. Zonggao Shi for his assistance in analyzing the tumor sequencing data, Dr. Catherine Lam, Dr. Kim E. Nichols, and Alise Blake for their ex- pertise on this case, and the patient and her family for consenting to participate in this study.
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