Progressive metastatic infantile fibrosarcoma with multiple acquired mutations

Abstract

Infantile fibrosarcoma is the most common soft-tissue sarcoma in children under the age of 1 yr and is defined molecularly by NTRK fusion proteins. This tumor is known to be locally invasive; however, although rare, metastases can occur. The NTRK fusion acts as a driver for tumor formation, which can be targeted by first- and second-generation TRK inhibitors. Although NTRK gatekeeper mutations have been well-described as mechanisms of resistance to these agents, alternative pathway mutations are rare. Here, we report the case of a patient with infantile fibrosarcoma treated with chemotherapy and TRK inhibition that developed metastatic, progressive disease with multiple acquired mutations, including TP53, SUFU, and an NTRK F617L gatekeeper mutation. Alterations in pathways of SUFU and TP53 have been widely described in the literature in other tumors; however, not yet in infantile fibrosarcoma. Although most patients have a sustained response to TRK inhibitors, a subset will go on to develop mechanisms of resistance that have implications for clinical management, such as in our patient. We hypothesize this constellation of mutations contributed to the patient's aggressive clinical course. Taken together, we report the first case of infantile fibrosarcoma with ETV6::NTRK3 and acquired SUFU, TP53, and NTRK F617L gatekeeper mutation along with detailed clinical course and management. Our report highlights the importance of genomic profiling in recurrent infantile fibrosarcoma to reveal actionable mutations, such as gatekeeper mutations, that can improve patient outcomes.

INTRODUCTION

Infantile fibrosarcoma (IFS) is a soft-tissue tumor of intermediate malignancy that is most common in children of <1 yr of age (Sultan et al. 2010). It most often presents in the extremities as a rapidly growing, nontender mass that is variable in size and consistency (Chung and Enzinger 1976; Soule and Pritchard 1977; Orbach et al. 2010). IFS has a low metastatic potential (<10%) and the prognosis is excellent, with overall survival >90% (Chung and Enzinger 1976; Orbach et al. 2016). Traditionally, surgery has been the mainstay of treatment, with chemotherapy being reserved for patients with metastatic or unresectable disease. More recently, targeted therapies have shown significant promise and their use in the upfront setting is being evaluated (Grier et al. 1985; Parida et al. 2013; Demetri et al. 2020).

IFS is characterized by a t(12;15)(p13;q25) translocation resulting in a ETV6::NTRK3 gene fusion (Bourgeois et al. 2000; Orbach et al. 2016). Expression of ETV6::NTRK3 leads to the constitutive activation of the MAPK and PI3K pathways, which contributes to uncontrolled cell growth and resistance to apoptosis resulting in tumor progression (Paraiso et al. 2010; Cocco et al. 2018). In NTRK fusion-containing tumor models, treatment with first-generation TRK inhibitors, such as larotrectinib, results in the inhibition of the MAPK, PI3K-AKT, PKC, and STAT3 pathways (Cocco et al. 2018). TRK inhibitors are typically associated with response rates of >75%, regardless of tumor histology (Hong et al. 2020). Although most patients have a sustained response to TRK inhibitors, a subset will go on to develop mechanisms of resistance that have implications for clinical management. The most common mechanism of resistance to first-generation inhibitors is the emergence of target mutations of the NTRK kinase domain resulting in steric hindrance (Cocco et al. 2019). Second-generation TRK inhibitors such as repotrectinib have been developed to overcome this resistance mechanism to first-line products. Repotrectinib is a potent small-molecule inhibitor of the ALK, ROS1, and TRK family that can circumvent the steric interference at the ATP binding pocket that leads to resistance from other agents. Alterations in alternative pathways have not been well-described. Here, we report the case of a patient with infantile fibrosarcoma treated with chemotherapy and TRK inhibition that developed metastatic, progressive disease along with multiple acquired mutations that we hypothesize contributed to his aggressive clinical course.

RESULTS

Clinical Presentation and Treatment Outcomes

The patient originally presented to medical care at the age of 5 mo after the mother noticed a small mass on the right buttocks. An ultrasound guided biopsy initially reported this lesion as a kaposiform hemangioendothelioma; thus, the lesion was embolized and the patient was started on prednisone. When the lesion progressed, vincristine and sirolimus were added and the original biopsy was reexamined. Upon reexamination, the diagnosis was changed to a cellular spindle cell neoplasm consistent with infantile fibrosarcoma (Fig. 1A,F).

Figure 1.

Morphologic and phenotypic progression of a case of recurrent and metastatic infantile fibrosarcoma with multiple acquired mutations. Initial biopsy showed a well-vascularized malignant fibroblastic neoplasm composed of a monomorphic population of spindled cells with eosinophilic cytoplasm and round-to-elongated nuclei (A); p53 stain was positive in <10% of tumor cell nuclei (F). Post-therapy sample (17 mo from initial diagnosis) showed dense collagenization and rare, atypical cells (B); p53 stain was positive in rare tumor cell nuclei (G). Increased cytological atypia over time and p53 overexpression in nearly all tumor nuclei was seen on specimens taken at 36 (C,H), 41 (D,I), and 43 (E,J) months from initial diagnosis. (A–E) Hematoxylin and eosin stain; (F–J) p53 immunohistochemical stain (3,3′-diaminobenzidine, nuclear positivity).

At that time, the tumor was not resectable and so the patient went on to receive chemotherapy consisting of six alternating cycles of vincristine, dactinomycin, cyclophosphamide (VAC) with ifosfamide, doxorubicin, and etoposide (IDE), as these were the only therapeutic options available where the patient was located. The tumor continued to enlarge on therapy, at which time he was referred for potential targeted therapy. At the time of referral, the patient was found to have a large right buttock mass but was otherwise clinically well. There was concern for severe speech delay at the time of presentation that was felt to be unrelated.

Baseline magnetic resonance imaging (MRI) revealed a large right buttock mass involving and expanding the space of the gluteal musculature, extending into the pelvis through the pyriform sinus, measuring 10.5 cm × 8.4 cm × 11.3 cm. The intrapelvic portion abutted and displaced the iliac neurovascular bundle and rectum. The tumor was markedly heterogeneous in signal intensity, with curvilinear T2 hypointense areas representing fibrosis and T2 hyperintense and enhancing areas reflecting the more cellular components. The mass did not demonstrate restricted diffusion. 18F-fluorodeoxyglucose positron emission tomography (FDG-PET)/computed tomography (CT) at this time demonstrated low-grade FDG uptake, which was felt to reflect the intrinsic metabolic activity of the tumor as opposed to treatment effect given the history of progression (Table 1). There was no evidence of metastatic disease; however, routine staging imaging did reveal a duplicated left renal collection system and a choroidal and arachnoid cyst of the brain, of no clinical significance.

Table 1.

Disease progress with imaging and treatment

The patient started treatment with larotrectinib 100 mg/m². The patient required a dose reduction to 75 mg/m² secondary to recurrent neutropenia and brief therapy interruptions due to other unrelated illness but otherwise tolerated the therapy well. The tumor showed a significant clinical response within 2 wk, and after 13 cycles of therapy MRI demonstrated significant interval reduction in size of the right gluteal mass measuring 1.0 cm × 1.5 cm × 1.2 cm (Table 1). Specifically, the imaging characteristics suggested a residual fibrotic mass without hypercellular components. A biopsy done at that time showed dense collagenization with only rare, atypical cells (Fig. 1B,G). There was no evidence of active disease. Based on these findings, the decision was made to continue therapy for an additional two cycles to confirm the near complete response and then therapy was stopped. The patient received 15 cycles of larotrectinib in total.

One month after stopping treatment with larotrectinib, there was clinical suspicion of local tumor regrowth. Repeat MRI demonstrated a new mildly heterogeneous T2 hyperintense and enhancing ovoid mass in the right gluteus maximus muscle. The mass measured 2.5 cm × 4.5 cm × 3.2 cm. Mild restricted diffusion was observed reflecting greater hypercellularity than the initial tumor. The PET/CT at this time demonstrated increased FDG uptake to a greater degree than the initial tumor (Table 1). There was no metastatic disease. He underwent a repeat biopsy of the mass, which showed a cellular and moderately pleomorphic spindle cell neoplastic proliferation with the composition ranging from small cells with round to ovoid nuclei to scattered cells with large, atypical nuclei and prominent nucleoli, with nuclear overexpression of p53. Based on these findings, and the history of a good response to larotrectinib, he restarted larotrectinib at the previously tolerated dose of 75 mg/m².

The patient initially exhibited a good clinical response; however, 6 mo after the resumption of larotrectinib the lesion again began to grow, with MRI showing an increased enhancing area in the primary tumor, which was concerning for progression of disease (Table 1). After reviewing potential therapeutic options of alternative chemotherapy, a second-generation TRK inhibitor, or aggressive local control, the decision was made to undergo surgical resection. The result of the surgery was a gross total resection. Pathology from the surgery demonstrated an extensively viable and infiltrative cellular neoplasm composed of spindle-to-oval cells arranged in fascicles with some degree of nuclear pleomorphism, numerous mitoses. The margins were focally positive at the sacral, superior, and inferior margins (Fig. 1D). By immunohistochemistry, the neoplastic cells showed diffuse p53 nuclear accumulation (mutant pattern; Fig. 1I). Postoperative imaging obtained 3 wk postprocedure revealed regrowth of the mass at the areas of the positive margins and so he underwent a second surgical resection to attempt to achieve a negative margin. The patient then went on to receive adjuvant proton radiation to a dose of 55.8 GyRBE to the resection bed with a 1-cm margin for subclinical disease, which he tolerated well. The reresection pathology showed tumor morphologically similar to the most recently resected tumor.

One month following radiotherapy the patient presented with fatigue, lethargy abnormal gait, and labs consistent with acute kidney injury. MRI demonstrated interval development of a lobulated heterogeneous mass in the right hemipelvis with a greater degree of diffusion restriction compared to prior recurrences. CT chest at this time demonstrated pulmonary nodules concerning for metastatic disease (Table 1). Notably, the imaging characteristics of increased diffusion restriction and FDG PET/CT avidity were different than his initial disease presentation. He underwent nephrostomy tube placement and ultrasound guided biopsy of the new pelvis mass. The pathology showed recurrent infantile fibrosarcoma, with diffuse nuclear staining for p53 (Fig. 1E,J). Given the larotrectinib resistance, he was consented to a phase I/II study of Repotrectinib (NCT04094610) at which time therapy is ongoing.

Genomic Analyses

At the time of initial diagnosis, fluorescence in situ hybridization testing was positive for ETV6 rearrangement and reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of the ETV6::NTRK3 fusion, confirming the diagnosis of infantile fibrosarcoma.

Whole-genome sequencing (WGS), whole-exome sequencing (WES), and transcriptome (RNA) sequencing was performed on the biopsy from the first recurrence specimen and showed an oncogenic in-frame fusion between ETV6 (NM_01987.4) exon 5 and NTRK3 (NM_001012338.2) exon 15, which retained an intact kinase domain of NTRK3; a TP53 (NM_000546.5) c.742C > T; p.Arg248Trp (R248W) gain-of-function variant (Xu et al. 2011; Klemke et al. 2021); and a single-nucleotide deletion in exon 7 of SUFU (NM_016169.3) c.846del; p.Glu283ArgfsTer30, that results in a frameshift of the protein coding sequence (beginning at amino acid 283) and creates a premature stop codon within the amino-terminal binding domain of this protein product. Gains of Chromosomes 8, 17, and 20, known to be recurrent in infantile fibrosarcoma (Mandahl et al. 1989; Schofield et al. 1994; Gupta et al. 2020), as well as copy neutral loss of heterozygosity of Chromosome 11, deletions involving Chromosomes 10 and 13q13.1-q14.2 (including the RB1 gene locus), and chromothripsis of Chromosome 15 were also identified. Notably, the NTRK3 locus on 15q25.3 was not affected by copy-number alterations resulting from the chromothripsis event. The R248W variant is present within the duplicated copy of Chromosome 17, which explains its high allele frequency (WGS, 96%; WES, 89%; RNA, 97%). The SUFU frameshift indel variant is also present in a high proportion of the sequencing reads in this sample (WGS, 85%; WES, 63%; RNA, 41%) because of the Chromosome 10 deletion event affecting the other copy of SUFU in tumor cells. As a result, no intact copy of SUFU was left in this tumor.

Comprehensive sequencing analysis (WGS, WES, and transcriptome; Table 2) carried out in the reresection specimen from the second recurrence event revealed a newly acquired NTRK3 (NM_001012338.2) c.1849T > C; p.F617L gatekeeper mutation in addition to the previously identified genomic alterations.

Table 2.

Genomic variants

Gene	Chromosome(s)	HGVS DNA reference	HGVS protein reference	Variant type	Predicted effect	Detected on first recurrence? Variant allele frequency/total reads	Detected on second recurrence? Variant allele frequency/total reads
ETV6::NTRK3	12 and 15	t(12;15)(p13.2;q25.3)hg19 Chr 12:12022903(+):: Chr 15:88483984(−)	n/a	ETV6::NTRK3 fusion	Oncogenic, sensitivity to TRK inhibitors	Yes	Yes
TP53	17	NM_000546.5: c.742C > T	p.Arg248Trp	Single-nucleotide substitution	Gain of function, oncogenic	Yes. WGS, 96%/70; WES, 89%/614; RNA, 97%	Yes. WGS, 76%/66; WES, 74%/890; RNA, 94%
SUFU	10	NM_016169.3: c.846del	p.Glu283ArgfsTer30	Frameshift deletion	Likely oncogenic	Yes. WGS, 85%/13; WES, 63%/162; RNA. 41%	Yes. WGS, 52%/33; WES, 43%/270; RNA, 30%
NTRK3	15	NM_001012338.2: c.1849T > C	p.Phe617Leu	Single-nucleotide substitution	Larotrectinib resistance	No	Yes. WGS, 22%/50; WES, 34%/630; RNA, 95%

Details of sequencing experiments are provided in Supplemental Table 1.

(HGVS) Human Genome Variation Society, (n/a) not applicable, (TRK) tyrosine receptor kinase, (WGS) whole-genome sequencing, (WES) whole-exome sequencing.

TP53 sequencing was retrospectively performed in the diagnostic biopsy specimen using an Archer VariantPlex Custom panel, which covers exons 1–11 of TP53, and was negative for the presence of the R248W variant, detected in the recurrence samples.

The patient underwent germline genetics evaluation given the history of recurrent infantile fibrosarcoma, duplicated left renal collection system, choroidal and arachnoid cysts, neutropenia, as well as macrocephaly and absent speech. Chromosome microarray and trio WES were performed on blood at a commercial laboratory (Ambry Genetics). Notably, the SUFU and TP53 variants reported in the tumor were confirmed to be absent in the germline. No single-nucleotide variants were reported in the nuclear or mitochondrial genes. The patient was found to have a pathogenic, heterozygous, de novo deletion of 16p11.2 spanning ∼655 kb. Although there is no known association between the 16p11.2 microdeletion and infantile fibrosarcoma, this deletion is likely to explain the patient's nononcologic findings of macrocephaly, speech impairment, renal anomalies, and brain cysts (Owen et al. 2018; Kim et al. 2020; Yang et al. 2020; Chung et al. 2021).

DISCUSSION

Here, we report a unique case of an infantile fibrosarcoma harboring a combination of genomic alterations that have not been previously described in this tumor type. In addition to the canonical ETV6::NTRK3 fusion and some aneuploidy changes commonly associated with IFS (Mandahl et al. 1989; Speleman et al. 1989; Schofield et al. 1994), the tumor developed pathogenic variants in TP53 and SUFU, followed by an NTRK3 gatekeeper (kinase domain) mutation, which likely contributed to the aggressive clinical course of the patient, with several recurrences and progressive disease. In this case, it is notable that the TP53 and SUFU variants were first detected after exposure to chemotherapy and larotrectinib, at the time of the first recurrence, whereas the NTRK3 gatekeeper mutation was detected at second recurrence after the second exposure to larotrectinib.

TP53 mutations are associated with unfavorable prognosis, tumor progression, recurrence, dedifferentiation, and high histologic grade in several sarcoma subtypes (Thoenen et al. 2019). The TP53 p.Arg248Trp (R248W) variant affects the conserved DNA binding domain of p53, which is a hotspot for missense mutations within the p53 protein, and alters the DNA binding ability of p53 resulting in gain-of-function activities whereby the p53 oncogenic protein exerts a dominant-negative effect over the remaining wild-type protein (Xu et al. 2011). Nuclear accumulation of mutant p53^R248W is postulated to affect DNA damage response, induce resistance to multiple CYP3A4-metabolized antineoplastic drugs (including etoposide) (Do et al. 2012; Xu et al. 2014), suppress apoptosis, and abrogate p53-mediated tumor suppression activities, promoting uncontrolled cell proliferation and aggressive pathological changes (Song et al. 2007; Jiang et al. 2015; Yamamoto and Iwakuma 2018; Zhou et al. 2019). Given that the histologic progression observed in the first recurrence specimen from our patient coincided with the detection of the R248W variant and nuclear overexpression of p53 by immunohistochemistry (IHC), we hypothesize that this variant may be a predisposing factor for the phenotypic transformation of the tumor. This assumption is supported by reports of small cell transformation in lung adenocarcinomas with TP53 and RB1 inactivation undergoing treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) (Lee et al. 2017).

Even though TP53 is the most mutated gene in human cancer, and TP53 variants have been reported in a handful of NTRK-fused tumors (Okamura et al. 2018; Keddy et al. 2021), alterations in this gene have not been described in infantile fibrosarcoma. However, Thorwarth et al. (2022) recently reported the presence of a somatic ARID1A frameshift variant (p.W1686Cfs*11) in an intrathoracic infantile fibrosarcoma. This tumor also developed a PIK3R1 in-frame (likely activating) deletion (p.F46_Q457del), followed by two NTRK3 resistance variants (solvent front p.G623R and the xDFG motif p.G696A) and failed to respond to therapy with VAC, larotrectinib, and selitrectinib. ARID1A functions as a tumor suppressor and collaborates with TP53 in the negative regulation of genes involved in cell-cycle progression, such as CDKN1A and SMAD3 (Guan et al. 2011). These findings suggest that TP53 pathway alterations acquired early during tumor development may provide survival advantages to the mutant clones and contribute to the survival and malignant progression of the tumor. In ETV6::NTRK3 fused tumors, mutant p53-mediated signaling likely exerts its oncogenic effect through STAT3 activation. STAT3 is one of the downstream effectors of wild-type p53 and is constitutively activated by oncogenic ETV6::NTRK3 fusions. Activated STAT3 and wild-type p53 negatively regulate each other. The mutant p53^R248W has been shown to form a highly stable complex with phosphorylated STAT3 that results in STAT3 activation (Schulz-Heddergott et al. 2018; Klemke et al. 2021). These transcription factors seem to collaborate to promote oncogenic transformation and malignant progression in several cancer types (Niu et al. 2005; Pham et al. 2020). As therapy with larotrectinib inhibits the aberrant activity of the STAT3 pathway (Doebele et al. 2015; Drilon et al. 2016), it is possible that the development of the R248W mutation in this tumor may represent a molecular mechanism aimed to readjust STAT3 expression levels to rescue and support cell proliferation and other oncogenic functions needed for tumor survival and progression. Interestingly, transcriptional expression of STAT3 was elevated in both recurrence specimens tested from this patient to levels higher than most solid tumors, including other infantile fibrosarcoma samples that were previously tested by our laboratory. However, the extent to which the copy gain of Chromosome 17 detected in this tumor may influence STAT3 dosage is uncertain.

Likewise, the SUFU truncating deletion detected in this sample has not been reported in patients with infantile fibrosarcoma. SUFU is a negative regulator of the hedgehog signaling pathway which is implicated in the development and progression of a variety of cancer types, such as medulloblastoma, glioblastoma, and colorectal carcinoma. A frameshift variant regarded as of uncertain significance in PTCH1, which is also in the hedgehog pathway, was reported in an NTRK1-fusion positive metastatic undifferentiated sarcoma in an adult patient (Hemming et al. 2020), which also harbored a somatic (likely inactivating) PTEN rearrangement, a KRAS G12V mutation and an NTRK1 G595R solvent-front mutation. Following unsuccessful therapy with larotrectinib, that patient reportedly showed durable response to selitrectinib. Cross-talk between the MAPK and hedgehog signaling pathways occurs in several types of cancer and contributes to clinical resistance to hedgehog pathway inhibitors; however, the potential role of SUFU in the pathogenesis and evolution of this tumor is uncertain.

The most common mechanisms of resistance to TRK inhibitors include NTRK kinase domain mutations and co-existing molecular alterations in the MAPK pathway (e.g., BRAF, KRAS, and MET genes) involved in reactivation of aberrant downstream signaling of NTRK (Okamura et al. 2018; Doebele 2019; Hemming et al. 2020). In the case reported herein, larotrectinib resistance can be attributed to the NTRK3 p.Phe617Leu (F617L) variant that was detected at a clonal level in the second recurrence sample of the tumor, although additional investigation is needed to determine if the TP53 and SUFU variants would play any synergistic role mediating resistance to NTRK inhibition in this tumor. The F617L variant has been reported in a 10-mo-old girl with an anterior mediastinal IFS harboring ETV6::NTRK3 fusion and another established NTRK3 resistance mutation, G623R (Oliver et al. 2021), as well as in a patient with gastrointestinal stromal tumor after disease progression with larotrectinib treatment (Hong et al. 2019), but in none of those instances concurrent alterations in the p53 or hedgehog signaling pathways were observed.

In conclusion, we report the first case of infantile fibrosarcoma with TP53 and SUFU mutations that correlated with recurrence after chemotherapy and the development of high-grade histologic changes and preceded the detection of the NTRK3 F617L gatekeeper resistance mutation. The clonal relationship between these variants could not be established by the sequencing analyses performed in the tumor, but based on the morphologic features observed on serial histologic assessments at different disease time points, they most likely contributed to disease progression and metastasis. Our report also emphasizes the importance of pursuing comprehensive genetic tumor testing for patients with disease progression, because such an approach could potentially guide clinically meaningful therapeutic decisions.

METHODS

p53 Immunohistochemistry

Immunohistochemical staining with p53 antibody (clone DO-7, Ventana Medical Systems) was performed using an autostainer (BenchMark ULTRA, Ventana Medical Systems) according to the manufacturer's instructions.

Fluorescence In Situ Hybridization

ETV6 break-apart fluorescence in situ hybridization (FISH) was performed on 4-micron slides from formalin-fixed, paraffin-embedded (FFPE) tumor tissue using the LSI ETV6 BA-20-ORGR probe (Empire Genomics) according to the manufacturer's protocols.

RT-PCR

RNA was isolated from FFPE tumor tissue using the RNeasy kit (QIAGEN) and cDNA was generated using SuperScript VILO cDNA Synthesis Kit (ThermoFisher Scientific). For the ETV6::NTRK3 fusion transcript, PCR amplification was performed using a fusion-specific primer pair (forward: 5′-ACCACATCATGGTCTCTGTCTC-3′; reverse: 5′-AGTTCTCGCTTCAGCACGAT-3′) and the Taqman probe: 5′-FAM-CATTGGGAGAATAGCAGATGTGCAGCAC-TAMRA-3′. The PCR was performed using an ABI 7500 Real Time PCR system (ThermoFisher Scientific).

Genomic Sequencing

Consent for clinical genomics sequencing of tumor and germline specimens was obtained at the patient's first initial visit by the admitting physician. In this case, the patient's tumor underwent clinical sequencing, and consent was obtained from the parents upfront according to routine practice. Tumor specimens were evaluated histopathologically to assess specimen adequacy. The recommended tumor purity is ≥40%. DNA extracted from frozen tumor tissue specimens and germline specimens using the Chemagic DNA Blood Kit special on a Chemagic 360 instrument (PerkinElmer) were used to construct libraries for WGS using Illumina TruSeq DNA LT PCR-Free Sample Kit and for WES using the Illumina TruSeq Exome Library Prep Kit (both from Illumina). RNA was extracted from the tumor samples and the Illumina TruSeq Stranded Total RNA LT Kit (Illumina) was used to generate libraries for transcriptome sequencing (RNA-seq). WGS, WES, and RNA-seq were performed on DNA and RNA extracted from the tumor specimen; WGS and WES were performed on DNA extracted from the patient's germline specimen. All libraries were sequenced using a paired-end 2 × 125 bp cycle protocol and sequencing by synthesis (SBS) technology on an Illumina NovaSeq 6000 instrument. For WGS, samples were sequenced to obtain a minimum of 40% of bases at 45× or greater depth of coverage throughout the coding regions (exons ±10 bp neighboring intronic regions). For WES, samples were sequenced to obtain a minimum of 65% of bases at 45× or greater depth of coverage. For RNA-seq, samples were sequenced to obtain a minimum of 15% of bases at 45× or greater depth of coverage. Variants should be present in a minimum of four reads to be reported. Equivocal variants should be present in two of the three methods (WGS, WES, RNA-seq) or be confirmed by an alternative method to be reported. Only coding regions (exons ±10 bp neighboring intronic regions) are evaluated. Data from WGS, WES, and RNA-seq were analyzed independently to validate the presence of lesions in two or more platforms. A combined analysis was also performed to provide higher coverage particularly in cases with tumor heterogeneity and low tumor purity and/or for regions of the genome that are difficult to sequence because of high guanine cytosine content. Sequencing data was analyzed via custom-designed bioinformatics pipelines on a SJCRH HIPAA-compliant high-performance computing system, using the hg19 (GRCh37) human genome reference sequence for alignment, as previously described (Rusch et al. 2018). Variants found in the tumor were compared to the matched germline to eliminate private benign polymorphisms. This assay detects copy-number variation, structural variation (tumor only), fusion transcripts (tumor only), single-nucleotide variants, small insertions, and deletions (indels). Data analysis was performed using publicly available software, publicly available databases, proprietary databases commercially available, and databases/software developed at St. Jude Children's Research Hospital. Variants are selected for review based on these resources, scientific literature, and other predefined variant features. A list of all variants and additional technical details on sequencing results found in these samples are available upon request.

TP53 Sequencing

Tumor specimens were evaluated histopathologically to assess specimen adequacy. DNA was extracted automatically from six 10-µm FFPE tumor tissue sections of each sample using the Maxwell 16 FFPE Tissue LEV DNA Purification Kit on a Maxwell 16 Research system (both Promega). DNA concentrations were determined with the Qubit HS DNA assay (Thermo Fisher Scientific) according to the manufacturers’ protocols. Target-enriched libraries were generated by Anchored Multiplex PCR (AMP) (Archer VariantPlex Custom panel) targeting exons 1–11 of the TP53 gene according to the manufacturer's protocol. Pooled samples were sequenced in a 2 × 151 bp MiSeq platform. Sequencing data was analyzed via the Archer Analysis software (version 6.2.1) on a St. Jude Children's Research Hospital HIPAA-compliant high-performance computing system, using the hg19 (GRCh37) human genome reference sequence for alignment. Single-nucleotide variants (SNVs), insertion/deletion (indel) variants, and internal tandem duplication (ITD) variant detection are included in the analysis. The limit of detection is >5% variant allele frequency for SNVs and indels.

ADDITIONAL INFORMATION

Data Deposition and Access

The data that support the findings of this study are available in the St. Jude Cloud at https://platform.stjude.cloud/data/samples, reference numbers SJST032952_D1, SJST032952_D2, and SJST032952_G1. The p. Arg248Trp TP53 and p.Glu283fs SUFU variants can be found in ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) under accession numbers SCV003762123.1 and SCV003762124.1, respectively.

Ethics Statement

Written consent was not obtained from family members because of patient and family availability. The study was reviewed and approved as exempt non-human subjects research by the Institutional Review Boards of St. Jude Children's Research Hospital (Institutional Review Board #00000029).

Author Contributions

L.V.F. and J.G. conceptualized the study. J.G., R.M., M.Ka., B.Se., and A.P. provided clinical care of the patient and clinical data. T.S., S.C.K., and R.R. performed histopathologic evaluation and provided histopathology data. L.V.F. analyzed and interpreted the molecular data. Z.S. developed methods for quality control assessment of sequencing data. L.V.F., M.Ka., R.M., and Z.R.A. wrote the original draft. T.S., S.C.K., M.K., R.R., B.Sh., and Z.R.A. assembled figures. All authors reviewed the writing and edited the manuscript. All authors have reviewed, edited, and approved the current version of the paper and its submission to CSH Molecular Case Studies.

Competing Interest Statement

The authors have declared no competing interest.