Refractory Alveolar Rhabdomyosarcoma in a 11-Year Old Male

$Refractory Alveolar Rhabdomyosarcoma in a 11-Year Old Male$

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Research Report for CSHL Molecular Case Studies

Refractory Alveolar Rhabdomyosarcoma in a 11-year old male

Fusion-Negative Alveolar Rhabdomyosarcoma

Cora A. Ricker1, Andrew D. Woods1, William Simonson2, Melvin Lathara3, Ganapati Srinivasa3, Erin R. Rudzinski4, Atiya Mansoor1, Robert G. Irwin2, Charles Keller1#, Noah E. Berlow1#

1 Children’s Cancer Therapy Development Institute, Beaverton, OR 97005 USA

2 Mary Bridge Hospital, Tacoma, WA 98403 USA

3 Omics Data Automation, Beaverton, OR 97005 USA

4 Department of Laboratories, Seattle Children's Hospital, Seattle, WA 98105 USA

# correspondence:

Noah Berlow PhD, 12655 SW Beaverdam Rd W, Beaverton OR 97005 USA, tel: 806-370-8119, fax: 270-675-3313, email: [email protected]

Charles Keller MD, 12655 SW Beaverdam Rd W, Beaverton OR 97005 USA, tel: 801-232-8038, fax: 270-675-3313, email: [email protected]

word count (4,144 words in main text, excluding Abstract, Methods, References and figure legends)

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Abstract

Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% five-year disease-free survival rate when a chromosomal fusion is present, and 40% five-year disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-year old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.

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Introduction

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma occurring in children with two major subtypes, embryonal and alveolar (Rudzinski et al. 2017). Embryonal rhabdomyosarcoma (ERMS) tends to have a more favorable prognosis and is mainly seen in the orbit, head, neck, and retroperitoneum (Rudzinski et al. 2017). Alveolar rhabdomyosarcoma (ARMS), on the other hand, has a less favorable prognosis and often involves the trunk and extremities

(Rudzinski et al. 2017). While PAX3:FOXO1 or PAX7:FOXO1 chimeric oncogenes are found in the majority of ARMS cases,

15% of rhabdomyosarcomas with available fusion data are negative for FOXO1 rearrangement (Rudzinski et al. 2017).

These cases are prognostically similar to embryonal rhabdomyosarcoma, which dictates their risk-based treatment

(Rudzinski et al. 2017). In general, fusion status serves as a better predictor of prognosis than histology (Rudzinski et al.

2017). The 5-year event-free survival (EFS) for fusion-negative ARMS is 29% (Rudzinski et al. 2017). Despite advancements in uncovering the clinical and mutational profiles of the myriad RMS subtypes, such as recurrent TP53 tumor suppressor gene loss of function in PAX fusion-negative (PFN) RMS, survival rates have not seen similar significant improvement (Breneman et al. 2003; Williams et al. 2004; Davis and Keller 2012; Malempati and Hawkins 2012; Rudzinski et al. 2017).

Several predisposition syndromes such as neurofibromatosis, Costello syndrome, and Li-Fraumeni syndrome have been linked to sarcomas (Farid and Ngeow 2016). Li-Fraumeni syndrome (LFS) is an autosomal dominant disorder commonly characterized by heterozygous germline mutations in TP53. LFS predisposes patients to a wide range of malignancies, including sarcomas, which account for 25-35% of all LFS-associated tumors and 1-10% of RMS (Olivier et al. 2003;

Gonzalez et al. 2009; Palmero et al. 2010; Ognjanovic et al. 2012). Data suggests that overall survival outcomes were improved with early detection of tumors through surveillance of patients with TP53 mutations (Ballinger et al. 2015).

However, detection of LFS can be challenging for clinicians. Presentation of clinical characteristics, surveillance strategies, and therapeutic interventions can all differ between patients given the wide range of malignancies associated with LFS.

Most TP53 mutations associated with LFS are missense mutations, which may or may not significantly affect protein function

(Olivier et al. 2003). The ambiguity of classifying these mutations as benign or deleterious can make clinicians reluctant to diagnose patients with LFS given the psychological ramifications that can come with diagnosis and active surveillance thereafter. The International Agency for Research on Cancer (IARC) TP53 Database has compiled TP53 mutations reported in published literature and can be used for further risk assessment (Hainaut et al. 1997). In the context of rhabdomyosarcoma, TP53 mutants have been associated with increased risk of second neoplastic malignancy (Pondrom et al. 2020) and associated with worsened overall survival (Casey et al. 2020), suggesting TP53 plays an important role in clinical progression and TP53 status should be investigated in the course of clinical decision making.

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Advanced stage or metastatic patients often receive intensified treatments, which historically show a 3-4% mortality rate from treatment-related toxicities (Crist et al. 2001). Molecularly-targeted therapies provide a promising approach for treatment of RMS patients as these therapies have the potential to reduce drug-induced toxicities compared to traditional chemotherapies. Here, we present a case of an 11-year-old male with fusion-negative ARMS who underwent standard of care followed by exhaustive precision medicine approaches to his progressive disease and a functional genomics characterization at end of life. Drug screening in conjunction with genetic analysis has provided insight to the biological status of the index case and potentially promising drug candidates for future preclinical and clinical studies.

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Results

Clinical Presentation

An 11-year old Caucasian male presented with a tender left upper arm mass. No recent injury was noted, but the patient had a history of prior supracondylar fracture of the distal humerus. Approximately 3 weeks later, an MRI of the humerus revealed a lesion within the left upper arm measuring approximately 2.6 x 2 cm in the axial plane and approximately 3.5 cm in the craniocaudal dimension within the triceps region of the left upper arm, possibly arising from the medial head of the triceps, with edema and enhancement down to the attachment of the mid-humeral shaft (Figure 1). The differential diagnosis included soft-tissue fibroma, myxomatous tumor, or neuromuscular mass. Soft tissue sarcoma was not excluded. Two weeks after presentation, a follow-up x-ray of the upper arm mass found that the humerus appeared to be normal without focal bony abnormality, cortical disruption, or periostitis (Supplemental Figures 1-2). Prominent soft tissue was found along the medial aspect of the distal portion of the upper arm corresponding in location to the known mass. The mass was also slightly seen dorsally on the internally rotated view. No calcifications or ossifications were identified in the soft tissues.

Four weeks after presentation, the mass had grown dramatically in size with increased pain. A biopsy of the primary left upper, posterior arm mass revealed a small round blue cell malignancy (Figure 2). Histology was described as a poorly differentiated malignant neoplasm arranged in loosely cohesive sheets of primitive round to polygonal cells with interruptions by few fibrous septa and scattered large vessels. Desmin was reported to be diffusively positive, tumor cell myogenin was positive (~60%), and cytokeratin was found negative. PTEN, BAF47 (INI1/SMARCB1), and BRG1 (SMARCA4) were positive. Focal areas of lower cellular cohesion created a vague papillary appearance. The individual tumor cells were reported to be pleomorphic, ranging from intermediate to large. The majority of cells had scant cytoplasm; however, rare cells had a moderate amount of densely eosinophilic cytoplasm with possible cross-striations (rhabdomyoblasts). Multiple scattered multinucleated giant cells were present which also contained deeply eosinophilic cytoplasm. Random cells had enlarged hyperchromatic and anaplastic nuclei. Numerous apoptoses and mitoses were seen. Rare, atypical mitotic figures were noted. The patient was diagnosed with a malignant neoplasm of connective and other soft tissue of upper limb, including shoulder. Tumor morphology suggested an ARMS tumor. However, fluorescence in situ hybridization (FISH) studies were negative for the classic FOXO1 t(2,13) or t(1:13) rearrangements and did not support a diagnosis of ARMS.

Bilateral bone marrow biopsies were performed, and were negative for small round blue cell morphology.

Five days after the biopsy, a follow-up post-resection multiplanar multisequential MRI scan of the left humerus was performed with and without intravenous contrast. The MRI revealed a heterogeneous mass much larger than the previous scan, measuring 5.2 x 6 x 16.8 cm (AP x TR x CC). At the more inferior aspect of the mass, a central area of low signal intensity was found on the T1 weight sequence with peripheral rim enhancement measuring 8 x 2.4 cm that might represent 5

Downloaded from molecularcasestudies.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press a central cystic component (possibly representing central necrosis or fluid collection given the recent surgery). The mass abutted the ulnar neurovascular bundle. Nonspecific, small left axillary lymph nodes were noted. Staging investigations through a chest CT revealed no lung metastasis.

Twelve days after diagnosis, the lymph nodes were biopsied and sent for histological analysis. The patient was found to have a metastatic neoplasm consistent with rhabdomyosarcoma in 4 out of 31 lymph nodes. The patient was classified as having stage III, intermediate risk group III RMS, N1, M0 according to the Children’s Oncology Group (COG) protocol. The patient began treatment with protocol ARST0531 consisting of 42 weeks of vincristine, adriamycin, cyclophosphamide

(VAC) and alternating with vincristine and irinotecan (VI). Radiation was started six weeks after diagnosis and given for a month and a half for local control (5040 cGy in 28fx at 180cGy/fx using 3DCRT). Complications of treatment included mucositis, clostridioides difficile infection, headache, and exacerbated neuropathy.

Sixteen months after diagnosis (six months after chemotherapy was completed), chest CT showed new lung masses, raising concerns of metastatic recurrence. No arm pain was noted. On the PET-CT scan, the fluorine-18-deoxyglucose (FDG) signal in the right lower lung lobe mass was most consistent with progressive metastatic disease. A new left lower lung lobe nodule further raised concern of metastatic disease, despite lack of discernable FDG activity in the left lower lobe.

Lobectomy of the right lower lobe revealed a small round blue cell tumor, consistent with metastatic rhabdomyosarcoma

(1.6 cm) with vascular invasion and positive bronchovascular margin. Clear margins were not obtained. The patient was diagnosed with relapsed stage IV ARMS.

Eighteen and a half months after diagnosis (two and a half months after relapse), the patient began reinduction chemotherapy with vinorelbine, cyclophosphamide, and temsirolimus (ARST-0921) and supplemented with glutamine and vitamin B2. Three months after starting reinduction chemotherapy, the patient completed 28 days of radiation to the right lung. Relapse treatment was given over a period of seven and a half months. At the end of relapse treatment, a full body

MRI and local CT showed stable disease. Possible nodules in the left lung and a mass in the lower left forearm were noted.

Two months after completing relapse treatment (twenty eight months after initial diagnosis), the patient enrolled onto clinical trial ADVL1515 and started treatment with prexasertib, a CHEK1 inhibitor. Radiographic scans one month after enrollment onto ADVL1515 showed new cancerous growth in the arms and lungs. Prexasertib treatment was stopped and the patient enrolled onto clinical trial ADVL1312 and began treatment with MK-1775, a WEE1 inhibitor. After one month of MK-1775 treatment, radiographic scans showed continued progression and therapy was stopped. The patient had also enrolled onto the Pediatric MATCH trial (APEC1621), but no targetable mutations were found from genomic sequencing.

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Thirty months after initial diagnosis the patient began daily oral pazopanib, a 5-day course of temozolomide, and radiotherapy to the left forearm and lungs for local control. A biopsy was performed for clinical trial eligibility determination and development of a patient-derived xenograft at Champions Oncology. One month later (thirty-one months after initial diagnosis) the patient was determined to be eligible for clinical trial NCT-02162732, a genomically-guided therapy trial for children with cancer. The patient began treatment with vorinostat and palbociclib and supplemented with myrrh oil. The patient was also treated with weekly thoracentesis.

Two weeks later, the patient presented to the ER with shortness of breath and chest pain. The necrotic tumor in the left arm was opened and debrided and the patient received IV clindamycin. A drain was placed for the patient’s malignant pleural effusion with loculation. A week later (thirty-two months after initial diagnosis), the patient reported a moderate increase in symptoms attributed to his disease over a 24-hour period. Doppler ultrasound showed acute occlusive venous thrombosis involving the right internal jugular vein, right subclavian vein, right axillary vein, and right cephalic vein, with additional nonocclusive thrombus in the right basilic vein (superior vena cava syndrome syndrome). CT chest scans showed near complete opacification of the right hemithorax (presumed malignant), extensive thrombosis, and an increased left lower lobe lesion measuring 3.9 cm. Pulmonary fluid increased with drainage reaching ~1.5 L/day, and the patient required increased support and spent more time on a bilevel positive airway pressure (BiPAP) machine. Medical treatment was not felt to be a viable option given the patient’s significant bleeding risk. Vorinostat and palbociclib, as well as supportive care, were discontinued by parental request. The patient was taken off BiPAP and passed away at the age of 14, approximately thirty- three months after initial diagnosis. The discharge diagnosis was progressive hypoxic respiratory failure, malignant pleural effusion, and relapsed rhabdomyosarcoma.

Genomic Analysis

Cytogenetics revealed 46,XY[20], normal male karyotype. Previous testing by OncoPlex identified a TP53 p.V172F (Tier

2A) mutation with associated loss of heterozygosity, suggesting that both copies of the gene are expected to be inactivated in the tumor tissue. FGFR1 amplification (Tier 2A/4A, log2 tumor/normal read ratio = 0.96 by whole exome sequencing

[WES]) and MYCL amplification (Tier 5A, log2 tumor/normal read ratio = 1.91 by WES) were both present. Targeted sequencing (Ambry Genetics) recently confirmed that the patient shared the same TP53 mutation (V172F) as his mother.

Two previous generations also had early onset breast cancer on the mother’s side (maternal grandmother and great grandmother).

To identify further genomic alterations and potential therapeutic targets, we sequenced and analyzed DNA and RNA isolated from a tumor tissue sample from the left arm, as well as DNA from patient-matched normal tissue. Tissue for sequencing

Downloaded from molecularcasestudies.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press experiments were provided post-mortem. After filtering for nonsynonymous mutations not identified in the database of single nucleotide polymorphisms (Sherry et al. 2001), 109 mutations were found. No somatic mutations were found in genes commonly mutated in PFN tumors, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR

(Stratton et al. 1989; Taylor et al. 2000; Taylor et al. 2009; Shern et al. 2014). However, two germline mutations were found in TP53, including the same variant found in the patient’s mother (c.514G>T p.Val172Phe) (Table 1) which is considered likely pathogenic for hereditary cancer-predisposing syndrome (ClinVar ID: 428909). Another missense variant in TP53

(c.215C>G p.Pro72Arg) was found to alter drug response in several neoplasms but found to not be associated with Li-

Fraumeni syndrome 1 (ClinVar ID: 12351). TP53 mutations have previously been associated with response to the majority

(six of eight) therapeutic regimens administered to the patient (Table 2). No somatic mutations were found in genes previously reported to be recurrently mutated in RMS (Shern et al. 2014); however, germline protein coding mutations were found in FGFR4 (c.28G>A p.Val10Ile; c.407C>T p.Pro136Leu) and nonsense mediated decay variants were found in NF1

(c.37T>C p.Ter13Arg).

In addition to the germline variants found in TP53, a germline disruptive inframe insertion variant was found in FAM83D

(c.345 347dupGGC p.Ala116dup), along with copy number gain in FAM83D (log2 ratio 0.7). Further, FAM83D is overexpressed (23.5-fold, 2.84 TPM vs. 0.1207 TPM) than median expression of the GTEx normal skeletal muscle tissue cohort (n=803). The clinical significance of the FAM83D mutation (c.345 347dupGGC p.Ala116dup) is at present unknown; however, FAM83D overexpression occurs frequently in hepatocellular carcinoma, ovarian cancer and metastatic lung adenocarcinomas (Inamura et al. 2007; Ramakrishna et al. 2010; Liao et al. 2015; Wang et al. 2015) and correlated with significantly decreased overall survival and metastatic relapse-free survival in breast cancer (Wang et al. 2013). Further, elevated FAM83D was found to correlate with TP53 mutations and genome instability (Walian et al. 2016).

Other somatic mutations were identified, including KDM2B and HCAR1. Both had truncating mutations and copy number loss (log2 ratio under -0.4). Several genes located on chromosome 1 were found to be mutated, amplified, and transcripts per million (TPM) over 100 or under 10 (Figure 3). Using RNA isolated from the tumor of the left arm, 10 gene fusions were identified using the STAR-Fusion analysis pipeline, with many fused genes located on chromosome 1 (Figure 3,

Supplemental Table 1). Notably, RNA sequencing data from the tumor sample procured following recurrence did not identify any of the typical gene fusion events associated with alveolar rhabdomyosarcoma, including PAX3:FOXO1,

PAX7:FOXO1, PAX3:NCOA1, PAX3:NCOA2, and PAX3:FOXO4. A second gene fusion analysis pipeline FusionCatcher also did not find evidence to support the presence of the ARMS-associated gene fusions (Supplemental Table 2). The lack of evidence of PAX3:FOXO1 in the patient’s tumor at diagnosis (confirmed by FISH analysis) and lack of any evidence to

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The presence of clustered fusion events suggests and 10 switches in copy number status suggests chromosome 1 may be chromothripsis-positive with high confidence event on chromosome 1(Bolkestein et al. 2020; Cortés-Ciriano et al. 2020).

Notably, the lesions appear over a small region of the genome, suggesting the clustered variants may be classified as a kataegis event. FGFR4, CCND2, and IGF2 were found to be the top genes overexpressed in PFN RMS cases in a previous study (Shern et al. 2014) and were also found to be overexpressed (12-fold, 16-fold, and 153-fold overexpression, respectively) in the patient’s tumor compared to a panel of normal skeletal muscle tissue samples (n=803) from the

Genotype-Tissue Expression (GTEx) project (Consortium 2013). A complete list of gene mutations and associated gene expression and copy number variation data is provided in Supplemental Table 3 and Supplemental Table 4.

Given the relative rarity of fusion-negative ARMS cases amongst the RMS clinical milieu, we investigated genomic similarities between the index case and other clinical RMS samples using a previously published hierarchical clustering dendrogram approach (Supplemental Figure 3) (Ricker et al. 2020). Similar to previous findings regarding genomic similarities between ERMS and fusion-negative ARMS (Williamson et al. 2010), CF-00449 clusters amongst ERMS samples, as do the majority of other fusion-negative ARMS samples. Taken together, the histopathology designation of

ARMS by multiple pathologists, the lack of evidence of fusions from FISH analysis and RNA sequencing analysis, and the genomic similarity to ERMS more so than ARMS, strongly support the diagnosis of CF-00449 as a fusion-negative ARMS.

Functional analyses

To identify molecular compounds that would inhibit tumor growth, we conducted a chemical screen consisting of a panel of

60 agents from a clinically relevant drug portfolio on cells harvested from the patient’s pleural fluid (Table 3, Supplemental

Figure 4). Several drugs were highly active, including panobinostat (IC50 = 6 nM, pan HDAC inhibitor), brefeldin A (IC50 =

21 nM, ATPase inhibitor), CUDC-907 (IC50 = 99 nM, HDAC1/2/3/10 + PI3Kα inhibitor), YM155 (IC50 = 9 nM, Survivin inhibitor), INK128 (IC50 = 5 nM, mTORC1/2), MK-1775 (IC50 = 79 nM, WEE1), thapsigargin (IC50 = 9 nM, SERCA), BMS-

754807 (IC50 = 66 nM, IGF1R + AURK inhibitor), JQ1(IC50 = 92 nM, BET (BRD4) inhibitor), and mithramycin A (IC50 = 89 nM, SP1 inhibitor). TP53 mutations were associated with response in multiple high and moderate sensitivity agents (Table

4). Chemical screening was performed using cells isolated pleural effusion fluid while the patient was still alive, and did not influence clinical decision making.

The patient received the nonselective class I/II HDAC inhibitor, vorinostat, with limited response. Although HDAC inhibitors panobinostat, CUDC-907, and entinostat were found to impair growth of tumor cells in vitro, the HDAC inhibitor CUDC-101 9

Downloaded from molecularcasestudies.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press did not demonstrate significant activity in vitro. Panobinostat is a pan-HDAC inhibitor with activity against all HDAC enzymes, while CUDC-907 inhibits class I-III HDAC genes plus phosphoinositide 3-kinase alpha (PI3K-alpha), and entinostat inhibits classes I and III. Meanwhile, CUDC-101 inhibits class I/II HDACs as well as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER). The discrepancy of response to tested HDAC inhibitors can potentially be explained by differences in their mechanism of action (Li and Seto 2016). These results suggest that inhibition of class I and II HDACs may not be sufficient for tumor suppression of PFN ARMS and targeting of other HDAC classes may have a more significant contribution to tumor survival. Further supporting this notion, panobinostat and entinostat were found to have different consequences in PAX3:FOXO1 ARMS (Bharathy et al. 2018).

The WEE1 inhibitor, MK-1775, which had high potency in vitro (IC50 = 79 nM), was administered to the patient under trial

ADVL1312 and did not prevent progression. Prexasertib (LY2606368) was also used to treat the patient under trial

ADVL1515 but did not suppress the patient’s tumor growth, consistent with the limited in vitro activity of prexasertib (IC50 >

10 µM).

Clinically, temsirolimus (mTOR inhibitor) in combination with vinorelbine and cyclophosphamide resulted in stable disease with potential tumor growth. INK128 (mTORC1/mTORC2 inhibitor) and BKM120 (IC50 = 198 nM, mTOR and PI3K inhibitor) both had high activity in tumor cells, while sirolimus (also an mTOR inhibitor) did not have activity in tumor cells in vitro.

BKM120 activity suggests that tumor growth and progression may be dependent on multiple pathways in PFN ARMS.

Additionally, agents associated with alteration of TP53 expression also demonstrated in vitro efficacy, including prima-1

(IC50 = 135 nM) and CUDC-305 (IC50 = 137 nM, HSP90 inhibitor). Previous studies have found that prima-1 reactivates

TP53 which induces high levels of apoptosis, and has demonstrated tumor reduction with no apparent toxicity in p53 mutant tumor xenografts in SCID mice (Bykov et al. 2002). Despite efforts in elucidating the mechanism of TP53 reactivation by prima-1, the mechanism remains largely unknown. Several factors, including hypoxic conditions and proteins present within and surrounding the cell, can affect the sensitivity of cancer cells to prima-1 (Rieber and Strasberg-Rieber 2012; Peng et al. 2013). Similarly, TP53 expression can be altered by HSP90 inhibitors (such as CUDC-305), and thus could potentially be beneficial for TP53 mutant patients (Lin et al. 2008).

Discussion

In this study, we have presented a case of an 11-year-old male with fusion negative alveolar rhabdomyosarcoma (ARMS) bearing germline TP53 mutations, one of which (c.514G>T, p.Val172Phe) is a hereditary mutation likely predisposing to Li-

Fraumeni syndrome (LFS). The index case patient presented with a tender left upper arm mass and no injury was initially 10

Downloaded from molecularcasestudies.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press noted. Tissue injury is widely reported to be a preceding factor in sarcoma development, with approximately 13% of patients with accidental injury to the extremities at risk of developing sarcoma (Olsson and Wagner 2017), likely due to activation of satellite cells to generate muscle precursor cells to repair the skeletal muscle (Shadrach and Wagers 2011). We suspect that the prior supracondylar fracture of the distal humerus was a preceding factor which potentially promoted sarcoma development in conjunction with germline TP53 mutations.

Our genomic findings were consistent with previous genetic findings on PFN ARMS cases, including overexpression of

FGFR4, CCND2, and IGF2 and significantly higher rates of non-synonymous somatic mutations than the PAX-gene fusion positive tumors (PFP) (17.8 vs. 6.4, p = .0002) (Shern et al. 2014). Whole-exome sequencing identified 109 nonsynonymous mutations for this case (approximately 3.6 mutations/MB), most consistent with a PFN genotype diagnosed at a later age. No somatic mutations in genes commonly mutated in PFN tumors were found in this case, including TP53,

HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000; Taylor et al.

2009; Shern et al. 2014). However, missense mutations were found in both TP53 and FGFR4 in the germline, as well as a nonsense-mediated decay mutation in NF1.

The biological significance of the FGFR4 germline variants and their contribution to sarcoma initiation, growth, and progression is largely unknown. Several previous studies have focused on the role of FGFR4 in RMS as a regulator of myogenic differentiation and muscle regeneration after injury. Somatic mutations in FGFR4 have been shown to contribute to the growth and metastasis of RMS, higher expression of FGFR4 correlates with poor survival, and FGFR4 suppresses phospho-Akt in RMS (Taylor et al. 2009). Germline variants in FGFR4 may play a similar role in promoting the invasion and metastasis of RMS.

The index case patient was treated with several targeted therapies, including temsirolimus (mTOR inhibitor), prexasertib

(CHEK1 inhibitor), MK01775 (WEE1 inhibitor), pazopanib (multi-kinase inhibitor specific for VEGFR, PDGFR, c-KIT and

FGFR), vorinostat (HDAC inhibitor), and palbociclib (CDK4/6 inhibitor). Functional testing of same or similar agents resulted in mixed concordance between in vitro response and clinical response. Patient tumor cells were highly sensitive to MK-

1775, while clinical use resulted in continued disease progression. Efficacy of WEE1 inhibition may have differed from the cells that were harvested from intrapleural fluid because of host factors (e.g., drug metabolism) (HogenEsch and Nikitin

2012). HDAC inhibitor vorinostat was also provided to the patient with limited response, although multiple HDAC inhibitors

(panobinostat, CUDC-907, and entinostat) were sensitive in vitro, while one HDAC inhibitor (CUDC-101) was not sensitive in vitro. CHEK1 inhibitor prexasertib was also used to treat the patient but did not suppress the patient’s tumor growth, reflecting in vitro response of prexasertib. mTOR inhibitor temsirolimus (mTOR inhibitor) plus vinorelbine and 11

Downloaded from molecularcasestudies.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press cyclophosphamide stabilized the patient’s disease. Two mTOR-associated agents (INK128 and BKM120) caused reduction in cell viability in vitro, while one mTOR agent (sirolimus) had limited in vitro efficacy.

TP53 interacting agents (prima-1 directly and CUDC-305 operating through HSP90) were also promising in vitro. Treatment with prima-1 refolds TP53 in mutated cells into the wildtype conformation and restores sequence-specific DNA binding

(Zhang et al. 2018). Other therapies that restore TP53 function, such as reactivating p53 and inducing tumor apoptosis

(RITA), may merit investigation for PFN ARMS. Additionally, repression of TP53 is thought to be controlled by the TP53- p21-DREAM-E2F/CHR pathway, which regulates FAM83D (Engeland 2018). Future studies should aim to further understand how germline variants in FAM83D coupled with TP53 germline mutations contribute to sarcoma initiation, growth, and progression. Understanding the biological and clinical significance of FAM83D could have predictive and prognostic implications across several cancer types and may be relevant for the index case patient, given FAM83D aberrations present in patient’s germline.

In summary, we have reported a PFN ARMS case with an inherited likely pathogenic germline TP53 mutation. The patient was resistant to several targeted therapies, demonstrating a need for improving our understanding of the etiology and pathogenesis of PFN ARMS and discovering effective therapeutic approaches with limited treatment-related toxicities. In this case study, several novel mutations were reported and potential therapeutic interventions were identified for further study to motivate preclinical and clinical studies that aim to improve survival for fusion-negative RMS.

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Methods

Primary cell culture

Approximately 2 liters of pleural fluid was collected from a chest drain catheter and immediately chilled on ice. Twenty-four hours after initial collection, the fluid was transferred to 50 mL conical vials and centrifuged at 1200 rpm for 10 minutes.

Resulting cell pellets were broken up with Gibco RPMI 1640 media and checked for viability with 0.4% Trypan Blue Stain, then counted on a Life Technologies Countess II cell counter. Three million viable cells were used for immediate drug screening. Remaining cells were added to 15 cm tissue culture plates containing Gibco RPMI 1640 media supplemented with 10% Fetal Bovine Serum and 1% penicillin/streptomycin. The plates were placed in a water-jacketed 37˚C incubator with 5% CO2. After 72 hours, the media was changed and the remaining cellular debris was removed. The semi-adherent cell culture continued to expand for a minimum of 18 passages. Every 2-5 passages, stocks were collected in cryovials containing an FBS/10% DMSO cryopreservation media and then slow cooled and stored in liquid nitrogen for future use.

Drug screen

Cells from culture were counted, resuspended in media, and plated into white walled 384-well plates containing pre-diluted known concentrations of various drugs. After 72 hours, an equal volume of Cell-Titer Glo 2.0 (G9243, Promega, Madison,

WI) was added to each well. The cells were then incubated at room temperature while rocking in the dark for 15 minutes.

Cell viability was measured by luminescence with the BioTek Synergy HT plate reader (BioTek, Winooski, VT). EC50 values were calculated using GraphPad Prism (San Diego, CA, USA).

Whole-exome sequencing

Tumor and matched normal exome sequencing data were analyzed for the presence of somatic point mutation, somatic functional and structural mutations, potential germline mutations, polynucleotide insertions and deletions, and gene copy number variation. Somatic mutations, variations, and indels were called using Genome Analysis Toolkit (GATK) Version 4.0 with strict calling criterion (Tumor logarithm, of odd (TLOD) scores above 6.3) with the GRCh38 human reference genome.

Gene copy number variations were identified using samtools and VarScan2 quantified as a log ratio of tumor copy to normal copy. Regions with a log ratio > 0.40 were called as gained, regions with a log ratio < -0.40 were called as lost. Genes overlapping gained or lost regions by at least 15% of the gene’s genomic region were called as gained or lost, respectively.

Sequencing coverage is provided in Supplemental Table 5.

RNA sequencing

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RNA sequencing data was analyzed for gene expression and gene fusion events. Transcriptome data was aligned to STAR

– derived human transcriptome from GRCh38 human reference genome. Normalized gene expression was quantified using

RSEM. Non – tumor gene expression data was not provided, thus region – specific, skeletal muscle tissue, gene expression data was accessed from the Genotype – Tissue Expression (GTEx project to serve as a population normal and to identify under expressed and overexpressed genes). Gene fusion events were identified using STAR-Fusion (Haas et al. 2019) and

FusionCatcher (Nicorici et al. 2014) to identify transcriptome reads around the junction of fused genes and identify probable gene fusion events. Both fusion analyses were also performed using the GRCh38 human reference genome. Sequencing coverage is provided in Supplemental Table 5.

Hierarchical Clustering

The hierarchical clustering analysis approach was published previously (Ricker et al. 2020). Briefly, CF-00449 sequencing data was clustered amongst a cohort of RMS samples collected from multiple sources. Unsupervised hierarchical clustering was performed using average-linkage clustering and Euclidean distance metric. Hierarchical clustering analysis was performed in RStudio Version 3.6.2.

Circos plots

Circos plots to visualize the collection of genomics and transcriptomics data were generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Genes identified as bearing variants, having increased gene copy number, and overexpressed. Also identified in blue are genes involved in identified gene fusion events, and genes identified as being of interest due to overarching genetic and transcriptomic features in the tumor samples.

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Figure Legends

Figure 1. Orthopedic MRI of the humerus. An ovoid lesion (red asterisk) was found within the triceps region of the left upper arm utilizing a 1.5 Tesla magnet with the following sequences: axial T2 Turbo Spin Echo (TSE) with fat saturation, axial T2 TSE pulse, axial T1 combination, and axial T1 TSE after 4.6 mL of gadolinium (+ C) (from top to bottom, left). The following sequences were also obtained: coronal T1 TSE + C with fat saturation, coronal T1 TSE with fat saturation, coronal

Short-TI Inversion Recovery (STIR) combination, and sagittal T1 TSE + C with fat saturation (from top to bottom, right). The lesion measured approximately 2.6 x 2 cm in the axial plane and approximately 3.5 cm in the craniocaudal dimension (top, left and top, right). The lesion demonstrated T1 hypointensity and heterogeneous, but T2 hyperintensity without significant fat saturation. Within the ovoid region, there are two punctate foci of STIR hypointensity (29/19) and T1 hypointensity.

Figure 2. Histology slides from alveolar rhabdomyosarcoma case. Hematoxylin and eosin (H&E) staining of the left forearm (A), right lower lobe of the lung (B), pleural fluid (C) showing a metastatic small round blue cell malignancy, 200x magnification of right lower lobe of the lung (D), 400x magnification of right lower lobe of the lung (E), and 200x magnification of myogenin staining (F). Scale bars 300 µM.

Figure 3. Circos plot. A Circos plot was generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Exome data was analyzed for somatic point mutation, indel, and copy number variation data, as denoted by the inner and middle rings, respectively. Gene expression from RNA sequencing data was plotted on the outer ring.

Genes listed in blue bear germline mutations, while genes listed in red bear somatic mutations.

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Tables

Table 1. Selected variants of known biological interest (aligned to GRCh38 reference genome)

Gene Source Chr Trans HGVS HGVS dbSNP COSMIC Cli Gen TP CNV Clinical Nam cript DNA Protein nV otyp M Log Significanc e ID Ref Ref ar e Ratio e ID TP53 Germline 17 ENST c.514 p.Val17 rs113169 COSM33783 428 Het 11.7 -0.33 Likely 00000 G>T 2Phe# 1043 54 909 pathogenic 26930 COSM33783 (Hereditary 5.8 55 cancer- COSM35483 predisposing 9 syndrome) COSM35484 0 COSM35484 1 COSM35484 2 COSM44240 TP53 Germline 17 ENST c.215 p.Pro72 rs104252 COSM25006 123 Het 11.7 -0.33 Drug 00000 C>G Arg 2 1 51 response 26930 COSM37661 (Li-Fraumeni 5.8 90 syndrome 1) COSM37661 91 COSM37661 92 COSM37661 93 Het, Heterozygous

# Mutation was also observed in the Oncoplex Panel.

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Table 2. Possible treatment-related genomic features

Protocol/ Therapeutic regimen Molecular target Genomic features clinical trial ARST0531 VAC (vincristine, N/A cyclophosphamide (TP53: PMID17388661, adriamycin, PMID16243804, PMID26438783) cyclophosphamide) + irinotecan (TP53, PMID25567130) alternating vincristine and irinotecan (VI) ARST091 vinorelbine, mTOR cyclophosphamide (TP53: PMID17388661, cyclophosphamide, PMID16243804, PMID26438783) temsirolimus ADVL1515 prexasertib CHEK1 N/A ADVL1312 MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793

ADVL1312 pazopanib tyrosine kinases, TP53: PMID26646755, PMID25669829 VEGF ADVL1312 temozolomide alkylating agent TP53: PMID21730979, PMID24248532 NCT02162732 vorinostat HDAC inhibitor TP53: PMID26009011, PMID25669829 (class I and class II)

NCT02162732 palbociclib CDK4/CDK6 inhibitor N/A (supplemented with myrrh oil); weekly thoracentesis

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Table 3. Drug screen validation using cells from pleural fluid

Drug Target IC50 INK128 mTORC1,2 5 1 nm - 100 nm Panobinostat pan HDAC 6 100 nm - 500 nm YM155 Survivin 9 500 nm - 1000 nm Thapsigargin SERCA 9 1000 nm - 5000 nm Brefeldin A ATPase 21 5000 nm - 10000 nm BMS-754807 IGF1R, AURK 66 MK-1775 Wee1 79 Mithramycin A SP1 89 JQ1 BET (BRD4) 92 CUDC907 HDAC1,2,3,10, PI3Kα 99 ABT-263 BCL-2, BCL-XL, BCL-W 113 Volasertib PLK1/2/3 134 Prima1 TP53 135 CUDC-305 hsp90 137 JIB4 JMJD 139 Entinostat HDAC1,3 141 UNC0642 G9a/GLP 143 Crizotinib c-MET, ALK 167 SGI-1776 PIM1/2/3 kinase 186 AZD 8931 EGFR, HER2/3 189 BKM120 PI3K, mTOR 198 Dinaciclib CDKs 200 AZD5363 AKT1-3 204 GSK126 EZH2 265 I-BET-762 BET (BRD1-4) 468 PIK-93 PI4KIII-beta, PI3K-alpha 561 AMG900 AURK-A,B,C >10000 Aprepitant NK1R >10000 AZD8931 EGFR, HER2/3 >10000 BIX 01294 G9a >10000 CHIR-99021 GSK-3α,β >10000 Cocoa CFTR >10000 CRM1 Inhibitor III CRM1, exportin 1 >10000 CUDC101 HDAC1,2, EGFR, HER >10000 CUDC-427 IAP >10000 DAPT γ-secretase (Aβ) >10000 Dasatinib Abl, Src and c-Kit >10000 Dorsomorphin AMPK/BMP >10000 EPZ-5676 DOTL1 >10000 Galusertinib TGFβ receptor I (TβRI) >10000 21

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GSK 923295 CENP-E >10000 GSK-J4 H3K27 >10000 LDN-193189 BMP, ALK2/3 >10000 LDN-212854 ACVR1/BMPRs >10000 LSD1 Inhibitor IV LSD1 >10000 LY2606368 Chk1 >10000 LY2874455 FGFR1-4 >10000 Masitinib KIT, PDGFRα/β >10000 Midostaurin Kinase >10000 MK8245 SCD1 >10000 Naproxen Cox 1,2 >10000 OSI-906 IGF1R, InsR >10000 Pemetraxed TS, DHFR, GARFT >10000 PF-573228 FAK >10000 PF670462 CK1D, CK1E >10000 PFI-3 SMARCA2/4 >10000 Red 25 DNMT >10000 Sirolimus mTOR >10000 Toceranib KIT, VEGF2, PDGFR-b >10000 Trametinib MEK1/2 >10000

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Table 4. Drug activity genomic-related features*

Drug Inhibitor Genomic-related features panobinostat pan HDAC N/A brefeldin A ATPase N/A

CUDC-907 HDAC 1,2,3,10, PI3Kα N/A YM155 Survivin inhibitor N/A INK128 mTORC1/2 N/A MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793

thapsigargin SERCA N/A BMS-754807 IGF1R, AURK N/A JQ1 BET (BRD4) N/A High activity mithramycin A SP1 N/A GSK126 EZH2 N/A CUDC-305 HSP90 N/A AZD5363 AKT1-3 TP53: PMID24694055, PMID17145876, PMID17455259 ABT-263 BCL-2, BCL-XL, BCL- TP53: PMID24694055 W I-BET-762 (BET) BRD1-4 N/A dinaciclib CDKs N/A entinostat HDAC 1,3 N/A crizotinib c-MET, ALK TP53: PMID24694055 BKM120 PI3K, mTOR N/A SGI-1776 PIM 1/2/3 kinase N/A volasertib PLK 1/2/3 N/A UNC0642 G9a/GLP N/A prima1 TP53 N/A AZD 8931 EGFR, HER 2/3 N/A Moderate activity JIB4 JMJD N/A

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FIGURE 1

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FIGURE 2

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FIGURE 3

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Additional Information

Data Deposition and Access

All raw data was deposited to our CuReFAST database. DNA and RNA sequencing data from the patient’s sample are available through the European Genome-Phenome Archive (EGA) under accession ID EGAS00001004828. For hierarchical clustering analysis, data from the Gene Expression Omnibus (GSM758578, GSE138269, GSM984615), EGA

(EGAS00001003981), and dbGaP (accession number: phs001121.v1.p1) were used. Champions Oncology data were received from the Champions TumorGraft database. Genomics data from PDX models from The Jackson Laboratory are available through the Mouse Tumor Biology Database (http://tumor.informatics.jax.org/mtbwi/pdxSearch.do).

Ethics Statement

The genetic studies and publication of clinical details was approved by the Children’s Cancer Therapy Development Institute

Institutional Review Board (IRB) and with full written informed consent obtained from parents of the proband.

Author Contributions

Study design: CK, CR

Data collection and analysis: CK, CAR, AW, AM, ER, NEB

Contributed materials: BS, RI, WS

Manuscript writing: CK, CAR, AW, NEB

Acknowledgements

We are tremendously grateful for all the patients who kindly donated their tumor tissue as part of our CuReFAST initiative.

This work was supported through Building Blocks to a Cure (1:1 match) crowdfunding campaign on Consano

(https://consano.org/projects/building-blocks-to-a-cure-11-match/), the Children’s Cancer Project, and the Sam Day

Foundation.

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Supplementary Information

Supplemental Figure Legends

Supplemental Figure 1. Orthopedic MRI scan with axial view. On the left, axial T2FS TSE, axial T2 TSE, T1 combo, and axial localizer from top to bottom. On the right, Axial T1fs TSE, T1FS TSE + C, T1FS TSE + C, and STIR combination.

Supplemental Figure 2. Orthopedic MRI scans with coronal and sagittal view. On the left, coronal T1FS TSE + C, coronal T1FS TSE, and coronal STIR combination. On the right, localizer coronal low, localizer sagittal low, and sagittal

T1FS TSE + C. On the bottom, coronal T1 combination.

Supplemental Figure 3. Hierarchical clustering of CF-00449 genomic data amongst RMS cohort samples. RMS samples clustered based on DNA mutation data and RNA expression data from multiple RMS clinical samples. Heatmaps represent gene expression in log2 scaled values, binary mutation status, and binary metadata status.

Supplemental Figure 4. Image of pleural fluid derived cell culture 72 hours after plating.

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SUPPLEMENTAL FIGURE 1

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SUPPLEMENTAL FIGURE 2

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SUPPLEMENTAL FIGURE 3

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SUPPLEMENTAL FIGURE 4

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Supplemental Table 1. Fusions identified from RNA sequencing via STAR-Fusion analysis (aligned to GRCh38 reference genome)

Fusion Name Junction Read Count Spanning Frag Count Splice Type

LRBA--SVBP 1471 215 ONLY REF SPLICE

FHL3--SVBP 862 148 ONLY REF SPLICE

FHL3--SVBP 266 148 ONLY REF SPLICE

KCNQ4--PPCS 164 22 ONLY REF SPLICE

FHL3--LRBA 75 28 INCL NON REF SPLICE

FHL3--LRBA 39 28 INCL NON REF SPLICE

KCNQ4--CCDC30 45 4 INCL NON REF SPLICE

MYOT--AC091979.1 23 7 ONLY REF SPLICE

FHL3--LRBA 16 28 INCL NON REF SPLICE

FHL3--LRBA 9 28 INCL NON REF SPLICE

RIMKLA--HIVEP3 6 0 INCL NON REF SPLICE

MYO1E--AC013652.1 5 0 ONLY REF SPLICE

MYOT--AC091979.1 3 7 ONLY REF SPLICE

Supplemental Table 2. Fusions identified from RNA sequencing via FusionCatcher analysis (aligned to GRCh38 reference genome)

Fusion Name Junction Read Spanning Frag Fusion Description Predicted Effect

Count Count

LRBA--SVBP 1496 64 cancer,t816,exon-exon in-frame

FHL3--SVBP 933 62 t518 UTR/CDS(truncated)

FHL3--SVBP 933 61 t518,exon-exon UTR/CDS(truncated)

FHL3--SVBP 933 34 t518 UTR/CDS(truncated)

FHL3--SVBP 933 34 t518,exon-exon UTR/CDS(truncated)

KCNQ4--PPCS 157 36 m129,t56,exon-exon out-of-frame

FHL3--LRBA 151 46 cancer,t18 UTR/CDS(truncated)

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FHL3--LRBA 151 29 cancer,t18 intronic/CDS(truncate

FHL3--LRBA 151 24 cancer,t18 UTR/CDS(truncated)

FHL3--LRBA 151 17 cancer,t18 UTR/CDS(truncated)

AL023284.2-- 44 26 pseudogene,t44 exonic(no-known-

CCDC30 CDS)/UTR

MYOT-- 29 21 lncrna,t9,exon-exon CDS(truncated)/exoni

AC091979.1 c(no-known-CDS)

CHRNG--EIF4E2 26 19 adjacent,t5,1K

gh,exon-exon

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Supplemental Table 3. Germline and somatic variants (aligned to GRCh38 reference genome, top interpretation)

HGV Clinic HGV C Trans S Varia al CNV Gene S Geno Sour dbSN Cosm ClinV h cript Prote nt Signif TPM log Name DNA type ce P ID ic ID ar ID r ID in Type icanc ratio Ref. Ref. e ENST0 c.27 00003 p.Leu misse 2.185 somat SCMH1 1 4C> Het NA 20.21 NA NA NA 26197. 92Ile nse 321 ic A 11 ENST0 c.26 00003 4 p.Lys frame somat DTNBP1 6 Het NA 11.69 -0.12 NA NA NA 38950. 265d 89fs shift ic 9 elGA ENST0 c.84 stop 00000 p.Tyr 0.118 somat VPS13D 1 15C Het gaine NA 5.9 NA NA NA 11700. 2805* 2 ic >A d 10 ENST0 p.Ter - COS 1 00004 c.37 stop Germl rs252 NF1 13Arg Hom NA 4.19 0.393 M412 NA 7 22121. T>C lost ine 5574 ext*? 8 9831 2 ENST0 00003 c.40 p.Pro misse 233.1 2.764 somat ZMPSTE24 1 Het NA NA NA NA 72759. C>T 14Ser nse 3 4 ic 3 c.16 16 ENST0 1623 - 1 00003 p.Val frame somat KDM2B delT Het NA 8.47 0.096 NA NA NA 2 77069. 539fs shift ic GAC 9 8 TTG G ENST0 c.87 GPATCH2 1 00002 2 p.Thr frame 0.118 somat Het NA 3.66 NA NA NA L 4 61530. 873d 291fs shift 9 ic 11 elCA ENST0 c.12 2 00003 p.Ser frame somat ANGPT4 47de Het NA 0.01 0.643 NA NA NA 0 81922. 416fs shift ic lC 4 ENST0 c.90 - 00002 p.Arg frame somat UCP1 4 0del Het NA 0 0.172 NA NA NA 62999. 300fs shift ic G 6 3 COS ENST0 M216 c.16 p.Arg rs779 00002 misse 336.8 1.745 somat 9987 P3H1 1 36C 546Tr Het NA 74419 NA 36040. nse 6 124 ic COS >T p 5 8 M216 9988 c.84 8 ENST0 900d 00002 p.Glu frame 0.259 somat VGF 7 elAG Het NA 0.33 NA NA NA 49330. 283fs shift 9 ic AGC 2 GCA CTC

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CTG GGC GGC TCC GAG GCG GGC GAG CGC CTT CTC CAG CAA GGG ENST0 c.52 00003 p.Phe frame 709.1 2.947 somat PPT1 1 8del Het NA NA NA NA 72779. 176fs shift 7 156 ic T 8 misse ENST0 c.44 p.His nse - 1 00005 somat G6PC 8C> 150A Het &splic NA 0.01 0.371 NA NA NA 7 85489. ic A sn e 5 1 region misse ENST0 c.25 p.Lys nse 00002 0.045 somat KCNT2 1 94A 865M Het &splic NA 0.17 NA NA NA 94725. 6 ic >T et e 13 region c.14 ENST0 42 00003 p.Phe frame 2.128 somat MFSD2A 1 1443 Het NA 30.35 NA NA NA 72809. 481fs shift 942 ic delT 5 T ENST0 c.68 stop 00003 p.Arg 0.045 somat GPR25 1 8C> Het gaine NA 0.06 NA NA NA 04244. 230* 6 ic T d 4 ENST0 c.12 p.Asp 00003 misse 0.570 somat ADCK5 8 25G 409A Het NA 3.23 NA NA NA 08860. nse 2 ic >A sn 10 ENST0 COS 1 00003 c.44 p.Leu misse somat SPHK1 Het NA 3.41 0.569 NA M641 NA 7 23374. T>C 15Pro nse ic 4553 8 ENST0 c.30 p.Ser 00003 frame 0.570 somat SCRIB 8 73de 1025f Het NA 46.58 NA NA NA 20476. shift 2 ic lT s 7 disrup c.33 ENST0 p.Cys tive 38de 1 00004 11 infram 0.492 somat FAAP100 lTG Het NA 9.16 NA NA NA 7 25898. Gly12 e 9 ic GGT 2 del deleti G on ENST0 c.67 p.Ala 2 00003 misse 0.499 somat BRWD1 15G 2239 Het NA 11.33 NA NA NA 1 33229. nse 5 ic >T Ser 6

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ENST0 c.51 p.Arg 00003 misse 0.570 somat ZNF696 8 7C> 173Tr Het NA 2.39 NA NA NA 30143. nse 2 ic T p 7 ENST0 c.50 - 1 00004 p.Val frame somat HCAR1 51de Het NA 0.01 0.096 NA NA NA 2 32564. 17fs shift ic lTG 9 2 ENST0 c.12 stop 1 00003 p.Gly 0.118 somat SIPA1L1 07G Het gaine NA 4.96 NA NA NA 4 58550. 403* 9 ic >T d 6 c.17 17 1760 delG GCG CTG CTG GCA ENST0 GCA 00003 p.Gly frame 0.187 somat KEL 7 TCA Het NA 0.29 NA NA NA 55265. 573fs shift 5 ic TGG 6 CCC ACG AGC TGT TGC ACA TCT T ENST0 c.32 stop - 1 00003 p.Gln somat PLIN5 2C> Het gaine NA 1.38 0.033 NA NA NA 9 81848. 108* ic T d 1 7 2A1I:A _118- struct c.35 drug - 1 A_210: ural germli rs116 22594 ERCC1 4T> NA Hom respo 35.83 0.218 NA 9 ENST0 intera ne 15 5 C nse 4 00000 ction 13807 disrup COS ENST0 tive c.54 rs145 M443 2 00001 p.Leu infram Benig 0.661 germli 19505 CTSA 56de Het 90.61 78382 915 0 91018. 19del e n 3 ne 9 lGCT 68 19505 9 deleti 9 on COS M174 ENST0 Patho c.17 9518 1 00001 p.Ile6 misse genic, 0.186 germli rs180 IL4R 8A> Het 8.57 COS 14666 6 70630. 0Val nse protec 2 ne 5010 G M690 6 tive 1247 14666 COS ENST0 p.Asn drug M117 00002 c.61 misse 0.307 germli rs928 82932 ABCB1 7 21As Het respo 0.11 8512 65724. A>G nse 1 ne 2564 7 p nse 82932 7 7

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ENST0 c.16 p.Ser COS 2 00003 misse 0.724 germli rs811 MROH8 03T> 535Pr Het NA 4.11 M498 NA 0 43811. nse 1 ne 5854 C o 4878 8 disrup rs534 c.34 ENST0 tive 81325 5 p.Ala COS 2 00002 infram 0.724 germli 9 FAM83D 347d 116du Het NA 2.9 M116 NA 0 17429. e 1 ne rs777 upG p 7792 8 inserti 28773 GC on 3 ENST0 COS c.11 p.Gln 1 00003 misse Patho 0.230 germli rs511 M156 APOA4 40G 380Hi Het 0 17905 1 57780. nse genic 9 ne 0 1077 >T s 4 17905 disrup c.46 rs749 ENST0 tive 1 p.Pro 16763 1 00004 infram 163.7 0.138 germli MDK 463d 154de Het NA rs773 NA NA 1 89525. e 2 9 ne elCC l 48386 5 deleti C 7 on rs128 Benig COS ENST0 07684 c.16 n/Like - M168 00002 p.Ala frame germli 85 28260 CLDN16 3 6del Hom ly 0.01 0.177 3974 64734. 56fs shift ne rs368 8 G benig 6 28260 2 23405 n 8 4 c.77 1 disrup 785d ENST0 p.Asp tive elTG rs780 COS 00003 257 infram 324.3 0.176 germli NCL 2 ATG Het NA 76604 M101 NA 22723. Asp2 e 5 9 ne AAG 3 7753 8 61del deleti ATG on ATG A ENST0 COS c.14 drug - 00002 p.Ile4 misse germli rs180 M500 MTRR 5 7A> Hom respo 8.47 0.138 7029 64668. 9Met nse ne 1394 1933 G nse 5 6 7029 rs397 seque ENST0 c.3- - 93412 1 nce 193.8 germli SNRPD2 00003 544d NA Het NA 0.391 9 NA NA 9 featur 5 ne 42669 upG 4 rs557 e 42289 conse c.69 p.Ala rvativ ENST0 0 230 e COS 2 00002 126.4 0.670 germli rs106 RALY 691i Gly23 Het infram NA M132 NA 0 46194. 8 1 ne 49600 nsA 1insS e 6800 7 GC er inserti on conse c.55 rs200 rvativ ENST0 6 p.Pro 07466 e COS 2 00002 561d 186 0.743 germli 1 TAF4 Hom infram NA 10.11 M473 NA 0 52996. elCC Gly18 5 ne rs200 e 2011 8 TGG 7del 50820 deleti C 3 on

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c.*9 splice *13+ donor 16de &splic lGG rs149 ENST0 e GAG - 99169 1 00005 region 161.5 germli EIF3G GTG NA Hom NA 0.098 2 NA NA 9 93066. &3 7 ne AGT 9 rs321 5 prime GAG 7685 UTR GAC &intro TGC n C COS M337 8354; COS M337 8355; COS M354 839;C ENST0 c.11 p.Val Likely - rs113 OSM 1 00002 misse germli 42890 TP53 8G> 40Ph Het patho 11.66 0.326 16910 35484 7 69305. nse ne 9 T e genic 9 43 0;CO 8 SM35 4841; COS M354 842;C OSM 44240 ;4289 09 COS M337 8354 COS M337 8355 COS M354 839 COS ENST0 c.51 p.Val Likely - rs113 M354 1 00002 misse germli 42890 TP53 4G> 172P Het patho 11.66 0.326 16910 840 7 69305. nse ne 9 T he genic 9 43 COS 8 M354 841 COS M354 842 COS M442 40 42890 9 ENST0 c.48 p.Asp - 00002 misse germli rs961 ANKRD31 5 27C 1609 Het NA 0.01 0.175 NA NA 74361. nse ne 098 >G Glu 9 3

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COS M112 7775 ENST0 COS c.82 p.Gln 1 00004 misse Patho 0.103 germli rs156 M112 PTPRJ 7A> 276Pr Het 0.83 8690 1 18331. nse genic 7 ne 6734 7776 C o 6 COS M373 6043 8690 COS M498 ENST0 c.53 p.Ser 6364 1 00003 misse protec germli rs817 TIRAP 9C> 180Le Het 1.48 0.225 COS 4467 1 92678. nse tive ne 7374 T u M498 7 6365 4467 ENST0 c.72 stop - rs121 1 00002 p.Trp Patho germli SLC7A7 6G> Het gaine 0.6 0.246 90867 NA 6214 4 85850. 242* genic ne A d 2 9 11 ENST0 c.34 drug 00002 p.Ile1 misse 0.006 germli rs180 NAT2 8 1T> Het respo 0 NA 723 86479. 14Thr nse 8 ne 1280 C nse 3 rs201 conse 42776 c.23 rvativ ENST0 9 71 p.Glu e - 1 00002 210.2 germli rs580 HSP90B1 2373 791de Het infram NA 0.077 NA NA 2 99767. 5 ne 0607 delG l e 3 9 rs869 AA deleti 21598 on 8 COS ENST0 drug - M400 00003 c.46 p.Gly misse germli rs104 22593 ADRB2 5 Het respo 1.48 0.131 3392 05988. G>A 16Arg nse ne 2713 7 nse 7 22593 5 7 ENST0 c.38 p.Thr - COS 00002 misse germli rs124 COL6A5 3 38A 1280 Het NA 0 0.130 M499 NA 65379. nse ne 88457 >C Pro 7 8804 10 ENST0 c.66 p.Gly COS 00003 misse 0.044 germli rs492 OR2B11 1 8G> 223A Het NA 0.01 M374 NA 18749. nse 3 ne 5663 A sp 6893 7 ENST0 c.14 p.Ser 00003 misse Patho 0.113 germli rs969 GLUD2 X 92T> 498Al Hom 0.86 NA 29936 28078. nse genic 1 ne 7983 G a 2 COS ENST0 c.12 p.Val drug M375 1 00000 misse - germli rs210 22596 CYP4F2 97G 433M Het respo 0.1 6415 9 11989. nse 0.139 ne 8622 9 >A et nse 22596 11 9 ENST0 c.11 p.Arg - COS 1 00004 misse germli rs388 KRT3 23C 375Gl Het NA 0.02 0.052 M595 NA 2 17996. nse ne 7954 >G y 48 9775 2

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Benig COS ENST0 c.47 p.Ser n, - M375 1 00003 misse germli rs780 13034 SLC29A3 3C> 158P Hom drug 1.7 0.118 1939 0 73189. nse ne 668 1 T he respo 5 13034 5 nse 1 ENST0 c.10 - 00005 p.Ile3 misse protec germli ADH1C 4 48A Het 0.16 0.179 rs698 NA 18180 15683. 50Val nse tive ne >G 8 5 ENST0 COS c.88 drug 00005 p.Ile2 misse 0.233 germli rs102 M543 TAS2R38 7 6A> Het respo 0 2906 47270. 96Val nse 6 ne 46939 8552 G nse 1 2906

Het, Heterozygous

Hom, Homozygous

Supplemental Table 4. Germline and somatic variants (aligned to GRCh38 reference genome, all interpretations)

Gene Chr HGVS HGVS Genot Variant Clinical TP CN Sourc dbS Cos Clin DNA Protein ype Type Signific M V e NP mic Var Ref. Ref. ance log ID ID ID rati o SCMH 1 c.274C> p.Leu92Ile Het missens NA 20.2 2.18 somati NA NA NA 1 A e 1 532 c 1 SCMH 1 c.304C> p.Leu102Ile Het missens NA 20.2 2.18 somati NA NA NA 1 A e 1 532 c 1 SCMH 1 c.91C>A p.Leu31Ile Het missens NA 20.2 2.18 somati NA NA NA 1 e 1 532 c 1 SCMH 1 c.133C> p.Leu45Ile Het missens NA 20.2 2.18 somati NA NA NA 1 A e 1 532 c 1 SCMH 1 c.91C>A p.Leu31Ile Het missens NA 20.2 2.18 somati NA NA NA 1 e 1 532 c 1 SCMH 1 c.91C>A p.Leu31Ile Het missens NA 20.2 2.18 somati NA NA NA 1 e 1 532 c 1 SCMH 1 c.133C> p.Leu45Ile Het missens NA 20.2 2.18 somati NA NA NA 1 A e 1 532 c 1 SCMH 1 c.91C>A p.Leu31Ile Het missens NA 20.2 2.18 somati NA NA NA 1 e 1 532 c 1 SCMH 1 c.274C> p.Leu92Ile Het missens NA 20.2 2.18 somati NA NA NA 1 A e 1 532 c 1 SCMH 1 c.91C>A p.Leu31Ile Het missens NA 20.2 2.18 somati NA NA NA 1 e 1 532 c 1

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SCMH 1 c.133C> p.Leu45Ile Het missens NA 20.2 2.18 somati NA NA NA 1 A e 1 532 c 1 NF1 17 c.37T>C p.Ter13Arg Hom stop lost NA 4.19 - Germli rs252 CO NA ext*? 0.39 ne 5574 SM4 38 129 831 NF1 17 c.1918T p.Ter640Ar Hom stop lost NA 4.19 - Germli rs252 CO NA >C gext*? 0.39 ne 5574 SM4 38 129 831 DTNBP 6 c.264 p.Lys89fs Het frameshi NA 11.6 - somati NA NA NA 1 265delG ft 9 0.12 c A DTNBP 6 c.264 p.Lys89fs Het frameshi NA 11.6 - somati NA NA NA 1 265delG ft 9 0.12 c A DTNBP 6 c.213 p.Lys72fs Het frameshi NA 11.6 - somati NA NA NA 1 214delG ft 9 0.12 c A DTNBP 6 c.159 p.Lys54fs Het frameshi NA 11.6 - somati NA NA NA 1 160delG ft 9 0.12 c A DTNBP 6 c.213 p.Lys72fs Het frameshi NA 11.6 - somati NA NA NA 1 214delG ft 9 0.12 c A DTNBP 6 c.159 p.Lys54fs Het frameshi NA 11.6 - somati NA NA NA 1 160delG ft 9 0.12 c A VPS13 1 c.8415C p.Tyr2805* Het stop NA 5.9 0.11 somati NA NA NA D >A gained 82 c VPS13 1 c.11877 p.Tyr3959* Het stop NA 5.9 0.11 somati NA NA NA D C>A gained 82 c VPS13 1 c.11952 p.Tyr3984* Het stop NA 5.9 0.11 somati NA NA NA D C>A gained 82 c ZMPST 1 c.40C>T p.Pro14Ser Het missens NA 233. 2.76 somati NA NA NA E24 e 13 44 c KDM2 12 c.1616 p.Val539fs Het frameshi NA 8.47 - somati NA NA NA B 1623del ft 0.09 c TGACTT 69 GG KDM2 12 c.1709 p.Val570fs Het frameshi NA 8.47 - somati NA NA NA B 1716del ft 0.09 c TGACTT 69 GG KDM2 12 c.1709 p.Val570fs Het frameshi NA 8.47 - somati NA NA NA B 1716del ft 0.09 c TGACTT 69 GG KDM2 12 c.1439 p.Val480fs Het frameshi NA 8.47 - somati NA NA NA B 1446del ft 0.09 c TGACTT 69 GG KDM2 12 c.1358 p.Val453fs Het frameshi NA 8.47 - somati NA NA NA B 1365del ft 0.09 c TGACTT 69 GG

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GPAT 14 c.872 p.Thr291fs Het frameshi NA 3.66 0.11 somati NA NA NA CH2L 873delC ft 89 c A GPAT 14 c.872 p.Thr291fs Het frameshi NA 3.66 0.11 somati NA NA NA CH2L 873delC ft 89 c A GPAT 14 c.872 p.Thr291fs Het frameshi NA 3.66 0.11 somati NA NA NA CH2L 873delC ft 89 c A GPAT 14 c.872 p.Thr291fs Het frameshi NA 3.66 0.11 somati NA NA NA CH2L 873delC ft 89 c A GPAT 14 c.872 p.Thr291fs Het frameshi NA 3.66 0.11 somati NA NA NA CH2L 873delC ft 89 c A GPAT 14 c.872 p.Thr291fs Het frameshi NA 3.66 0.11 somati NA NA NA CH2L 873delC ft 89 c A ANGP 20 c.1247d p.Ser416fs Het frameshi NA 0.01 0.64 somati NA NA NA T4 elC ft 3 c UCP1 4 c.900del p.Arg300fs Het frameshi NA 0 - somati NA NA NA G ft 0.17 c 26 P3H1 1 c.1636C p.Arg546Tr Het missens NA 336. 1.74 somati rs779 CO NA >T p e 86 512 c 7441 SM2 4 95 169 987; CO SM2 169 988 P3H1 1 c.1636C p.Arg546Tr Het missens NA 336. 1.74 somati rs779 CO NA >T p e 86 512 c 7441 SM2 4 95 169 987; CO SM2 169 988 P3H1 1 c.1636C p.Arg546Tr Het missens NA 336. 1.74 somati rs779 CO NA >T p e 86 512 c 7441 SM2 4 95 169 987; CO SM2 169 988 P3H1 1 c.557C> p.Ala186Val Het missens NA 336. 1.74 somati rs779 CO NA T e 86 512 c 7441 SM2 4 95 169 987; CO SM2 169 988 P3H1 1 c.52G>A p.Ala18Thr Het missens NA 336. 1.74 somati NA CO NA e 86 512 c SM5 4 846 714; CO 44

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SM5 846 715 P3H1 1 c.52G>A p.Ala18Thr Het missens NA 336. 1.74 somati NA CO NA e 86 512 c SM5 4 846 714; CO SM5 846 715 P3H1 1 c.52G>A p.Ala18Thr Het missens NA 336. 1.74 somati NA CO NA e 86 512 c SM5 4 846 714; CO SM5 846 715 P3H1 1 c.52G>A p.Ala18Thr Het missens NA 336. 1.74 somati NA CO NA e 86 512 c SM5 4 846 714; CO SM5 846 715 VGF 7 c.848 p.Glu283fs Het frameshi NA 0.33 0.25 somati NA NA NA 900delA ft 99 c GAGCG CACTC CTGGG CGGCT CCGAG GCGGG CGAGC GCCTT CTCCA GCAAG GG VGF 7 c.848 p.Glu283fs Het frameshi NA 0.33 0.25 somati NA NA NA 900delA ft 99 c GAGCG CACTC CTGGG CGGCT CCGAG GCGGG CGAGC GCCTT CTCCA GCAAG GG VGF 7 c.848 p.Glu283fs Het frameshi NA 0.33 0.25 somati NA NA NA 900delA ft 99 c GAGCG CACTC CTGGG CGGCT CCGAG 45

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GCGGG CGAGC GCCTT CTCCA GCAAG GG PPT1 1 c.528del p.Phe176fs Het frameshi NA 709. 2.94 somati NA NA NA T ft 17 715 c 6 PPT1 1 c.441del p.Phe147fs Het frameshi NA 709. 2.94 somati NA NA NA T ft 17 715 c 6 PPT1 1 c.126del p.Phe42fs Het frameshi NA 709. 2.94 somati NA NA NA T ft 17 715 c 6 PPT1 1 c.132del p.Phe44fs Het frameshi NA 709. 2.94 somati NA NA NA T ft 17 715 c 6 PPT1 1 c.291del p.Phe97fs Het frameshi NA 709. 2.94 somati NA NA NA T ft 17 715 c 6 PPT1 1 c.216del p.Phe72fs Het frameshi NA 709. 2.94 somati NA NA NA T ft 17 715 c 6 PPT1 1 c.441del p.Phe147fs Het frameshi NA 709. 2.94 somati NA NA NA T ft 17 715 c 6 PPT1 1 c.441del NA Het structur NA 709. 2.94 somati NA NA NA T al 17 715 c interacti 6 on PPT1 1 c.441del NA Het structur NA 709. 2.94 somati NA NA NA T al 17 715 c interacti 6 on PPT1 1 c.441del NA Het structur NA 709. 2.94 somati NA NA NA T al 17 715 c interacti 6 on PPT1 1 c.441del NA Het structur NA 709. 2.94 somati NA NA NA T al 17 715 c interacti 6 on G6PC 17 c.448C> p.His150As Het missens NA 0.01 - somati NA NA NA A n e&splice 0.37 c region 15 G6PC 17 c.487C> p.His163As Het missens NA 0.01 - somati NA NA NA A n e&splice 0.37 c region 15 KCNT2 1 c.2594A p.Lys865Me Het missens NA 0.17 0.04 somati NA NA NA >T t e&splice 56 c region KCNT2 1 c.2522A p.Lys841Me Het missens NA 0.17 0.04 somati NA NA NA >T t e&splice 56 c region KCNT2 1 c.2372A p.Lys791Me Het missens NA 0.17 0.04 somati NA NA NA >T t e&splice 56 c region

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MFSD2 1 c.1442 p.Phe481fs Het frameshi NA 30.3 2.12 somati NA NA NA A 1443del ft 5 894 c TT 2 MFSD2 1 c.1403 p.Phe468fs Het frameshi NA 30.3 2.12 somati NA NA NA A 1404del ft 5 894 c TT 2 MFSD2 1 c.935 p.Phe312fs Het frameshi NA 30.3 2.12 somati NA NA NA A 936delT ft 5 894 c T 2 GPR25 1 c.688C> p.Arg230* Het stop NA 0.06 0.04 somati NA NA NA T gained 56 c ADCK5 8 c.1225G p.Asp409As Het missens NA 3.23 0.57 somati NA NA NA >A n e 02 c SPHK1 17 c.44T>C p.Leu15Pro Het missens NA 3.41 0.56 somati NA CO NA e 9 c SM6 414 553 SCRIB 8 c.3073d p.Ser1025fs Het frameshi NA 46.5 0.57 somati NA NA NA elT ft 8 02 c SCRIB 8 c.3073d p.Ser1025fs Het frameshi NA 46.5 0.57 somati NA NA NA elT ft 8 02 c SCRIB 8 c.2830d p.Ser944fs Het frameshi NA 46.5 0.57 somati NA NA NA elT ft 8 02 c SCRIB 8 c.58delT p.Ser20fs Het frameshi NA 46.5 0.57 somati NA NA NA ft 8 02 c FAAP1 17 c.33 p.Cys11 Het disruptiv NA 9.16 0.49 somati NA NA NA 00 38delTG Gly12del e 29 c GGTG inframe deletion BRWD 21 c.6715G p.Ala2239S Het missens NA 11.3 0.49 somati NA NA NA 1 >T er e 3 95 c ZNF69 8 c.517C> p.Arg173Tr Het missens NA 2.39 0.57 somati NA NA NA 6 T p e 02 c ZNF69 8 c.517C> p.Arg173Tr Het missens NA 2.39 0.57 somati NA NA NA 6 T p e 02 c HCAR1 12 c.50 p.Val17fs Het frameshi NA 0.01 - somati NA NA NA 51delTG ft 0.09 c 69 SIPA1L 14 c.1207G p.Gly403* Het stop NA 4.96 0.11 somati NA NA NA 1 >T gained 89 c SIPA1L 14 c.1207G p.Gly403* Het stop NA 4.96 0.11 somati NA NA NA 1 >T gained 89 c SIPA1L 14 c.1207G p.Gly403* Het stop NA 4.96 0.11 somati NA NA NA 1 >T gained 89 c SIPA1L 14 c.3781T p.Ser1261P Het missens NA 4.96 0.11 somati NA NA NA 1 >C ro e 89 c SIPA1L 14 c.3781T p.Ser1261P Het missens NA 4.96 0.11 somati NA NA NA 1 >C ro e 89 c SIPA1L 14 c.2206T p.Ser736Pr Het missens NA 4.96 0.11 somati NA NA NA 1 >C o e 89 c SIPA1L 14 c.3844T p.Ser1282P Het missens NA 4.96 0.11 somati NA NA NA 1 >C ro e 89 c KEL 7 c.1717 p.Gly573fs Het frameshi NA 0.29 0.18 somati NA NA NA 1760del ft 75 c GGCGC TGCTG GCAGC ATCATG GCCCA 47

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CGAGC TGTTG CACAT CTT PLIN5 19 c.322C> p.Gln108* Het stop NA 1.38 - somati NA NA NA T gained 0.03 c 31 ERCC1 19 c.354T> NA Hom structur drug 35.8 - germli rs116 NA 2259 C al respons 3 0.21 ne 15 45 interacti e 84 on ERCC1 19 c.354T> NA Hom structur drug 35.8 - germli rs116 NA 2259 C al respons 3 0.21 ne 15 45 interacti e 84 on ERCC1 19 c.354T> NA Hom structur drug 35.8 - germli rs116 NA 2259 C al respons 3 0.21 ne 15 45 interacti e 84 on ERCC1 19 c.354T> NA Hom structur drug 35.8 - germli rs116 NA 2259 C al respons 3 0.21 ne 15 45 interacti e 84 on CTSA 20 c.54 p.Leu19del Het disruptiv Benign 90.6 0.66 germli rs145 CO 1950 56delGC e 1 13 ne 7838 SM4 59 T inframe 268 439 deletion 15;1 950 59 CTSA 20 c.108 p.Leu37del Het disruptiv Benign 90.6 0.66 germli rs145 CO 1950 110delG e 1 13 ne 7838 SM4 59 CT inframe 268 439 deletion 15;1 950 59 CTSA 20 c.54 p.Leu19del Het disruptiv Benign 90.6 0.66 germli rs145 CO 1950 56delGC e 1 13 ne 7838 SM4 59 T inframe 268 439 deletion 15;1 950 59 CTSA 20 c.108 p.Leu37del Het disruptiv Benign 90.6 0.66 germli rs145 CO 1950 110delG e 1 13 ne 7838 SM4 59 CT inframe 268 439 deletion 15;1 950 59 CTSA 20 c.108 p.Leu37del Het disruptiv Benign 90.6 0.66 germli rs145 CO 1950 110delG e 1 13 ne 7838 SM4 59 CT inframe 268 439 deletion 15;1 950 59 CTSA 20 c.108 p.Leu37del Het disruptiv Benign 90.6 0.66 germli rs145 CO 1950 110delG e 1 13 ne 7838 SM4 59 CT inframe 268 439 deletion 15;1 950 59

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CTSA 20 c.54 p.Leu19del Het disruptiv Benign 90.6 0.66 germli rs145 CO 1950 56delGC e 1 13 ne 7838 SM4 59 T inframe 268 439 deletion 15;1 950 59 IL4R 16 c.178A> p.Ile60Val Het missens Pathoge 8.57 0.18 germli rs180 CO 1466 G e nic, 62 ne 5010 SM1 6 protectiv 749 e 518; CO SM6 901 247; 146 66 IL4R 16 c.223A> p.Ile75Val Het missens Pathoge 8.57 0.18 germli rs180 CO 1466 G e nic, 62 ne 5010 SM1 6 protectiv 749 e 518; CO SM6 901 247; 146 66 IL4R 16 c.223A> p.Ile75Val Het missens Pathoge 8.57 0.18 germli rs180 CO 1466 G e nic, 62 ne 5010 SM1 6 protectiv 749 e 518; CO SM6 901 247; 146 66 IL4R 16 c.223A> p.Ile75Val Het missens Pathoge 8.57 0.18 germli rs180 CO 1466 G e nic, 62 ne 5010 SM1 6 protectiv 749 e 518; CO SM6 901 247; 146 66 IL4R 16 c.223A> p.Ile75Val Het missens Pathoge 8.57 0.18 germli rs180 CO 1466 G e nic, 62 ne 5010 SM1 6 protectiv 749 e 518; CO SM6 901 247; 146 66 IL4R 16 c.223A> p.Ile75Val Het missens Pathoge 8.57 0.18 germli rs180 CO 1466 G e nic, 62 ne 5010 SM1 6 protectiv 749 e 518; 49

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CO SM6 901 247; 146 66 ABCB1 7 c.61A>G p.Asn21Asp Het missens drug 0.11 0.30 germli rs928 CO 8293 e respons 71 ne 2564 SM1 27 e 178 512; 829 327 ABCB1 7 c.61A>G p.Asn21Asp Het missens drug 0.11 0.30 germli rs928 CO 8293 e respons 71 ne 2564 SM1 27 e 178 512; 829 327 ABCB1 7 c.61A>G p.Asn21Asp Het missens drug 0.11 0.30 germli rs928 CO 8293 e respons 71 ne 2564 SM1 27 e 178 512; 829 327 ABCB1 7 c.61A>G p.Asn21Asp Het missens drug 0.11 0.30 germli rs928 CO 8293 e respons 71 ne 2564 SM1 27 e 178 512; 829 327 MROH 20 c.1603T p.Ser535Pr Het missens NA 4.11 0.72 germli rs811 CO NA 8 >C o e 41 ne 5854 SM4 984 878 MROH 20 c.1615T p.Ser539Pr Het missens NA 4.11 0.72 germli rs811 CO NA 8 >C o e 41 ne 5854 SM4 984 878 MROH 20 c.1525T p.Ser509Pr Het missens NA 4.11 0.72 germli rs811 CO NA 8 >C o e 41 ne 5854 SM4 984 878 MROH 20 c.406T> p.Ser136Pr Het missens NA 4.11 0.72 germli rs811 CO NA 8 C o e 41 ne 5854 SM4 984 878 MROH 20 c.1528T p.Ser510Pr Het missens NA 4.11 0.72 germli rs811 CO NA 8 >C o e 41 ne 5854 SM4 984 878 MROH 20 c.709A> p.Ile237Val Het missens NA 4.11 0.72 germli rs626 NA 8 G e 41 ne 2124 1 MROH 20 c.721A> p.Ile241Val Het missens NA 4.11 0.72 germli rs626 NA 8 G e 41 ne 2124 1 MROH 20 c.631A> p.Ile211Val Het missens NA 4.11 0.72 germli rs626 NA 8 G e 41 ne 2124 1

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MROH 20 c.739A> p.Ile247Val Het missens NA 4.11 0.72 germli rs626 NA 8 G e 41 ne 2124 1 MROH 20 c.93 p.His32fs 2-Jan frameshi NA 4.11 0.72 germli rs114 CO NA 8 93+1ins ft&splice 41 ne 6721 SM1 AGTGC region 4 026 CGGCC 494; GCGGG CO GCCCT SM1 GTCTG 592 TAAG 566; CO SM1 592 567; CO SM4 603 440 MROH 20 c.170 p.Ser57fs 2-Jan frameshi NA 4.11 0.72 germli rs114 CO NA 8 171insA ft&stop 41 ne 6721 SM1 GTGCC gained 4 026 GGCCG 494; CGGGG CO CCCTG SM1 TCTGTA 592 AG 566; CO SM1 592 567; CO SM4 603 440 MROH 20 c.182 p.Ser61fs 2-Jan frameshi NA 4.11 0.72 germli rs114 CO NA 8 183insA ft&stop 41 ne 6721 SM1 GTGCC gained 4 026 GGCCG 494; CGGGG CO CCCTG SM1 TCTGTA 592 AG 566; CO SM1 592 567; CO SM4 603 440 MROH 20 c.200 p.Ser67fs 2-Jan frameshi NA 4.11 0.72 germli rs114 CO NA 8 201insA ft&stop 41 ne 6721 SM1 GTGCC gained 4 026 GGCCG 494; CGGGG CO CCCTG SM1 TCTGTA 592 AG 566; CO 51

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SM1 592 567; CO SM4 603 440 MROH 20 c.93 p.His32fs 2-Jan frameshi NA 4.11 0.72 germli rs114 CO NA 8 93+1ins ft&splice 41 ne 6721 SM1 AGTGC region 4 026 CGGCC 494; GCGGG CO GCCCT SM1 GTCTAT 592 AAG 566; CO SM1 592 567; CO SM4 603 440 MROH 20 c.170 p.Ser57fs 2-Jan frameshi NA 4.11 0.72 germli rs114 CO NA 8 171insA ft&stop 41 ne 6721 SM1 GTGCC gained 4 026 GGCCG 494; CGGGG CO CCCTG SM1 TCTATA 592 AG 566; CO SM1 592 567; CO SM4 603 440 MROH 20 c.182 p.Ser61fs 2-Jan frameshi NA 4.11 0.72 germli rs114 CO NA 8 183insA ft&stop 41 ne 6721 SM1 GTGCC gained 4 026 GGCCG 494; CGGGG CO CCCTG SM1 TCTATA 592 AG 566; CO SM1 592 567; CO SM4 603 440 MROH 20 c.200 p.Ser67fs 2-Jan frameshi NA 4.11 0.72 germli rs114 CO NA 8 201insA ft&stop 41 ne 6721 SM1 GTGCC gained 4 026 GGCCG 494; CGGGG CO 52

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CCCTG SM1 TCTATA 592 AG 566; CO SM1 592 567; CO SM4 603 440 FAM83 20 c.345 p.Ala116du Het disruptiv NA 2.9 0.72 germli rs534 CO NA D 347dup p e 41 ne 8132 SM1 GGC inframe 59;rs 167 insertion 7772 792 8773 3 FAM83 20 c.345 p.Ala116du Het disruptiv NA 2.9 0.72 germli rs534 CO NA D 347dup p e 41 ne 8132 SM1 GGC inframe 59;rs 167 insertion 7772 792 8773 3 FAM83 20 c.255 p.Ala86dup Het disruptiv NA 2.9 0.72 germli rs534 CO NA D 257dup e 41 ne 8132 SM1 GGC inframe 59;rs 167 insertion 7772 792 8773 3 APOA4 11 c.1140G p.Gln380Hi Het missens Pathoge 0 0.23 germli rs511 CO 1790 >T s e nic 09 ne 0 SM1 5 561 077; 179 05 APOA4 11 c.440G> p.Ser147As Hom missens NA 0 0.23 germli rs510 NA A n e 09 ne 4 MDK 11 c.461 p.Pro154del Hom disruptiv NA 163. 0.13 germli rs749 NA 463delC e 72 89 ne 1676 CC inframe 3;rs7 deletion 7348 3867 MDK 11 c.460 p.Pro154fs Hom frameshi NA 163. 0.13 germli rs749 NA 463delC ft 72 89 ne 1676 CCC 3;rs7 7348 3867 CLDN1 3 c.166del p.Ala56fs Hom frameshi Benign/ 0.01 - germli rs128 CO 2826 6 G ft Likely 0.17 ne 0768 SM1 08 benign 76 485;r 683 s368 974; 2340 282 54 608 CLDN1 3 c.166del p.Ala56fs Hom frameshi Benign/ 0.01 - germli rs128 CO 2826 6 G ft Likely 0.17 ne 0768 SM1 08 benign 76 485;r 683 s368 974; 2340 282 54 608

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CLDN1 3 c.166G> p.Ala56Pro Hom missens Benign 0.01 - germli rs321 CO 3444 6 C e 0.17 ne 4506 SM3 45 76 759 932; 344 445 CLDN1 3 c.166G> p.Ala56Pro Hom missens Benign 0.01 - germli rs321 CO 3444 6 C e 0.17 ne 4506 SM3 45 76 759 932; 344 445 NCL 2 c.771 p.Asp257 Het disruptiv NA 324. 0.17 germli rs780 CO NA 785delT Asp261del e 35 69 ne 7660 SM1 GATGA inframe 43 017 AGATG deletion 753 ATGA MTRR 5 c.147A> p.Ile49Met Hom missens drug 8.47 - germli rs180 CO 7029 G e respons 0.13 ne 1394 SM5 e 85 001 933; 702 9 MTRR 5 c.66A>G p.Ile22Met Hom missens drug 8.47 - germli rs180 CO 7029 e respons 0.13 ne 1394 SM5 e 85 001 933; 702 9 MTRR 5 c.66A>G p.Ile22Met Hom missens drug 8.47 - germli rs180 CO 7029 e respons 0.13 ne 1394 SM5 e 85 001 933; 702 9 MTRR 5 c.66A>G p.Ile22Met Hom missens drug 8.47 - germli rs180 CO 7029 e respons 0.13 ne 1394 SM5 e 85 001 933; 702 9 MTRR 5 c.66A>G p.Ile22Met Hom missens drug 8.47 - germli rs180 CO 7029 e respons 0.13 ne 1394 SM5 e 85 001 933; 702 9 MTRR 5 c.132A> p.Ile44Met Hom missens drug 8.47 - germli rs180 CO 7029 G e respons 0.13 ne 1394 SM5 e 85 001 933; 702 9 MTRR 5 c.66A>G p.Ile22Met Hom missens drug 8.47 - germli rs180 CO 7029 e respons 0.13 ne 1394 SM5 e 85 001 933; 702 9

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MTRR 5 c.90A>G p.Ile30Met Hom missens drug 8.47 - germli rs180 CO 7029 e respons 0.13 ne 1394 SM5 e 85 001 933; 702 9 MTRR 5 c.132A> p.Ile44Met Hom missens drug 8.47 - germli rs180 CO 7029 G e respons 0.13 ne 1394 SM5 e 85 001 933; 702 9 MTRR 5 c.66A>G p.Ile22Met Hom missens drug 8.47 - germli rs180 CO 7029 e respons 0.13 ne 1394 SM5 e 85 001 933; 702 9 MTRR 5 c.66A>G p.Ile22Met Hom missens drug 8.47 - germli rs180 CO 7029 e respons 0.13 ne 1394 SM5 e 85 001 933; 702 9 MTRR 5 c.66A>G p.Ile22Met Hom missens drug 8.47 - germli rs180 CO 7029 e respons 0.13 ne 1394 SM5 e 85 001 933; 702 9 SNRP 19 c.3- NA Het sequenc NA 193. - germli rs397 NA D2 544dup e 85 0.39 ne 9341 G feature 14 29;rs 5574 2289 SNRP 19 c.3- NA Het sequenc NA 193. - germli rs283 NA D2 579A>T e 85 0.39 ne 9627 feature 14 3 RALY 20 c.690 p.Ala230 Het conserv NA 126. 0.67 germli rs106 CO NA 691insA Gly231insS ative 48 01 ne 4960 SM1 GC er inframe 0 326 insertion 800 RALY 20 c.492 p.Ala164 Het conserv NA 126. 0.67 germli rs106 CO NA 493insA Gly165insS ative 48 01 ne 4960 SM1 GC er inframe 0 326 insertion 800 RALY 20 c.642 p.Ala214 Het conserv NA 126. 0.67 germli rs106 CO NA 643insA Gly215insS ative 48 01 ne 4960 SM1 GC er inframe 0 326 insertion 800 RALY 20 c.642 p.Ala214 Het conserv NA 126. 0.67 germli rs106 CO NA 643insA Gly215insS ative 48 01 ne 4960 SM1 GC er inframe 0 326 insertion 800 RALY 20 c.697G> p.Gly233Se Het missens NA 126. 0.67 germli rs202 NA A r e 48 01 ne 0833 85 RALY 20 c.499G> p.Gly167Se Het missens NA 126. 0.67 germli rs202 NA A r e 48 01 ne 0833 85 55

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RALY 20 c.649G> p.Gly217Se Het missens NA 126. 0.67 germli rs202 NA A r e 48 01 ne 0833 85 RALY 20 c.649G> p.Gly217Se Het missens NA 126. 0.67 germli rs202 NA A r e 48 01 ne 0833 85 TAF4 20 c.556 p.Pro186 Hom conserv NA 10.1 0.74 germli rs200 CO NA 561delC Gly187del ative 1 35 ne 0746 SM4 CTGGC inframe 61;rs 732 deletion 2005 011 0820 3 EIF3G 19 c.*9 NA Hom splice NA 161. - germli rs149 NA *13+16d donor&s 57 0.09 ne 9916 elGGGA plice 89 92;rs GGTGA region& 3217 GTGAG 3 prime 685 GACTG UTR&int CC ron TP53 17 c.514G> p.Val172Ph Het missens Likely 11.6 - germli rs113 CO 4289 T e e pathoge 6 0.32 ne 1691 SM3 09 nic 69 043 378 354; CO SM3 378 355; CO SM3 548 39; CO SM3 548 40; CO SM3 548 41; CO SM3 548 42; CO SM4 424 0;42 890 9 TP53 17 c.514G> p.Val172Ph Het missens Likely 11.6 - germli rs113 CO 4289 T e e pathoge 6 0.32 ne 1691 SM3 09 nic 69 043 378 354; CO SM3 378 355; CO SM3 548 56

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39; CO SM3 548 40; CO SM3 548 41; CO SM3 548 42; CO SM4 424 0;42 890 9 TP53 17 c.514G> p.Val172Ph Het missens Likely 11.6 - germli rs113 CO 4289 T e e pathoge 6 0.32 ne 1691 SM3 09 nic 69 043 378 354; CO SM3 378 355; CO SM3 548 39; CO SM3 548 40; CO SM3 548 41; CO SM3 548 42; CO SM4 424 0;42 890 9 TP53 17 c.514G> p.Val172Ph Het missens Likely 11.6 - germli rs113 CO 4289 T e e pathoge 6 0.32 ne 1691 SM3 09 nic 69 043 378 354; CO SM3 378 355; CO SM3 548 57

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39; CO SM3 548 40; CO SM3 548 41; CO SM3 548 42; CO SM4 424 0;42 890 9 TP53 17 c.118G> p.Val40Phe Het missens Likely 11.6 - germli rs113 CO 4289 T e pathoge 6 0.32 ne 1691 SM3 09 nic 69 043 378 354; CO SM3 378 355; CO SM3 548 39; CO SM3 548 40; CO SM3 548 41; CO SM3 548 42; CO SM4 424 0;42 890 9 TP53 17 c.118G> p.Val40Phe Het missens Likely 11.6 - germli rs113 CO 4289 T e pathoge 6 0.32 ne 1691 SM3 09 nic 69 043 378 354; CO SM3 378 355; CO SM3 548 59

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39; CO SM3 548 40; CO SM3 548 41; CO SM3 548 42; CO SM4 424 0;42 890 9 TP53 17 c.235G> p.Val79Phe Het missens Likely 11.6 - germli rs113 CO 4289 T e pathoge 6 0.32 ne 1691 SM3 09 nic 69 043 378 354; CO SM3 378 355; CO SM3 548 39; CO SM3 548 40; CO SM3 548 41; CO SM3 548 42; CO SM4 424 0;42 890 9 TP53 17 c.397G> p.Val133Ph Het missens Likely 11.6 - germli rs113 CO 4289 T e e pathoge 6 0.32 ne 1691 SM3 09 nic 69 043 378 354; CO SM3 378 355; CO SM3 548 61

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39; CO SM3 548 40; CO SM3 548 41; CO SM3 548 42; CO SM4 424 0;42 890 9 TP53 17 c.397G> p.Val133Ph Het missens Likely 11.6 - germli rs113 CO 4289 T e e pathoge 6 0.32 ne 1691 SM3 09 nic 69 043 378 354; CO SM3 378 355; CO SM3 548 39; CO SM3 548 40; CO SM3 548 41; CO SM3 548 42; CO SM4 424 0;42 890 9 TP53 17 c.37G>T p.Val13Phe Het missens Likely 11.6 - germli rs113 CO 4289 e pathoge 6 0.32 ne 1691 SM3 09 nic 69 043 378 354; CO SM3 378 355; CO SM3 548 62

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39; CO SM3 548 40; CO SM3 548 41; CO SM3 548 42; CO SM4 424 0;42 890 9 TP53 17 c.481G> p.Val161Ph Het missens Likely 11.6 - germli rs113 CO 4289 T e e pathoge 6 0.32 ne 1691 SM3 09 nic 69 043 378 354; CO SM3 378 355; CO SM3 548 39; CO SM3 548 40; CO SM3 548 41; CO SM3 548 42; CO SM4 424 0;42 890 9 TP53 17 c.514G> p.Val172Ph Het missens Likely 11.6 - germli rs113 CO 4289 T e e pathoge 6 0.32 ne 1691 SM3 09 nic 69 043 378 354; CO SM3 378 355; CO SM3 548 63

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39; CO SM3 548 40; CO SM3 548 41; CO SM3 548 42; CO SM4 424 0;42 890 9 TP53 17 c.37G>T p.Val13Phe Het missens Likely 11.6 - germli rs113 CO 4289 e pathoge 6 0.32 ne 1691 SM3 09 nic 69 043 378 354; CO SM3 378 355; CO SM3 548 39; CO SM3 548 40; CO SM3 548 41; CO SM3 548 42; CO SM4 424 0;42 890 9 TP53 17 c.37G>T p.Val13Phe Het missens Likely 11.6 - germli rs113 CO 4289 e pathoge 6 0.32 ne 1691 SM3 09 nic 69 043 378 354; CO SM3 378 355; CO SM3 548 64

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39; CO SM3 548 40; CO SM3 548 41; CO SM3 548 42; CO SM4 424 0;42 890 9 TP53 17 c.397G> p.Val133Ph Het missens Likely 11.6 - germli rs113 CO 4289 T e e pathoge 6 0.32 ne 1691 SM3 09 nic 69 043 378 354; CO SM3 378 355; CO SM3 548 39; CO SM3 548 40; CO SM3 548 41; CO SM3 548 42; CO SM4 424 0;42 890 9 TP53 17 c.397G> p.Val133Ph Het missens Likely 11.6 - germli rs113 CO 4289 T e e pathoge 6 0.32 ne 1691 SM3 09 nic 69 043 378 354; CO SM3 378 355; CO SM3 548 65

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39; CO SM3 548 40; CO SM3 548 41; CO SM3 548 42; CO SM4 424 0;42 890 9 TP53 17 c.397G> p.Val133Ph Het missens Likely 11.6 - germli rs113 CO 4289 T e e pathoge 6 0.32 ne 1691 SM3 09 nic 69 043 378 354; CO SM3 378 355; CO SM3 548 39; CO SM3 548 40; CO SM3 548 41; CO SM3 548 42; CO SM4 424 0;42 890 9 TP53 17 c.397G> p.Val133Ph Het missens Likely 11.6 - germli rs113 CO 4289 T e e pathoge 6 0.32 ne 1691 SM3 09 nic 69 043 378 354; CO SM3 378 355; CO SM3 548 66

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39; CO SM3 548 40; CO SM3 548 41; CO SM3 548 42; CO SM4 424 0;42 890 9 TP53 17 c.215C> p.Pro72Arg Het missens drug 11.6 - germli rs104 CO 1235 G e respons 6 0.32 ne 2522 SM2 1 e 69 500 61; CO SM3 766 190; CO SM3 766 191; CO SM3 766 192; CO SM3 766 193; 123 51 TP53 17 c.215C> p.Pro72Arg Het missens drug 11.6 - germli rs104 CO 1235 G e respons 6 0.32 ne 2522 SM2 1 e 69 500 61; CO SM3 766 190; CO SM3 766 191; CO SM3 766 192; CO SM3 766 67

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193; 123 51 TP53 17 c.215C> p.Pro72Arg Het missens drug 11.6 - germli rs104 CO 1235 G e respons 6 0.32 ne 2522 SM2 1 e 69 500 61; CO SM3 766 190; CO SM3 766 191; CO SM3 766 192; CO SM3 766 193; 123 51 TP53 17 c.215C> p.Pro72Arg Het missens drug 11.6 - germli rs104 CO 1235 G e respons 6 0.32 ne 2522 SM2 1 e 69 500 61; CO SM3 766 190; CO SM3 766 191; CO SM3 766 192; CO SM3 766 193; 123 51 TP53 17 c.215C> p.Pro72Arg Het missens drug 11.6 - germli rs104 CO 1235 G e respons 6 0.32 ne 2522 SM2 1 e 69 500 61; CO SM3 766 190; CO SM3 766 191; CO 68

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SM3 766 192; CO SM3 766 193; 123 51 TP53 17 c.215C> p.Pro72Arg Het missens drug 11.6 - germli rs104 CO 1235 G e respons 6 0.32 ne 2522 SM2 1 e 69 500 61; CO SM3 766 190; CO SM3 766 191; CO SM3 766 192; CO SM3 766 193; 123 51 TP53 17 c.215C> p.Pro72Arg Het missens drug 11.6 - germli rs104 CO 1235 G e respons 6 0.32 ne 2522 SM2 1 e 69 500 61; CO SM3 766 190; CO SM3 766 191; CO SM3 766 192; CO SM3 766 193; 123 51 TP53 17 c.215C> p.Pro72Arg Het missens drug 11.6 - germli rs104 CO 1235 G e respons 6 0.32 ne 2522 SM2 1 e 69 500 61; CO SM3 766 69

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190; CO SM3 766 191; CO SM3 766 192; CO SM3 766 193; 123 51 TP53 17 c.215C> p.Pro72Arg Het missens drug 11.6 - germli rs104 CO 1235 G e respons 6 0.32 ne 2522 SM2 1 e 69 500 61; CO SM3 766 190; CO SM3 766 191; CO SM3 766 192; CO SM3 766 193; 123 51 TP53 17 c.98C>G p.Pro33Arg Het missens drug 11.6 - germli rs104 CO 1235 e respons 6 0.32 ne 2522 SM2 1 e 69 500 61; CO SM3 766 190; CO SM3 766 191; CO SM3 766 192; CO SM3 766 193; 123 51

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TP53 17 c.98C>G p.Pro33Arg Het missens drug 11.6 - germli rs104 CO 1235 e respons 6 0.32 ne 2522 SM2 1 e 69 500 61; CO SM3 766 190; CO SM3 766 191; CO SM3 766 192; CO SM3 766 193; 123 51 TP53 17 c.215C> p.Pro72Arg Het missens drug 11.6 - germli rs104 CO 1235 G e respons 6 0.32 ne 2522 SM2 1 e 69 500 61; CO SM3 766 190; CO SM3 766 191; CO SM3 766 192; CO SM3 766 193; 123 51 TP53 17 c.215C> p.Pro72Arg Het missens drug 11.6 - germli rs104 CO 1235 G e respons 6 0.32 ne 2522 SM2 1 e 69 500 61; CO SM3 766 190; CO SM3 766 191; CO SM3 766 192; 71

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CO SM3 766 193; 123 51 TP53 17 c.98C>G p.Pro33Arg Het missens drug 11.6 - germli rs104 CO 1235 e respons 6 0.32 ne 2522 SM2 1 e 69 500 61; CO SM3 766 190; CO SM3 766 191; CO SM3 766 192; CO SM3 766 193; 123 51 TP53 17 c.98C>G p.Pro33Arg Het missens drug 11.6 - germli rs104 CO 1235 e respons 6 0.32 ne 2522 SM2 1 e 69 500 61; CO SM3 766 190; CO SM3 766 191; CO SM3 766 192; CO SM3 766 193; 123 51 TP53 17 c.98C>G p.Pro33Arg Het missens drug 11.6 - germli rs104 CO 1235 e respons 6 0.32 ne 2522 SM2 1 e 69 500 61; CO SM3 766 190; CO SM3 72

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766 191; CO SM3 766 192; CO SM3 766 193; 123 51 TP53 17 c.98C>G p.Pro33Arg Het missens drug 11.6 - germli rs104 CO 1235 e respons 6 0.32 ne 2522 SM2 1 e 69 500 61; CO SM3 766 190; CO SM3 766 191; CO SM3 766 192; CO SM3 766 193; 123 51 ANKR 5 c.4827C p.Asp1609 Het missens NA 0.01 - germli rs961 NA D31 >G Glu e 0.17 ne 098 59 ANKR 5 c.4998C p.Asp1666 Het missens NA 0.01 - germli rs961 NA D31 >G Glu e 0.17 ne 098 59 ANKR 5 c.2104G p.Asp702As Het missens NA 0.01 - germli rs142 CO NA D31 >A n e 0.17 ne 2698 SM4 59 830 36; CO SM4 830 37 ANKR 5 c.2104G p.Asp702As Het missens NA 0.01 - germli rs142 CO NA D31 >A n e 0.17 ne 2698 SM4 59 830 36; CO SM4 830 37 ANKR 5 c.755 p.Met252de Het disruptiv NA 0.01 - germli rs105 CO NA D31 757delT l e 0.17 ne 6385 SM5 GA inframe 59 4;rs7 713 deletion 115; 73

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9633 CO 9850 SM5 713 116 ANKR 5 c.755 p.Met252de Het disruptiv NA 0.01 - germli rs105 CO NA D31 757delT l e 0.17 ne 6385 SM5 GA inframe 59 4;rs7 713 deletion 9633 115; 9850 CO SM5 713 116 PTPRJ 11 c.827A> p.Gln276Pr Het missens Pathoge 0.83 0.10 germli rs156 CO 8690 C o e nic 37 ne 6734 SM1 127 775; CO SM1 127 776; CO SM3 736 043; 869 0 PTPRJ 11 c.827A> p.Gln276Pr Het missens Pathoge 0.83 0.10 germli rs156 CO 8690 C o e nic 37 ne 6734 SM1 127 775; CO SM1 127 776; CO SM3 736 043; 869 0 PTPRJ 11 c.827A> p.Gln276Pr Het missens Pathoge 0.83 0.10 germli rs156 CO 8690 C o e nic 37 ne 6734 SM1 127 775; CO SM1 127 776; CO SM3 736 043; 869 0 PTPRJ 11 c.827A> p.Gln276Pr Het missens Pathoge 0.83 0.10 germli rs156 CO 8690 C o e nic 37 ne 6734 SM1 127 775; CO SM1 74

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127 776; CO SM3 736 043; 869 0 PTPRJ 11 c.977G> p.Arg326Gl Het missens NA 0.83 0.10 germli rs150 CO NA A n e 37 ne 3185 SM3 752 397; CO SM3 752 398; CO SM3 752 399 PTPRJ 11 c.977G> p.Arg326Gl Het missens NA 0.83 0.10 germli rs150 CO NA A n e 37 ne 3185 SM3 752 397; CO SM3 752 398; CO SM3 752 399 PTPRJ 11 c.977G> p.Arg326Gl Het missens NA 0.83 0.10 germli rs150 CO NA A n e 37 ne 3185 SM3 752 397; CO SM3 752 398; CO SM3 752 399 PTPRJ 11 c.977G> p.Arg326Gl Het missens NA 0.83 0.10 germli rs150 CO NA A n e 37 ne 3185 SM3 752 397; CO SM3 752 398; CO SM3 752 399 PTPRJ 11 c.2616G p.Glu872As Het missens NA 0.83 0.10 germli rs475 CO NA >C p e 37 ne 2904 SM4 998 272; 75

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CO SM4 998 273 PTPRJ 11 c.2616G p.Glu872As Het missens NA 0.83 0.10 germli rs475 CO NA >C p e 37 ne 2904 SM4 998 272; CO SM4 998 273 PTPRJ 11 c.2616G p.Glu872As Het missens NA 0.83 0.10 germli rs475 CO NA >C p e 37 ne 2904 SM4 998 272; CO SM4 998 273 TIRAP 11 c.539C> p.Ser180Le Het missens protectiv 1.48 0.22 germli rs817 CO 4467 T u e e 5 ne 7374 SM4 986 364; CO SM4 986 365; 446 7 TIRAP 11 c.539C> p.Ser180Le Het missens protectiv 1.48 0.22 germli rs817 CO 4467 T u e e 5 ne 7374 SM4 986 364; CO SM4 986 365; 446 7 TIRAP 11 c.539C> p.Ser180Le Het missens protectiv 1.48 0.22 germli rs817 CO 4467 T u e e 5 ne 7374 SM4 986 364; CO SM4 986 365; 446 7 TIRAP 11 c.539C> p.Ser180Le Het missens protectiv 1.48 0.22 germli rs817 CO 4467 T u e e 5 ne 7374 SM4 986 364; CO SM4 986 365; 446 7 76

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TIRAP 11 c.539C> NA Het structur protectiv 1.48 0.22 germli rs817 CO 4467 T al e 5 ne 7374 SM4 interacti 986 on 364; CO SM4 986 365; 446 7 TIRAP 11 c.539C> NA Het structur protectiv 1.48 0.22 germli rs817 CO 4467 T al e 5 ne 7374 SM4 interacti 986 on 364; CO SM4 986 365; 446 7 TIRAP 11 c.539C> NA Het structur protectiv 1.48 0.22 germli rs817 CO 4467 T al e 5 ne 7374 SM4 interacti 986 on 364; CO SM4 986 365; 446 7 TIRAP 11 c.539C> NA Het structur protectiv 1.48 0.22 germli rs817 CO 4467 T al e 5 ne 7374 SM4 interacti 986 on 364; CO SM4 986 365; 446 7 SLC7A 14 c.726G> p.Trp242* Het stop Pathoge 0.6 - germli rs121 NA 6214 7 A gained nic 0.24 ne 9086 62 79 SLC7A 14 c.726G> p.Trp242* Het stop Pathoge 0.6 - germli rs121 NA 6214 7 A gained nic 0.24 ne 9086 62 79 SLC7A 14 c.726G> p.Trp242* Het stop Pathoge 0.6 - germli rs121 NA 6214 7 A gained nic 0.24 ne 9086 62 79 SLC7A 14 c.726G> p.Trp242* Het stop Pathoge 0.6 - germli rs121 NA 6214 7 A gained nic 0.24 ne 9086 62 79 SLC7A 14 c.726G> p.Trp242* Het stop Pathoge 0.6 - germli rs121 NA 6214 7 A gained nic 0.24 ne 9086 62 79 SLC7A 14 c.726G> p.Trp242* Het stop Pathoge 0.6 - germli rs121 NA 6214 7 A gained nic 0.24 ne 9086 62 79

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NAT2 8 c.341T> p.Ile114Thr Het missens drug 0 0.00 germli rs180 NA 723 C e respons 68 ne 1280 e NAT2 8 c.590G> p.Arg197Gl Het missens drug 0 0.00 germli rs179 CO 722 A n e respons 68 ne 9930 SM3 e 763 234; 722 NAT2 8 c.200G> p.Arg67Gln Het missens drug 0 0.00 germli rs179 CO 722 A e respons 68 ne 9930 SM3 e 763 234; 722 NAT2 8 c.803G> p.Arg268Ly Het missens NA 0 0.00 germli rs120 CO NA A s e 68 ne 8 SM4 998 006 NAT2 8 c.413G> p.Arg138Ly Het missens NA 0 0.00 germli rs120 CO NA A s e 68 ne 8 SM4 998 006 HSP90 12 c.2371 p.Glu791del Het conserv NA 210. - germli rs201 NA B1 2373del ative 25 0.07 ne 4277 GAA inframe 73 69;rs deletion 5800 607;r s869 2159 88 HSP90 12 c.421 p.Glu141del Het conserv NA 210. - germli rs201 NA B1 423delG ative 25 0.07 ne 4277 AA inframe 73 69;rs deletion 5800 607;r s869 2159 88 ADRB2 5 c.46G>A p.Gly16Arg Het missens drug 1.48 - germli rs104 CO 2259 e respons 0.13 ne 2713 SM4 37 e 17 003 392; 225 937 ADRB2 5 c.79G>C p.Glu27Gln Hom missens NA 1.48 - germli rs104 CO NA e 0.13 ne 2714 SM4 17 159 614 COL6A 3 c.3838A p.Thr1280P Het missens NA 0 - germli rs124 CO NA 5 >C ro e 0.13 ne 8845 SM4 07 7 998 804 COL6A 3 c.3838A p.Thr1280P Het missens NA 0 - germli rs124 CO NA 5 >C ro e 0.13 ne 8845 SM4 07 7 998 804 COL6A 3 c.4430G p.Cys1477S Hom missens NA 0 - germli rs149 CO NA 5 >C er e 0.13 ne 7312 SM4 07 157 259

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COL6A 3 c.4430G p.Cys1477S Hom missens NA 0 - germli rs149 CO NA 5 >C er e 0.13 ne 7312 SM4 07 157 259 COL6A 3 c.4765T p.Ser1589P Hom missens NA 0 - germli rs168 CO NA 5 >C ro e 0.13 ne 2749 SM4 07 7 157 260 COL6A 3 c.4765T p.Ser1589P Hom missens NA 0 - germli rs168 CO NA 5 >C ro e 0.13 ne 2749 SM4 07 7 157 260 COL6A 3 c.6551A p.Gln2184A Het missens NA 0 - germli rs988 NA 5 >G rg e 0.13 ne 3988 07 COL6A 3 c.6563A p.Gln2188A Het missens NA 0 - germli rs988 NA 5 >G rg e 0.13 ne 3988 07 COL6A 3 c.392A> p.Gln131Ar Het missens NA 0 - germli rs988 NA 5 G g e 0.13 ne 3988 07 COL6A 3 c.68A>G p.Gln23Arg Het missens NA 0 - germli rs988 NA 5 e 0.13 ne 3988 07 COL6A 3 c.1316A p.Gln439Ar Het missens NA 0 - germli rs988 NA 5 >G g e 0.13 ne 3988 07 COL6A 3 c.6602G p.Gly2201A Hom missens NA 0 - germli rs819 NA 5 >A sp e 0.13 ne 085 07 COL6A 3 c.6614G p.Gly2205A Hom missens NA 0 - germli rs819 NA 5 >A sp e 0.13 ne 085 07 COL6A 3 c.443G> p.Gly148As Hom missens NA 0 - germli rs819 NA 5 A p e 0.13 ne 085 07 COL6A 3 c.119G> p.Gly40Asp Hom missens NA 0 - germli rs819 NA 5 A e 0.13 ne 085 07 COL6A 3 c.1367G p.Gly456As Hom missens NA 0 - germli rs819 NA 5 >A p e 0.13 ne 085 07 COL6A 3 c.6802G p.Glu2268* Het stop NA 0 - germli rs115 NA 5 >T gained 0.13 ne 3758 07 67 COL6A 3 c.6814G p.Glu2272* Het stop NA 0 - germli rs115 NA 5 >T gained 0.13 ne 3758 07 67 COL6A 3 c.643G> p.Glu215* Het stop NA 0 - germli rs115 NA 5 T gained 0.13 ne 3758 07 67 COL6A 3 c.319G> p.Glu107* Het stop NA 0 - germli rs115 NA 5 T gained 0.13 ne 3758 07 67 COL6A 3 c.1567G p.Glu523* Het stop NA 0 - germli rs115 NA 5 >T gained 0.13 ne 3758 07 67

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COL6A 3 c.7667G p.Ser2556A Het missens NA 0 - germli rs322 CO NA 5 >A sn e 0.13 ne 117 SM3 07 846 199; CO SM3 846 200 COL6A 3 c.7679G p.Ser2560A Het missens NA 0 - germli rs322 CO NA 5 >A sn e 0.13 ne 117 SM3 07 846 199; CO SM3 846 200 COL6A 3 c.1508G p.Ser503As Het missens NA 0 - germli rs322 CO NA 5 >A n e 0.13 ne 117 SM3 07 846 199; CO SM3 846 200 COL6A 3 c.1184G p.Ser395As Het missens NA 0 - germli rs322 CO NA 5 >A n e 0.13 ne 117 SM3 07 846 199; CO SM3 846 200 COL6A 3 c.7760d p.Leu2587f Het frameshi NA 0 - germli rs113 CO NA 5 elT s ft 0.13 ne 5579 SM4 07 6;rs3 589 9809 977; 1810 CO SM4 589 978 COL6A 3 c.7772d p.Leu2591f Het frameshi NA 0 - germli rs113 CO NA 5 elT s ft 0.13 ne 5579 SM4 07 6;rs3 589 9809 977; 1810 CO SM4 589 978 COL6A 3 c.1601d p.Leu534fs Het frameshi NA 0 - germli rs113 CO NA 5 elT ft 0.13 ne 5579 SM4 07 6;rs3 589 9809 977; 1810 CO SM4 589 978 COL6A 3 c.1277d p.Leu426fs Het frameshi NA 0 - germli rs113 CO NA 5 elT ft 0.13 ne 5579 SM4 07 6;rs3 589 977; 80

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9809 CO 1810 SM4 589 978 OR2B1 1 c.668G> p.Gly223As Het missens NA 0.01 0.04 germli rs492 CO NA 1 A p e 43 ne 5663 SM3 746 893 OR2B1 1 c.592G> p.Val198Me Hom missens NA 0.01 0.04 germli rs669 NA 1 A t e 43 ne 5302 OR2B1 1 c.389T> p.Ile130Ser Het missens NA 0.01 0.04 germli rs115 CO NA 1 G e 43 ne 8341 SM3 0 751 085 OR2B1 1 c.23delT p.Phe8fs Het frameshi NA 0.01 0.04 germli rs353 CO NA 1 ft 43 ne 0598 SM5 0;rs3 713 9773 051 3455 GLUD2 X c.1492T p.Ser498Al Hom missens Pathoge 0.86 0.11 germli rs969 NA 2993 >G a e nic 31 ne 7983 6 CYP4F 19 c.1297G p.Val433Me Het missens drug 0.1 - germli rs210 CO 2259 2 >A t e respons 0.13 ne 8622 SM3 69 e 9 756 415; 225 969 CYP4F 19 c.1297G p.Val433Me Het missens drug 0.1 - germli rs210 CO 2259 2 >A t e respons 0.13 ne 8622 SM3 69 e 9 756 415; 225 969 KRT3 12 c.1123C p.Arg375Gl Het missens NA 0.02 - germli rs388 CO NA >G y e 0.05 ne 7954 SM5 248 959 775 KRT3 12 c.396 p.Gly133 Het disruptiv NA 0.02 - germli rs148 CO NA 413delA Gly138del e 0.05 ne 5311 SM4 GGGGC inframe 248 42;rs 774 TGGTG deletion 7511 168 GCTTT 8318 GG 2 SLC29 10 c.473C> p.Ser158Ph Hom missens Benign, 1.7 - germli rs780 CO 1303 A3 T e e drug 0.11 ne 668 SM3 41 respons 85 751 e 939; 130 341 SLC29 10 c.715G> p.Val239Ile Hom missens Benign 1.7 - germli rs225 NA 1303 A3 A e 0.11 ne 2996 44 85 SLC29 10 c.976A> p.Ile326Val Hom missens Benign 1.7 - germli rs248 NA 1303 A3 G e 0.11 ne 7068 47 85 ADH1C 4 c.1048A p.Ile350Val Het missens protectiv 0.16 - germli rs698 NA 1818 >G e e 0.17 ne ;1818 0 98 0

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TAS2R 7 c.886A> p.Ile296Val Het missens drug 0 0.23 germli rs102 CO 2906 38 G e respons 36 ne 4693 SM5 e 9 438 552; 290 6 TAS2R 7 c.785C> p.Ala262Val Het missens NA 0 0.23 germli rs172 CO NA 38 T e 36 ne 6866 SM1 504 06 Het, Heterozygous

Hom, Homozygous

Supplemental Table 5. Sequencing coverage

Data type Coverage Tumor Whole Exome 100x coverage Matched Normal Whole Exome 50x coverage Whole Transcriptome 40M Reads

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Refractory Alveolar Rhabdomyosarcoma in a 11-year old male

Cora Ricker, Andrew Woods, William Simonson, et al.

Cold Spring Harb Mol Case Stud published online January 12, 2021 Access the most recent version at doi:10.1101/mcs.a005983

Supplementary http://molecularcasestudies.cshlp.org/content/suppl/2021/01/14/mcs.a005983.D Material C1

Published online January 12, 2021 in advance of the full issue.

Accepted Peer-reviewed and accepted for publication but not copyedited or typeset; accepted Manuscript manuscript is likely to differ from the final, published version. Published onlineJanuary 12, 2021in advance of the full issue.

Creative This article is distributed under the terms of the Commons http://creativecommons.org/licenses/by-nc/4.0/, which permits reuse and License redistribution, except for commercial purposes, provided that the original author and source are credited. Email Alerting Receive free email alerts when new articles cite this article - sign up in the box at the Service top right corner of the article or click here.

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