Metastatic Pediatric Sclerosing Epithelioid Fibrosarcoma

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running title: Case study of a young child with sclerosing epithelioid fibrosarcoma

Andrew D. Woods1#, Reshma Purohit1, Laura Crocker Mitchell2, John R. Collier1, Katherine A. Collier1, Melvin Lathara1,3, Katrina Learned4, Olena Vaske4, Heather Geiger5, Kazimierz O. Wrzeszczynski5, Vaidehi Jobanputra5, Ganapati Srinivasa3, Erin R. Rudzinski6, Kimberly Whelan2, Elizabeth Beierle2, Sheri L. Spunt7, Charles Keller1*#, Aman Wadhwa2*#

1 Children’s Cancer Therapy Development Institute, Beaverton, OR 97005 USA 2 University of Alabama at Birmingham, Birmingham, AL 35233 USA 3 Omics Data Automation, Beaverton, OR 97005 USA 4 University of California Santa Cruz, Santa Cruz, CA 95064 USA 5 New York Genome Center, New York, NY 10013 USA 6 Seattle Children’s Hospital, Seattle, WA 98105 USA 7 Stanford University School of Medicine, Palo Alto, CA 94304 USA

# contributed equally * corresponding authors:

Charles Keller MD, Children's Cancer Therapy Development Institute, 12655 SW Beaverdam Road West, Beaverton OR 97005 USA, Tel (970) 239-4296, Fax (970) 237-6388, email: [email protected]

Aman Wadhwa MD, MSPH, University of Alabama at Birmingham, 1600 7th Avenue South, Lowder Suite 500 Birmingham, AL 35233 USA, Tel (205) 638-2143, Fax (205) 975-1941, email: [email protected]

word count: 4099 words in main text, excluding abstract, methods, references and figure legends

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Abstract

Sclerosing epithelioid fibrosarcoma (SEF) is a rare and aggressive soft tissue sarcoma thought to originate in fibroblasts of the tissues comprising tendons, ligaments and muscles.

Minimally responsive to conventional cytotoxic chemotherapies, greater than 50% of SEF patients experience local recurrence and/or metastatic disease. SEF is most commonly discovered in middle-aged and elderly adults, but also rarely in children. A common gene fusion occurring between the EWSR1 and CREB3L1 genes has been observed in 80-90% of SEF cases. We describe here the youngest SEF patient reported to date (a 3-year-old Caucasian male) who presented with numerous bony and lung metastases. Additionally, we perform a comprehensive literature review of all SEF-related articles published since the disease was first characterized.

Finally, we describe the generation of an SEF primary cell line, the first such culture to be reported.

The patient described here experienced persistent disease progression despite aggressive treatment including multiple resections, radiotherapy and numerous chemotherapies and targeted therapeutics. Untreated and locally recurrent tumor and metastatic tissue were sequenced by whole genome, whole exome, and deep transcriptome next generation sequencing with comparison to a patient-matched normal blood sample. Consistent across all sequencing analyses was the disease-defining EWSR1-CREB3L1 fusion as a single feature consensus. We provide an analysis of our genomic findings and discuss potential therapeutic strategies for SEF.

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Introduction

Sclerosing epithelioid fibrosarcoma (SEF) is a rare and aggressive soft-tissue sarcoma,

first characterized in 1995 by Dr. Jeanne Meis-Kindblom et al (Meis-Kindblom et al., 1995) who

described the disease as a variant of low-grade fibromyxoid sarcoma (LGFMS). More recently, hybrid tumors have been described which share morphologic and gene fusion variants of both

SEF and LGFMS (Arbajian et al., 2017). Despite these shared characteristics, SEF is much more

aggressive with a relatively high incidence of local recurrence (>50% of cases), distant metastatic

spread (40-80% of cases) and mortality (25-57%) (Meis-Kindblom et al., 1995, Antonescu et al.,

2001). Due to the rarity of SEF, little information about effective treatment regimens exists.

Depending upon staging, most patients either undergo surgery alone or also receive systemic chemotherapy and radiotherapy to affected sites. Rates of response to traditional chemotherapy are low and even those receiving aggressive therapy fare poorly (Chew et al., 2018), especially those with metastatic disease at presentation.

The lack of specific phenotypic differentiation and immunohistochemical signature as well as morphologic similarity of SEF to LGFMS and hybrid-SEF/LGFMS tumors makes the pathologic diagnosis challenging. The aggressive nature of SEF makes accurate identification critical so that patients may receive appropriate care. Microscopically, pure SEF is characterized by small round or ovoid epithelioid fibroblast-like cells with pale or clear cytoplasm arranged in strands, cords, nests and sheets, embedded in a densely sclerotic and hyalinized collagenous stroma (Meis-

Kindblom et al., 1995, Antonescu et al., 2001). Genetically, classic SEF is characterized by fusion rearrangements, the most common being EWSR1-CREB3L1, and rarely FUS-CREB3L1 or

ESWR1-CREB3L2/3 (Wang et al., 2012b, Arbajian et al., 2017, Dewaele et al., 2017). We note that EWSR1 rearrangement may suggest a diagnosis of Ewing sarcoma (EWS), yet the morphologic and immunohistochemical profile of SEF differs from that of EWS. SEF typically demonstrates positive staining for CD24, MUC4 and vimentin and displays fibroblastic features

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(Jiao et al., 2002, Antonescu et al., 2001, Meis-Kindblom et al., 1995, Doyle et al., 2012). SEF has no obvious sex predilection and most commonly affects middle-aged and elderly adults but

rarely, children. Here, we describe the case of a child with SEF, the youngest so far reported in

the literature to our knowledge. This report follows a partial case report of this patient’s

histomorphology (Kurtz et al., 2021) but extends that report by presenting here a comprehensive

molecular analysis, the first description of a SEF cell culture and review of the pediatric and non-

pediatric disease features.

Results

Clinical Presentation and family history

A 3-year-old boy presented to the emergency room (ER) with left leg pain. Plain

radiographs of the left lower extremity showed a proximal left fibular complex cystic lesion with

poorly defined margins, thought to represent a bone cyst. However, follow-up magnetic resonance

imaging (MRI) 3 weeks later demonstrated T1 hypo-intense and T2 hyper-intense areas within

the cyst, raising concern for malignancy. A computed tomography (CT) guided biopsy was

performed which showed sheets of mononuclear cells and an abundance of multinucleated giant

cells upon microscopic examination, without evidence of malignancy.

Three months after symptom onset, the child suffered a fall and presented to the ER with

pain in the contralateral (right) leg, leading to further imaging that showed a right distal femur

pathologic fracture through another, larger bone cyst. Given these two cystic lesions, the patient

underwent a skeletal survey which highlighted additional bony lesions in the lower extremities, a

single lesion in the right 6th rib and right proximal humerus along with a soft-tissue density in the abdomen. A subsequent CT of the neck, chest, abdomen and pelvis showed a large mass (10.5 x 6.9 cm) arising from the superior pole of the left kidney and numerous metastatic nodules in both lungs, the largest of which was 1.8 cm in maximal diameter.

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An open renal biopsy yielded pink and grey soft tissue that microscopically showed tumor

cells arranged in small cohesive nests in some areas and sheets of cells in other areas.

Immunostaining of the biopsy specimen revealed the malignant cells to be diffusely positive for vimentin, focally positive for EMA and Cyclin D1 staining was observed in scattered clusters of tumor cells with a variable degree of nuclear staining. The cell membrane stained weakly for CD99 and the nuclei stained positively for BRG1 (SMARCA4) and INI-1 (SMARCB1), and negative for

SMA, Melan-A, HMB-95 and S-100 protein. A diagnosis of clear cell sarcoma of the kidney

(CCSK) was considered and samples were sent to a renal pathologist for review following which,

CCSK was ruled out. Fluorescence in-situ hybridization (FISH) break apart probes were negative for CIC-rearrangement but revealed a 3’ telomeric deletion in the EWSR1 gene. Bilateral bone marrow biopsies from the anterior iliac crests were negative for malignancy. Vincristine, doxorubicin and cyclophosphamide chemotherapy was started while pending a final diagnosis given the tumor burden and concerns for disease progression based on symptoms.

Six weeks after the renal biopsy, a next generation panel genetic sequencing report from

FoundationMedicine® identified an EWSR1/CREB3L1 fusion, NOTCH1 splice site variant

(5167+1G>A – sub clonal), and a TMEM30A mutation (Y134fs*1); microsatellite status (MS) was

stable and a low tumor mutational burden (2 Mutations/Mb) was documented. In light of the

EWSR1-CREB3L1 fusion, the diagnosis of sclerosing epithelioid fibrosarcoma (SEF) was made,

and treatment was changed to doxorubicin and ifosfamide, followed by radiation to the right femur,

left fibula metastasis and the left kidney (37.5/37.5/45 Gy, 15 fractions) tumor per Children’s

Oncology Group protocol ARST0332 Arm D (Spunt et al., 2020). Seven months from symptom

onset, the patient underwent curettage surgery of the right femur lesion.

Two weeks post curettage surgery, the patient underwent a left radical

nephroureterectomy. The histopathology of the residual tumor consisted of nests and cords of

epithelioid cells set within a background of densely hyalinized sclerotic matrix. The lesional cells

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were small to medium sized and contained pale eosinophilic cytoplasm. The nuclei were bland and round or angulated and showed a small nucleolus. Cytoplasmic MUC4 immunostaining was intense in the cytoplasm of the tumor cells.

Three months later (eight months from start of therapy) and after 5 cycles (21 days each) of doxorubicin and ifosfamide, the patient underwent bilateral thoracotomy with complete wedge resection of 8 lung lesions. All lesions showed similar morphological features: well defined lesions with pushing borders, a bland nodular and densely hyalinized fibrous appearance, and nests and cords of small epithelioid cells (Figure 1A and 1B). Some areas displayed a more cellular spindled appearance with mild pleomorphic cytologic features consisting of nuclear enlargement and mild hyperchromasia. Immunohistochemical staining was positive for CD24 (Figure 1C) and negative for cleaved NOTCH1 (Figure 2D). Staining was positive for JAGGED1 (upstream of

NOTCH) and weakly positive for HEY1 (downstream of NOTCH) (Figure 1E and 1F). One month later, the patient underwent resection of a left fibula lesion.

A surveillance CT scan (chest, abdomen and pelvis) four weeks later showed new

metastatic involvement of multiple bones of the skull, ribs and extremities. The patient was given

palliative radiation therapy to selected lesions and started on pazopanib which had demonstrated activity in other soft tissue sarcomas.

Four months from first recurrence and 1 month after completing radiation, CT scans of the chest showed a new nodular density in the right upper lobe and pazopanib was discontinued.

Ahead of planned resection of the lung nodule and following appropriate informed consent discussions, the patient was enrolled on an IRB-approved institutional registry study designed to identify targetable mutations in recurrent pediatric tumors via RNA sequencing and pathway analysis. RNA sequencing of the lung metastasis showed high expression of CDK1 and

NOTCH3. Chemotherapy was changed to trabectedin based on this agent’s activity in other translocation-related sarcomas. Further disease progression after 3 cycles of trabectedin

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prompted treatment with gemcitabine and docetaxel, another regime known to be active in many soft tissue sarcomas. A subsequent positron emission tomography (PET) scan showed continued progression in bony sites (Figure 2).

At this point the patient was enrolled on a phase I Children’s Oncology Group clinical trial

(ADVL1615) and completed 4 treatment cycles of pevonedistat (a NEDD8 activating enzyme inhibitor) with irinotecan and temozolomide. Disease progression in several bony lesions was observed and treatment was discontinued. Persistent post-chemotherapy nausea, vomiting, headaches, and retro-orbital pain led to a follow up MRI that identified new leptomeningeal spread of disease. The patient then underwent 5 days of palliative whole brain radiation. Following radiation, the patient started treatment with compassionate use I-131 omburtamab, a radio- labelled antibody against B78H9/CD276 which is widely expressed on most sarcomas with some benefit in patients with central nervous system neuroblastoma (Kramer et al., 2017) and administered intraventricularly through an Ommaya reservoir. The patient tolerated two infusions of I-131 omburtamab; however, roughly 6 weeks after the second infusion, the child passed away from progression of CNS disease.

Literature review

A comprehensive literature search led to 59 papers published between 1995 and 2021 describing 230 cases of pure SEF (Imada et al., 2017, Wojcik et al., 2014, Stockman et al., 2014,

Dong et al., 2018, Dewaele et al., 2017, Shenoy et al., 2019, Rekhi et al., 2011, Tomimaru et al.,

2009, Elkins and Wakely, 2011, Righi et al., 2015, Kilaikode et al., 2013, Reid et al., 1996,

Hindermann and Katenkamp, 2003, Hanson et al., 2001, Zhang and Chou, 2018, Xu et al., 2016,

Bai et al., 2013, Xia et al., 2020, Donner et al., 2000, Ogose et al., 2004, Gisselsson et al., 1998,

Wang et al., 2012b, Carvalho et al., 2017, Smith et al., 2008, Popli et al., 2018, Kanno et al., 2009,

Patterson et al., 2017, Jiao et al., 2002, Eyden et al., 1998, Puerta Roldán et al., 2013, Leisibach

et al., 2012, Tsuchido et al., 2010, Sassi et al., 2008, Monarca et al., 2013, Bell et al., 2016,

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Grunewald et al., 2010, Argani et al., 2015, Ertoy Baydar et al., 2015, Ohlmann et al., 2015,

Christensen et al., 1997, Chow et al., 2004, Boudová et al., 2001, Bilsky et al., 2000, Folk et al.,

2007, Battiata and Casler, 2005, Arya et al., 2001, Arbajian et al., 2017, Kao et al., 2020, Perez et al., 2019, Zhang et al., 2019, Laliberte et al., 2018, Antonescu et al., 2001, Meis-Kindblom et al., 1995, Choi et al., 2007, Warmke and Meis, 2021, Kosemehmetoglu et al., 2020, Ding et al.,

2020). Patient ages ranged from 9-87 years. Primary and metastatic sites are shown in Figure 3.

Primary tumor locations were reported most commonly in the lower limb or limb girdle (28.3%) and the trunk (18.7%). Other primary tumor sites included the brain, head and neck, soft tissue, lung or pleura, pancreas, kidney, bone, upper limb or limb girdle and liver. The most common site

of metastasis was the lung or pleura (42.1%). Interestingly, while rare as a primary site, the second most common metastatic site was bone (28.6%). Other sites of metastasis were rare but included the trunk, upper limb or limb girdle, lower limb or limb girdle, pancreas, lymph nodes, brain, heart, liver, kidney and soft tissues.

Genomic analyses

The patient described in this report had multiple sequencing analyses, allowing for comparisons between different metastatic sites and across the timeline of disease progression

(Table 1). Tissues resected from the primary kidney tumor, a lung metastasis, a bone metastasis

and a blood-matched patient normal sample were DNA and RNA sequenced and analyzed.

Whole genome, whole exome and deep transcriptome sequencing was performed. This

sequencing dataset was analyzed for both pathogenic germline alterations and actionable

(druggable) targets based on the presence of somatic missense mutations altering the function

of the original gene, evidence of copy number gain, and level of gene expression (Table 2 and

Supplemental Tables S1-5). Circos plot representations of genomic alterations discovered within each dataset are provided in Supplemental Figures S1-4. Detailed fusion calling data is provided in Supplemental Tables S6-7.

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The most common/recurrent genetic change was the EWSR1-CREB3L1 fusion. Studies by Mertens et al (Arbajian et al., 2017) suggest that this fusion alone can dramatically alter the

normal fibroblast transcriptional program, upregulating CD24 and CD276 amongst other genes.

Indeed, CD24 and CD276 were clearly upregulated across this patient’s sequencing samples.

The average CD276 TPM was 178, compared to a normal CD276 TPM average expression of

19.9 (as determined by the Genome Tissue Expression (GTEx) project). Average CD24 TPM across this patient’s sequencing samples was 170. For comparison, 12 epithelioid sarcoma patient samples and cell lines were probed for CD24 expression levels, resulting in an average

CD24 TPM level of 34.7 and a median TPM level of 3.7 (data not shown). SIGLEC10 was of interest as a known cell-cell interactor for CD24 (Barkal et al., 2019), as well as NOTCH1 whose

signaling pathway has been observed to be activated when CD24 is expressed (Tang et al.,

2018). The TP53 tumor suppressor gene was mutated in the femur metastasis sample, and TP53-

axis genes MDM2, MDM4, and CDKN2A (ARF) were overexpressed in the primary tumor and

lung metastasis compared to healthy comparison tissue.

Primary cell culture

Post-mortem tumor tissue samples were collected by rapid autopsy. A small section of tumor tissue from the left lung was minced by hand and digested with a Miltenyi Gentle Macs tumor dissociation system. Resultant cultures were maintained in Gibco’s AmnioMAX C-100

complete medium, a specialized media developed for the short-term culture of human amniotic

fluid cells. Cell cultures retained a fibroblastic morphology displaying elongated (spindle-shaped)

processes extending out from the ends of cell bodies as well as flattened and oval nuclei (Figure

4A). To distinguish this cell culture from normal fibroblasts, we performed polymerase chain

reaction (PCR) analysis to detect the EWSR1-CREB3L1 gene fusion (Figure 4B), short tandem

repeat (STR) (Figure 4C) and drug screen validations (Figure 4D).

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Discussion

SEF lacks a specific phenotypic differentiation and immunohistochemical signature and

shares clinical and morphological features with several differential cancer types, challenging

diagnosis in the absence of genetic data. SEF most commonly occurs in the lower limb or limb

girdle of middle-aged or elderly adults yet also presents in a wide range of alternative anatomical

sites and across a wide range of ages (Meis-Kindblom et al., 1995, Antonescu et al., 2001, Kao et al., 2020, Wang et al., 2012b). The case presented here represents a primary SEF tumor of the kidney in conjunction with multiple bony metastases discovered in a very young patient. This case emphasizes the importance of considering an SEF diagnosis for patients of all ages and tumor locations.

Multiple sequencing analyses allowed for a complete modeling of the genomic landscape of this particular SEF patient. Given the patient’s young age, one could hypothesize that the genetic abnormalities presented here are most likely disease-related and less likely to be from potentially confounding factors often observed in adults, such as lifestyle or long-term exposure to carcinogens. Common to all analyses was the EWSR1-CREB3L1 fusion. A list of genes which were abnormal across different sequencing analyses is shown in Table 2.

CD24

The CD24 gene is primarily expressed by immune cells and has been shown to be

overexpressed in a variety of human cancers including SEF, but not on human erythrocytes.

Arbajain et al showed that increased expression of CD24 in SEF is a direct result of the EWSR1-

CREB3L1 gene fusion event (Arbajian et al., 2017). As expected, CD24 gene expression was

detected in this patient’s transcriptome sequencing. CD24 is a highly glycosylated GPI-anchored

cell surface molecule which resembles cell surface mucin (Kristiansen et al., 2004). CD24 has

been associated with poor prognosis (Lee et al., 2009), is a cancer stem cell marker (Ortiz-

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Montero et al., 2018), and has been shown to regulate tumor cell proliferation, migration and invasion (Baumann et al., 2005). CD24 has been suggested as a therapeutic target in several

cancer types (Salnikov et al., 2013).

CD24 has multiple binding partners including P-selectin and Siglecs. In ovarian and breast

cancers, the CD24-P-selectin interaction has been shown to support the rolling of tumor cells on endothelial cells and the adhesion of tumor cells to platelets and mesothelium (Aigner et al., 1998,

Carroll et al., 2018), suggesting a role in metastasis. Recently, CD24 was shown to interact with

SIGLEC10 on tumor associated macrophages to provide a ‘don’t eat me’ signal, leading to a lack of clearance by the immune system (Barkal et al., 2019). Barkal et al suggested that antibodies which disrupt the interaction between CD24 and SIGLEC10 have therapeutic potential. A variety of antibody treatments have been studied to treat human disease, including the use of naked antibodies, bispecific antibodies and conjugated antibodies. Besides antibody treatment, Sagiv et al (2006) showed that overexpressed CD24 can be down-regulated to normal levels after short- and long-term exposure to the selective COX2 inhibitor, celecoxib (Sagiv et al., 2006).

NOTCH1

The NOTCH signaling pathway is dysregulated in the majority of human cancers. In

previous studies, CD24 expression was shown to accompany NOTCH signaling pathway

activation (Tang et al., 2018) and NOTCH1 inhibition has been shown to decrease CD24

transcription in esophageal adenocarcinoma (Wang et al., 2014b). In the patient presented in this

report, a NOTCH1 splice site variant (c.5167+1G>A) was observed in the primary kidney and metastatic lung tumors, possibly linked to the observed CD24 overexpression. The c.5167+1G>A variant is an intronic substitution listed as pathogenic (FATHMM prediction score=0.99) in the

Catalog of Somatic Mutations in Cancer (COSMIC) database. NOTCH1 signaling has been implicated in a wide-range of oncogenic activities including tumor growth, survival, and

metastasis, and has been shown to be important for cancer stem cell self-renewal, differentiation,

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proliferation, survival, and migration (Venkatesh et al., 2018, Wang et al., 2012a). In this patient’s

lung metastasis and primary kidney samples, numerous NOTCH1 target genes were found to be

overexpressed compared to normal organ-specific tissue collected within the GTEx project, most

notably CyclinD1 (CCND1) which was 45-fold overexpressed in the lung metastasis and 85-fold

overexpressed in the primary kidney tumor (Table 3). Cyclin D1 is a regulator of CDK4/6 which

in turn regulates cell cycle transition from G1 to S phase. Overexpression of Cyclin D1 leads to

rapid cellular growth (Qie and Diehl, 2016). NOTCH signaling has been shown to positively

regulate MTOR pathway activity and several NOTCH target genes within the PI3K–AKT-mTOR

pathway were overexpressed in this patient’s tumor samples (PIK3R1, PIK3R2, AKT1 and

MTOR). As previously noted, SEF tumors display a propensity to metastasize, oftentimes

progressing to widely metastatic disease and resulting in poor patient outcomes. NOTCH1 target

gene HEY1 has been shown to play a fundamental role in tumor vasculature development and

angiogenesis (Wang et al., 2014a), and was 53-fold overexpressed in this patient’s kidney tumor

(data not shown) compared to normal GTEx kidney tissue. No overexpression was observed in

the lung metastasis sample. HEY1 paralog genes HEY2 and HES1 were overexpressed in both

kidney and lung samples. Within the endothelium, crosstalk between NOTCH and VEGF was

shown to be necessary for productive tumor angiogenesis (Gu et al., 2012), and interruption of

this interaction resulted in decreased tumor perfusion and enhanced tumor growth inhibition in

animal models (Miles et al., 2014). Multiple targets within the NOTCH signaling pathway are

considered druggable with small molecule inhibitors, including inhibitors of NOTCH itself, CDK4/6,

PI3K and mTOR. Clinical trials targeting NOTCH are currently underway using gamma secretase

inhibitors, NOTCH receptor antibody treatment and NOTCH transcription complex inhibitors

(Moore et al., 2020).

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TP53

The TP53 tumor suppressor gene has been shown to be involved in a variety of human

cancers (Hollstein et al., 1994, Levine, 1997). TP53 gene axis members include MDM2, MDM4,

and CDKN2A (ARF) genes amongst others (Ozenne et al., 2010). MDM2 overexpression has

been observed previously in SEF patients (Jiao et al., 2002). In the patient described in this report,

TP53 dysregulation and MDM2 overexpression were observed in the primary tumor and multiple metastatic samples (Table 4). Two of the TP53 mutations (p.Pro301fs and p.Pro72Arg) observed in this patient’s femur metastasis sample have been characterized as pathogenic (Malapelle et

al., 2017) and associated with a poor drug response (Kim et al., 2009, Khrunin et al., 2010),

respectively. When compared to GTEx normal tissue, the lung metastasis sample showed a 4 to

5-fold greater MDM2 expression and the kidney tumor showed a 15-fold greater expression.

MDM2 and TP53 form an autoregulatory feedback loop where TP53 regulates MDM2 transcription and MDM2 regulates TP53 activity (Wu et al., 1993). MDM2 ubiquitinates TP53 for proteasomal degradation and works as a “brake” against normal TP53 tumor suppressor activities

(Haupt et al., 1997, Picksley and Lane, 1993, Honda et al., 1997). Increased MDM2 levels

therefore result in decreased wild-type TP53 tumor suppressive activities. Several MDM2

inhibitors have advanced to clinical trials and could be considered as a potential therapeutic

approach for SEF patients with MDM2 overexpression.

A CD24-TP53 gene axis has recently been established in prostate cancer, where CD24

expression is associated with aggressive and metastatic disease. Interestingly, mutant KRAS‐

induced upregulation of CD24 enhances prostate cancer bone metastasis (Weng et al., 2019).

We note that the second most common observed metastatic site in SEF is bone, creating the

intriguing hypothesis that a similar biologic mechanism underlies the propensity for bone

metastasis in SEF. Studies by Wang et al have demonstrated that CD24 can inhibit CDKN2A

(ARF) binding to NPM, leading to a decrease in CDKN2A (ARF) which therefore increases MDM2

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levels and decreases activity of TP53 and its downstream gene target, P21 (Wang et al., 2015).

These researchers also observed a higher rate of TP53 mutation associated with increased CD24 mRNA expression and suggest the possibility of restoring TP53 tumor-suppressor function while simultaneously disabling mutant TP53 oncogenic activity. Three out of the 4 most common TP53 mutants (p53R273H, p53V143A and p53R280T) in CD24 siRNA-silenced cells were almost equally efficient at suppressing colony formation as wild-type TP53. In contrast, TP53 mutants which retained CD24 expression failed to reduce colony formation (Wang et al., 2015). Further studies by Zhang et al confirmed the restoration of TP53 tumor suppressive activities in CD24-silenced

TP53-mutants and demonstrated a role for CD24 in tumor progression and metastasis by showing an abnormal nuclear accumulation of TP53 mutants in CD24 expressing prostate cancer cells

(Zhang et al., 2016).

Copy Number Variation

Increased copy number variation has been observed previously in SEF and has been proposed

to explain SEF’s aggressive nature as compared to the closely-related LGFMS which has a better

prognosis and has been observed to display only minimal copy number variation (Arbajian et al.,

2017). The primary kidney tumor sample in this report strongly suggests loss of 10q, 11p and chromosome 22 (Supplemental Figure S1), concurrent with previous observations within larger cohorts of SEF patients (Arbajian et al., 2017).

Cell culture

Tumor tissue collected at autopsy resulted in a primary cell culture with fibroblastic features

(Figure 4A) which was named “EZ-PZ”. The resultant EZ-PZ cell culture continued to grow

robustly until passage 20 before growth rates declined. To distinguish EZ-PZ from normal

fibroblasts, PCR, STR and drug screen analyses were performed. PCR results showed clear

bands with three different primer sets measuring 1) the fusion breakpoint, 2) a portion of the fusion

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and 3) the entire fusion, indicating that the SEF primary culture EZ-PZ contains the disease- defining EWSR1-CREB3L1 gene fusion while the fusion is absent from normal human fibroblast

BJ5TA cells (Figure 4B). The fusion sequence and primers used for PCR amplification are provided in Supplemental Table S8. STR results show no cross-contamination between EZ-PZ and known cell lines and establish a baseline for future testing and cell culture validation (Figure

4C). Drug screens showed activity against EZ-PZ in 8 out of 60 compounds tested, with IC50

concentrations of <5µM (Figure 4D). Interestingly, EZ-PZ was sensitive to INK128 (IC50=0.45µM), a novel TORC1/2 inhibitor, yet was not sensitive (IC50=>10µM) to the other MTOR inhibitors within

the screen (sirolimus and BKM120). We speculate that INK128’s observed potency is due to dual targeting of both TORC1 and TORC2, and that targeting TORC1 only or MTOR+PI3K is insufficient to reduce EZ-PZ’s cell viability. Three compounds within the drug screen (bortezomib, dasatinib and nabucasin) demonstrated activity against the normal human fibroblast cell line

BJ5TA but showed reduced or no activity against EZ-PZ. We suggest that the SEF cell culture generally displays a drug resistant phenotype, an observation consistent with this patients clinical

response and which is supported by the lack of activity observed in 52 of 60 oncogenic inhibitors

used within the screen.

As this case report demonstrates, SEF is an aggressive tumor which is oftentimes resistant

to conventional chemo- and targeted-therapies. SEF can occur across a wide variety of sites and

age groups. SEF is a fusion-driven cancer, the most common fusion event being the EWSR1-

CREB3L1 gene rearrangement. CD24, MDM2 and NOTCH1 represent potential therapeutic

targets for SEF, although further study of these targets in a SEF-specific context is warranted.

The EZ-PZ primary cell culture which resulted from this patient’s tumor tissue should provide a

study model to perform functional assays which will shed light upon the efficacy of the proposed

genetically-determined therapeutic targets.

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Methods

Immunohistochemistry

Immunohistochemistry was performed with the following antibodies and dilutions: Anti-CD24

antibody (1:100) (ab199140, Abcam, Cambridge, MA), cleaved Notch1 (1:400) (#4147, Cell

Signaling Technology, Danvers, MA), anti-JAGGED1 (1:100) (sc-390177, Santa Cruz

Biotechnology, Dallas, TX) and anti-HEY1 (1:150) (ab235173, Abcam).

Genetic sequencing

Genetic sequencing was performed at New York Genome Center, Foundation One Medicine, MD

Anderson Cancer Center and Covance by Lab Corp. DNA sequencing was performed with at

least 100X coverage, paired-end reads. DNA sequencing was performed on an Ilumina HiSeq X

or DNBseq. RNA sequencing was performed on an Illumina HiSeq 2500. Variant and sequencing

coverage tables are presented in Supplemental tables S4-5.

Genomic and transcriptomic analysis

Genetic sequencing analysis was performed in the following manner: whole exome and whole genome sequencing data was analyzed for the presence of somatic point mutations, 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 > 6.3). Gene copy number variations were identified using

Samtools and VarScan2 quantified as a log ratio of tumor copy to normal copy using the GRCh38 human reference genome. 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.

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NOTCH target gene expression analysis was performed by Omics Data Automation and

Children’s Cancer Therapy Development Institute.

Literature review

To determine common site locations of primary and metastatic SEF tumors, search terms

“sclerosing epithelioid fibrosarcoma”, “CD24”, “EWSR1”, and “EWSR1-CREB3L1” were entered into PubMed and Google search engines. Papers which did not define SEF primary location were excluded. Of the included studies, those that mentioned metastatic disease were used to determine metastatic site locations.

Clinical presentation

Medical history was gathered from patient medical records and discussions with the patient’s family and medical oncologist. Clinical molecular diagnostic whole genome and transcriptome sequencing was performed at the New York Genome Center as previously

described (Wrzeszczynski et al., 2018).

Cell culture generation and validation

Autopsy tumor samples were collected with informed consent (study # ccTDI-IRB-1).

Tumor tissue was minced by hand and processed in a GentleMacs dissociator with the

GentleMacs tumor dissociation kit (130-093-235 and 130-095-929, Miltenyi Biotec GmbH,

Bergisch Gladbach, Germany) according to manufacturer’s protocol. Resultant cultures were maintained in AmnioMAX C-100 complete medium (Gibco, Thermo Fisher Scientific). All cell cultures were maintained in a humidified incubator at 37˚C supplemented with 5% CO2. Short

Tandem Repeat (STR) validation and mycoplasma testing was performed on all cell cultures.

STR analysis was performed with the Promega PowerPlex16HS Assay: 15 Autosomal Loci, X/Y,

with a random match probability of approximately 1 in 1.83x1017.

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For PCR testing, DNA was collected with the PureLink Genomic DNA mini kit (K182002,

Invitrogen, Carlsbad, CA). Dye-based qPCR was performed with DreamTaq Green PCR master mix (K1081, Thermo Scientific, Waltham, MA). Twenty-five µL of master mix, forward and reverse primers, nuclease-free water and 2-4µL of DNA for each sample was made up on ice and then loaded onto a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA) and run for 35 cycles with an annealing temperature of 60°C. Samples were then loaded into a 2% agarose gel and placed on an electrophoresis apparatus for 90 minutes at 155V. To determine resultant band size, a 1Kb Plus DNA ladder (Invitrogen, 10787018) was loaded onto the gel with the samples.

After electrophoresis, the gel was placed in ethidium bromide (ETBR) and placed on a rocker for

10 minutes at medium speed, then washed with TAE buffer for 5 minutes. Bands were visualized with a Model SA-1000 (red) Personal Gel Imaging System SR737 (Alpha Innotech, San Leandro,

CA).

Chemical screens were conducted using an investigator-selected 60-agent drug screen.

Each agent was tested in triplicate at 4 dosage points. Initial drug stocks were diluted in appropriate solvents, then plated onto Nunc™ 384-Well Polystyrene White Microplates (164610,

Thermo Fisher Scientific) with DMSO and media-only controls. 2000 cells/well were added to the drug plates with a Multi-Flo liquid dispenser (BioTek, Winooski, VT), then placed in a humidified incubator for 72 hours at 37˚C supplemented with 5% CO2. Cell viability (ATP) was measured

using the CellTiter-Glo® Luminescent Cell Viability Assay (G7573, Promega, Madison, WI)

following manufacturer’s protocol. Output was read with a Synergy HT plate reader (BioTek).

Additional information

Data deposition and access

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Sequencing data within this paper is deposited in the European Genome-phenome Archive

(EGA). Study accession number is EGAS00001005214. Dataset accession number is

EGAD00001007515.

Table of abbreviations

SEF – Sclerosing Epithelioid Fibrosarcoma

CT – Computed Tomography

FISH – Fluorescence in situ hybridization

MS – Microsatellite Status

PET – Positron Emission Tomography

MRI – Magnetic Resonance Imaging

STR – Short Tandem Repeats

PCR – Polymerase Chain Reaction

TPM – Transcripts Per Million

Keywords

CD24, sclerosing epithelioid fibrosarcoma, genetic profiling, pediatric rare tumors

Ethics Statement

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All human tissue samples were acquired through the Cancer Registry for Familial and Sporadic

Tumors (CuRe-FAST) tumor banking program. All patients enrolled in CuRe-FAST provided written informed consent. All aspects of the study were reviewed and approved by the Children’s

Cancer Therapy Development Institute (cc-TDI) Institutional Review Board (Advarra, study # cc-

TDI-IRB-1).

Competing Interests Statement

The authors have no competing interests.

Author Contributions

Andrew Woods, Reshma Purohit, Laura Mitchell, Aman Wadhwa, and Charles Keller – wrote

manuscript

Sheri Spunt – edited manuscript for intellectual content

Katrina Learned, Olena Vaske – provided genetic sequencing data

Heather Geiger, Kazimierz Wrzeszczynski and Vaidehi Jobanputra – provided sequencing data

and performed genomic analysis

Melvin Lathara and Ganapati Srinivasa – performed genomic analysis

Elizabeth Beierle and Aman Wadhwa – provided tissue samples

John Collier, Katherine Collier and Aman Wadhwa – provided patient information and case history

Erin R. Rudzinski – performed histopathology

All authors – approved final version of manuscript

Funding

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Funding for this project was provided by an anonymous private foundation and the Rucker Collier

Foundation. https://www.ruckercollierfoundation.com

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Figure 1. A, Morphological diagnostic immunochemistry image with hematoxylin and eosin (H&E)

staining of the right posterior upper lung nodule biopsy. Circumscribed tumor adjacent to normal

tissue (top left corner) showing spindle to sclerotic tumor. The round spaces are thought to be

entrapped/residual alveolar spaces. Scalebar = 300µM. B, H&E closeup showing spindle to

sclerotic tumor. Scalebar = 200µM. C, Immunohistochemistry (IHC) staining for CD24. Scalebar

= 300µM. D, IHC staining for cleaved NOTCH1. Scalebar = 100µM. E, IHC staining for JAGGED1

(upstream of NOTCH). Scalebar = 300µM. IHC staining for HEY1 (downstream of NOTCH).

Scalebar = 100µM.

Figure 2. Radiology images showing progression of lung and numerous skeletal metastases over

a 16 week period while enrolled on clinical trial ADVL1615 (pevonedistat, irinotecan and

temozolomide). Left panel images represent metastatic sites at the time of trial enrollment. Right

panel images show disease progression at all sites after 4 treatment cycles. A, Progression of rib,

pelvic, femur and tibia metastases. B, Progression of the right femur metastasis. C, Growth in the

skull lesion. D, Progression of the lung metastases.

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Figure 3. Locations of primary and metastatic SEF tumors. Altogether, 230 cases reported in the literature from 1995-2021 were reviewed. A, Eleven primary tumor sites were reported, the most common being the lower limb or limb girdle (28.3%) and the trunk (18.7%). B, Eighty-eight patients reported metastasis to 12 sites, the most common being the lung (42.1%) and bone (28.6%).

Figure 4. SEF cell culture EZ-PZ. A, Brightfield confocal image of SEF cell culture EZ-PZ at 10X

magnification, scale bar = 100 µM. B, PCR indicating the presence of a EWSR1-CREB3L1 gene

fusion in the EZ-PZ cell culture. BJ5TA is a normal human fibroblast cell culture which shows an

absence of the EWSR1-CREB3L1 fusion. Three primer sets captured the fusion breakpoint, the

majority and the entirety of the gene fusion. C, Baseline STR analysis of the cell culture. Results

did not match to any known cell lines. The cell culture was free of mycoplasma. D, Drug screen

results from the EZ-PZ and BJ5TA cell cultures. Heatmap indicates IC50 values and shows a general increase in resistance and sensitivity of the SEF cell culture to TORC 1/2 inhibition.

Table Legends

Table 1. Location and type of tissue used for various sequencing analyses. WES: Whole Exome

Sequencing, WGS: Whole Genome Sequencing, NM: Matched Normal, MS: Microsatellite

Instability Status, TMB: Tumor Mutation Burden. Microsatellite instability status is a measure of genetic hypermutability due to impaired DNA mismatch repair. Tumor Mutation Burden is a measure of total mutations found in the DNA of cancer cells.

Table 2. Compiled common sequencing results from all samples. Of the 256 total genetic events observed, 33 were found to be common to multiple samples. The human reference genome used was GRCh38.

Table 3. NOTCH1 target gene expression levels reported as Transcripts Per Million (TPM).

Analysis was performed by Omics Data Automation and Children’s Cancer Therapy Development

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Institute. Primary kidney and lung metastasis TPM levels were compared to normal kidney and lung tissue TPM’s (GTEx), respectively.

Table 4. TP53 axis sequencing results showing mutations, copy number variations, and expression levels of TP53 axis genes TP53, MDM2/4, and CDKN2A (ARF). The human reference genome used was GRCh38. NYG=New York Genome Center, FO=Foundation One,

TH=Treehouse Childhood Cancer Initiative, ODA=Omics Data Automation. MS=Microsatellite

Instability Status, S=Stable, US=Unstable.

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Figures Figure 1. Immunohistochemistry staining from right posterior upper lung nodule biopsy.

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Figure 2. Lung and skeletal metastases progression over a 16-week period.

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Figure 3. Locations of SEF primary tumors (A) and metastatic sites (B).

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Figure 4. SEF cell culture, validation and drug screen.

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Tables

Table 1. Samples used for various sequencing analyses.

Post Dx Sample type (months) Sequencing type MS status TMB primary kidney biopsy 0 DNA WES stable 39 Muts primary kidney resection 5 DNA WES, WGS, RNA, NM DNA stable 39 Muts femur metastasis 11 DNA WES unstable 31 Muts lung metastasis 13 DNA WES, WGS, RNA, NM DNA stable 30 Muts normal blood N/A DNA WGS N/A N/A

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Table 2. Consensus longitudinal sequencing results.

HGVS PMID CNV Median Gene (# of analyses) Variant type Transcript ID HGVS DNA protein reference (log2) TPM Samples

EWSR1-CREB3L1 (7) Fusion Kidney Bx, Primary kidney, Lung relapse CREB3L1 (4) ENST00000527342.1 Loss, Gain 181.3 Primary kidney, Lung relapse EWSR1 (4) ENST00000331029.11 c.*1093T>A Loss, Gain 252.7 Primary kidney, Lung relapse CD24 (3) ENST00000606017.1 290 Primary kidney, Lung relapse BCR (2) ENST00000305877.12 c.3012+2059delG Loss 43.7 Primary kidney, Lung relapse CARS (2) ENST00000278224.13 Loss 23.7 Primary kidney, Lung relapse EP300(2) ENST00000263253.8 c.95-8921delT Loss 128.3 Primary kidney, Lung relapse EXT2 (2) ENST00000343631.3 Loss 47.6 Primary kidney, Lung relapse FANCF (2) ENST00000327470.5 Loss 2.5 Primary kidney, Lung relapse GON4L (2) Substitution - Missense ENST00000271883.9 c.4615C>G Q1539E 116.3 Primary kidney, Lung relapse HCAR3 (2) Substitution - Missense ENST00000528880.2 c.1127C>G A376G 0.65 Primary kidney, Lung relapse HRAS (2) ENST00000311189.7 Loss 34.5 Primary kidney, Lung relapse IGL (2) Loss Primary kidney, Lung relapse IRF1 (2) ENST00000245414.8 V175A Kidney Bx, Primary kidney LMO1 (2) ENST00000335790.7 Loss 0 Primary kidney, Lung relapse LMO2 (2) ENST00000290246.10 Loss 15.58 Primary kidney, Lung relapse MKL1 (2) Substitution - Missense ENST00000355630.7 c.902G>A Loss 39.26 Primary kidney, Lung relapse MLL2 (2) P2210L Kidney Bx, Primary kidney MYH9 (2) ENST00000216181.10 c.*5992G>A Loss 636.5 Primary kidney, Lung relapse MYOD1 (2) ENST00000250003.3 Loss 0 Primary kidney, Lung relapse NF2 (2) ENST00000334961.11 c.199-7820G>A Loss 45.47 Primary kidney, Lung relapse NOTCH1 (2) Splice site (subclonal) ENST00000277541.7 c. 5167+1G>A 21798893 16.48 Kidney Bx, Lung relapse NTRK1 (2) ENST00000358660.3 P171S Kidney Bx, Primary kidney NUP98 (2) ENST00000324932.11 Loss 43.1 Primary kidney, Lung relapse PATZ1 (2) ENST00000215919.3 Loss 25.89 Primary kidney, Lung relapse PDGFB (2) ENST00000331163.10 Loss 20.85 Primary kidney, Lung relapse PLCG2 (2) ENST00000359376.7 K1019E Kidney Bx, Primary kidney PRDM1 (2) ENST00000369089.3 K532Q Kidney Bx, Primary kidney ROS1 (2) ENST00000368507.7 R2269* 25186949 Kidney Bx, Primary kidney 34

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SEPT5 (2) ENST00000383045.7 Loss 102.5 Primary kidney, Lung relapse SIGLEC10 (2) Substitution - Missense ENST00000339313.9 c.430C>A Q144K 26925973 25.95 Primary kidney, Lung relapse SPARC (2) ENST00000231061.8 Gain 20457 Primary kidney, Lung relapse TMEM30A (2) Insertion - Frameshift ENST00000230461.10 c.401dupA Y134fs 30.46 Kidney Bx, Primary kidney WT1 (2) ENST00000332351.7 c.1002-8172dupT Loss 0.89 Primary kidney, Lung relapse

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Table 3. NOTCH1 target gene expression levels (TPM). gene lung tumor lung normal fold change (lung) kidney tumor kidney normal fold change (kidney) HEY2 68.24 5.4 12.64 66.93 2.287 29.27 HES1 236.15 72.71 3.25 440.28 66.71 6.6 CCND1 2855.08 63.51 44.95 1951.83 23.08 84.57 GATA3 12.1 6.22 1.95 99.06 22.15 4.47 MYC 179.66 55.01 3.27 51.42 7.855 6.55 PIK3R1 217.46 44.53 4.88 165.19 9.379 17.61 PIK3R2 95.57 22.27 4.29 107.66 20.85 5.16 AKT1 393.74 80.7 4.88 461.23 27.57 16.73 MTOR 27.99 15.39 1.82 49.52 8.864 5.59 BCL2 40.65 6.354 6.4 55.54 4.632 11.99 TCF3 96.49 24.83 3.89 177.55 13.24 13.41 NOTCH3 268.34 79.77 3.36 155.75 18.13 8.59 EP300 119.55 33.78 3.54 137.1 11.53 11.89

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Table 4. TP53 axis sequencing results.

ClinVar HGVS CNV TMB Post Dx Data Gene Chr Variant Transcript ID dbSNP ID ID HGVS protein (log2) TPM (Muts) MS Sample type (months) source Analysis Data type CDKN2A (ARF) 9 ENST00000304494.9 0.0176 10 39 S Primary kidney 5 NYG ODA WGS, RNA, NM MDM2 12 ENST00000258148.11 0.1897 117 39 S Primary kidney 5 NYG ODA WGS, RNA, NM MDM4 1 ENST00000367179.7 0.3419 166 39 S Primary kidney 5 NYG ODA WGS, RNA, NM TP53 17 ENST00000269305.8 0.2565 121 39 S Primary kidney 5 NYG ODA WGS, RNA, NM CDKN2A (ARF) 9 frameshift ENST00000498124.1 c.460delA p.Met154fs 31 US Femur relapse 11 MDA ODA WES CDKN2A (ARF) 9 frameshift ENST00000304494.9 c.64_65delCG p.Arg22fs 31 US Femur relapse 11 MDA ODA WES CDKN2A (ARF) 9 frameshift ENST00000530628.2 c.115delG p.Ala39fs 31 US Femur relapse 11 MDA ODA WES MDM2 12 frameshift ENST00000258148.11 c.730_731insT p.Glu244fs 31 US Femur relapse 11 MDA ODA WES MDM2 12 frameshift ENST00000258149.9 c.895_896insT p.Glu299fs 31 US Femur relapse 11 MDA ODA WES MDM2 12 frameshift ENST00000299252.8 c.367_368insT p.Glu123fs 31 US Femur relapse 11 MDA ODA WES MDM2 12 frameshift ENST00000350057.9 c.802_803insT p.Glu268fs 31 US Femur relapse 11 MDA ODA WES MDM2 12 frameshift ENST00000360430.6 c.292_293insT p.Glu98fs 31 US Femur relapse 11 MDA ODA WES MDM2 12 frameshift ENST00000393415.7 c.736_737insT p.Glu246fs 31 US Femur relapse 11 MDA ODA WES MDM2 12 frameshift ENST00000258148.11 c.1147delC p.Asn384fs 31 US Femur relapse 11 MDA ODA WES MDM2 12 frameshift ENST00000258149.9 c.1312delC p.Asn439fs 31 US Femur relapse 11 MDA ODA WES MDM2 12 frameshift ENST00000299252.8 c.784delC p.Asn263fs 31 US Femur relapse 11 MDA ODA WES MDM2 12 frameshift ENST00000348801.6 c.616delC p.Asn207fs 31 US Femur relapse 11 MDA ODA WES MDM2 12 frameshift ENST00000350057.9 c.1219delC p.Asn408fs 31 US Femur relapse 11 MDA ODA WES MDM2 12 frameshift ENST00000360430.6 c.709delC p.Asn238fs 31 US Femur relapse 11 MDA ODA WES MDM2 12 frameshift ENST00000393410.5 c.550delC p.Asn185fs 31 US Femur relapse 11 MDA ODA WES MDM2 12 frameshift ENST00000393413.7 c.475delC p.Asn160fs 31 US Femur relapse 11 MDA ODA WES MDM2 12 frameshift ENST00000517852.5 c.211delC p.Asn72fs 31 US Femur relapse 11 MDA ODA WES MDM4 1 frameshift ENST00000367180.5 c.90dupA p.Leu31fs 31 US Femur relapse 11 MDA ODA WES MDM4 1 frameshift ENST00000367179.7 c.377delT p.Leu126fs 31 US Femur relapse 11 MDA ODA WES MDM4 1 frameshift ENST00000367182.7 c.722delT p.Leu241fs 31 US Femur relapse 11 MDA ODA WES MDM4 1 frameshift ENST00000614459.4 c.428delT p.Leu143fs 31 US Femur relapse 11 MDA ODA WES MDM4 1 frameshift ENST00000367179.7 c.588dupA p.Phe197fs 31 US Femur relapse 11 MDA ODA WES MDM4 1 frameshift ENST00000367182.7 c.933dupA p.Phe312fs 31 US Femur relapse 11 MDA ODA WES MDM4 1 frameshift ENST00000444261.1 c.267dupA p.Phe90fs 31 US Femur relapse 11 MDA ODA WES MDM4 1 frameshift ENST00000454264.6 c.783dupA p.Phe262fs 31 US Femur relapse 11 MDA ODA WES MDM4 1 frameshift ENST00000612738.4 c.264dupA p.Phe89fs 31 US Femur relapse 11 MDA ODA WES MDM4 1 frameshift ENST00000614459.4 c.639dupA p.Phe214fs 31 US Femur relapse 11 MDA ODA WES TP53 17 missense ENST00000269305.8 rs1042522 12351 c.215C>G p.Pro72Arg 31 US Femur relapse 11 MDA ODA WES TP53 17 missense ENST00000610292.4 rs1042522 12351 c.98C>G p.Pro33Arg 31 US Femur relapse 11 MDA ODA WES TP53 17 frameshift ENST00000269305.8 c.140delC p.Pro47fs 31 US Femur relapse 11 MDA ODA WES TP53 17 frameshift ENST00000610292.4 c.23delC p.Pro8fs 31 US Femur relapse 11 MDA ODA WES TP53 17 frameshift ENST00000269305.8 rs876660726 233908 c.902delC p.Pro301fs 31 US Femur relapse 11 MDA ODA WES TP53 17 frameshift ENST00000504290.5 rs876660726 233908 c.506delC p.Pro169fs 31 US Femur relapse 11 MDA ODA WES TP53 17 frameshift ENST00000610292.4 rs876660726 233908 c.785delC p.Pro262fs 31 US Femur relapse 11 MDA ODA WES TP53 17 frameshift ENST00000610623.4 rs876660726 233908 c.425delC p.Pro142fs 31 US Femur relapse 11 MDA ODA WES TP53 17 frameshift ENST00000615910.4 rs876660726 233908 c.869delC p.Pro290fs 31 US Femur relapse 11 MDA ODA WES TP53 17 frameshift ENST00000269305.8 c.636dupT p.Arg213fs 31 US Femur relapse 11 MDA ODA WES TP53 17 frameshift ENST00000504290.5 c.240dupT p.Arg81fs 31 US Femur relapse 11 MDA ODA WES TP53 17 frameshift ENST00000514944.5 c.357dupT p.Arg120fs 31 US Femur relapse 11 MDA ODA WES TP53 17 frameshift ENST00000610292.4 c.519dupT p.Arg174fs 31 US Femur relapse 11 MDA ODA WES 37

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TP53 17 frameshift ENST00000615910.4 c.603dupT p.Arg202fs 31 US Femur relapse 11 MDA ODA WES TP53 17 frameshift ENST00000618944.4 c.159dupT p.Arg54fs 31 US Femur relapse 11 MDA ODA WES - CDKN2A (ARF) 9 ENST00000304494.9 0.0481 14 30 S Lung relapse 13 NYG ODA WGS, RNA, NM CDKN2A (ARF) 9 ENST00000304494.9 13 Lung relapse 13 TH ODA RNA MDM2 12 ENST00000258148.11 0.129 144 30 S Lung relapse 13 NYG ODA WGS, RNA, NM MDM2 12 ENST00000258148.11 99 Lung relapse 13 TH ODA RNA MDM4 1 ENST00000367179.7 0.1851 131 30 S Lung relapse 13 NYG ODA WGS, RNA, NM MDM4 1 ENST00000367179.7 24 Lung relapse 13 TH ODA RNA TP53 17 ENST00000269305.8 0.1395 111 30 S Lung relapse 13 NYG ODA WGS, RNA, NM TP53 17 ENST00000269305.8 35 Lung relapse 13 TH ODA RNA

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Supplemental Figure Legends

Supplemental Figures S1-4. Circos plot representations of sequenced tumor sites. Center plot lines show

gene fusions, inner ring shows germline and somatic mutations, middle ring shows copy number gain and loss,

outer ring shows gene expression levels, and outer rim shows chromosome position.

Supplemental Table Legends

Supplemental Tables S1-3. Sequencing results from all analyses. Bold text indicates common thread genetic events which were detected in multiple samples. Tables are presented as a time course: Table S1 is data from the initial biopsy (time point: 0 months), Table S2 is data from nephrectomy (time point: 5 months), and Table

S3 is data from the lung metastasis (time point: 13 months). A total of 256 events were observed, 33 of which were common to multiple analyses (shown in Table 2).

Supplemental Table S4. Variant table for compiled genes.

Supplemental Table S5. Sequencing coverage table for compiled genes.

Supplemental Tables S6-7. Detailed fusion calling data from the 3 fusion catchers used in the ODA analyses

(STAR fusion, Arriba and Fusion Catcher) showing evidence level of fusion presence as detected by each caller. Multiple fusions in the lung metastasis sample were detected by one caller yet were not detected at the threshold levels by the remaining callers, leading to the exclusion of these fusions from the Circos plots.

Supplemental Table S8. EWSR1-CEREB3L1 fusion sequence and PCR primer sequences used to probe for

the EWSR1-CREB3L1 gene fusion. Orange text indicates EWSR1 gene, green text indicates CREB3L1 gene.

Supplemental Cover art submission

Cover Art Image 1. Abstract painting representation of SEF H&E slide from right posterior upper lung nodule biopsy (Figure 1). Original artwork by Luke Avery. Photography by Debbie Vetter.

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Supplemental Figure S1. Primary kidney tumor, NYG WGS data.

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Supplemental Figure S2. Lung metastasis, NYG WGS data.

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Supplemental Figure S3. Lung metastasis (RNA only), Treehouse data.

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Supplemental Figure S4. Femur metastasis (DNA only), MDA data.

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Supplemental Table S1. Compiled sequencing results from initial kidney biopsy (0 months).

TMB Data Gene Chr Variant type HGVS DNA HGVS protein CNV TPM (Muts) MS source Analysis Data type

DUSP9 Loss 30 S FO FO DNA WES EWSR1-CREB3L1 Fusion 30 S FO FO DNA WES IRF1 V175A 30 S FO FO DNA WES MLL2 P2210L 30 S FO FO DNA WES NOTCH1 Splice site (subclonal) 30 S FO FO DNA WES NTRK1 P171S 30 S FO FO DNA WES PLCG2 K1019E 30 S FO FO DNA WES PRDM1 K532Q 30 S FO FO DNA WES ROS1 R2269 30 S FO FO DNA WES TMEM30A Y134fs*1 30 S FO FO DNA WES

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Supplemental Table S2. Compiled sequencing results from kidney nephrectomy (5 months).

TMB Data Gene Chr Variant type HGVS DNA HGVS protein CNV TPM (Muts) MS source Analysis Data type

ATRX R1803H 30 S FO FO DNA WES EWSR1-CREB3L1 Fusion 30 S FO FO DNA WES IRF1 V175A 30 S FO FO DNA WES KMT2C (MLL3) L804V 30 S FO FO DNA WES MLL2 P2210L 30 S FO FO DNA WES NTRK1 P171S 30 S FO FO DNA WES PLCG2 K1019E 30 S FO FO DNA WES PRDM1 K532Q 30 S FO FO DNA WES ROS1 R2269 30 S FO FO DNA WES SPEN R620Q 30 S FO FO DNA WES AHCTF1 1 Splice Donor Site c.1277+2T>C NYG NYG DNA WGS, RNA, NM AQR 15 Substitution - Missense c.1523C>T p.Ala508Val NYG NYG DNA WGS, RNA, NM ATP2B3 Loss NYG NYG DNA WGS, RNA, NM BCR Loss NYG NYG DNA WGS, RNA, NM BMPR1A Loss NYG NYG DNA WGS, RNA, NM CARS Loss NYG NYG DNA WGS, RNA, NM CHD7 8 Substitution - Missense c.5588C>A p.PRO1863Gln NYG NYG DNA WGS, RNA, NM CLTCL1 Loss NYG NYG DNA WGS, RNA, NM CREB3L1 Loss NYG NYG DNA WGS, RNA, NM DND1 5 Substitution - Missense c.1000G>A p.Glu334Lys NYG NYG DNA WGS, RNA, NM EP300 Loss NYG NYG DNA WGS, RNA, NM EWSR1 Loss NYG NYG DNA WGS, RNA, NM EWSR1-CREB3L1 Fusion c.902G>A p.Gly301Glu NYG NYG DNA WGS, RNA, NM EXT2 Loss NYG NYG DNA WGS, RNA, NM FANCF Loss NYG NYG DNA WGS, RNA, NM FAS Loss NYG NYG DNA WGS, RNA, NM FGFR2 Loss NYG NYG DNA WGS, RNA, NM FOXP1 Gain NYG NYG DNA WGS, RNA, NM

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GON4L 1 Substitution - Missense c.4615C>G p.Gln1539Glu NYG NYG DNA WGS, RNA, NM GPR32 19 Substitution - Missense c.994A>C p.Thr332Pro NYG NYG DNA WGS, RNA, NM HCAR3 12 Substitution - Missense c.1127C>G p.Ala376Gly NYG NYG DNA WGS, RNA, NM HRAS Loss NYG NYG DNA WGS, RNA, NM IGL Loss NYG NYG DNA WGS, RNA, NM KIAA1598 Loss NYG NYG DNA WGS, RNA, NM LILRB1 19 Substitution - Missense c.955G>A p.Ala319Thr NYG NYG DNA WGS, RNA, NM LMO1 Loss NYG NYG DNA WGS, RNA, NM LMO2 Loss NYG NYG DNA WGS, RNA, NM MCAM 11 Substitution - Missense c.1079G>A p.Ser360Asn NYG NYG DNA WGS, RNA, NM MKL1 22 Substitution - Missense NYG NYG DNA WGS, RNA, NM MKL1 Loss NYG NYG DNA WGS, RNA, NM MYH9 Loss NYG NYG DNA WGS, RNA, NM MYOD1 Loss NYG NYG DNA WGS, RNA, NM NBPF9 1 Substitution - Missense c.755A>G p.Asn252Ser NYG NYG DNA WGS, RNA, NM NF2 Loss NYG NYG DNA WGS, RNA, NM NFKB2 Loss NYG NYG DNA WGS, RNA, NM NT5C2 Loss NYG NYG DNA WGS, RNA, NM NUP98 Loss NYG NYG DNA WGS, RNA, NM NUTM2A Loss NYG NYG DNA WGS, RNA, NM PATZ1 Loss NYG NYG DNA WGS, RNA, NM PDGFB Loss NYG NYG DNA WGS, RNA, NM PTEN Loss NYG NYG DNA WGS, RNA, NM SEPT5 Loss NYG NYG DNA WGS, RNA, NM SIGLEC10 19 Substitution - Missense c.430C>A p.Gln144Lys NYG NYG DNA WGS, RNA, NM SMARCB1 Loss NYG NYG DNA WGS, RNA, NM SUFU Loss NYG NYG DNA WGS, RNA, NM TCF7L2 Loss NYG NYG DNA WGS, RNA, NM TLX1 Loss NYG NYG DNA WGS, RNA, NM TMEM30A 6 Insertion - Frameshift c.401dupA p.Tyr134fs NYG NYG DNA WGS, RNA, NM UGT2B11 4 Substitution - Missense c.1442A>G p.His481Arg NYG NYG DNA WGS, RNA, NM VTI1A Loss NYG NYG DNA WGS, RNA, NM WT1 Loss NYG NYG DNA WGS, RNA, NM ANG 14 Gain 136 39 S NYG ODA DNA WGS, RNA, NM ATP6V1G1 9 Gain 105 39 S NYG ODA DNA WGS, RNA, NM CALM1 14 Gain 233 39 S NYG ODA DNA WGS, RNA, NM 46

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DDX5 17 Gain 1131 39 S NYG ODA DNA WGS, RNA, NM EIF4A2 3 Gain 205 39 S NYG ODA DNA WGS, RNA, NM EIF5 2 Gain 132 39 S NYG ODA DNA WGS, RNA, NM FNBP4 11 Gain 275 39 S NYG ODA DNA WGS, RNA, NM IRF2BP2 1 Gain 265 39 S NYG ODA DNA WGS, RNA, NM KHDC4 1 Gain 105 39 S NYG ODA DNA WGS, RNA, NM MALAT1 11 Gain 10830 39 S NYG ODA DNA WGS, RNA, NM MCL1 1 Gain 260 39 S NYG ODA DNA WGS, RNA, NM MIR1248 3 Gain 552 39 S NYG ODA DNA WGS, RNA, NM PLK2 5 Gain 239 39 S NYG ODA DNA WGS, RNA, NM PNN 14 Gain 282 39 S NYG ODA DNA WGS, RNA, NM PNRC1 6 Gain 149 39 S NYG ODA DNA WGS, RNA, NM RN7SL2 14 Gain 1719 39 S NYG ODA DNA WGS, RNA, NM RNU11-2P 17 Gain 113 39 S NYG ODA DNA WGS, RNA, NM RNU4-1 12 Gain 141 39 S NYG ODA DNA WGS, RNA, NM RNU4-2 12 Gain 1282 39 S NYG ODA DNA WGS, RNA, NM RNU5A-1 15 Gain 764 39 S NYG ODA DNA WGS, RNA, NM RNU5B-1 15 Gain 1092 39 S NYG ODA DNA WGS, RNA, NM RPL35A 3 Gain 155 39 S NYG ODA DNA WGS, RNA, NM SBDS 7 Gain 127 39 S NYG ODA DNA WGS, RNA, NM SNORA31 13 Gain 111 39 S NYG ODA DNA WGS, RNA, NM SNORA63 3 Gain 785 39 S NYG ODA DNA WGS, RNA, NM SNORA70G 12 Gain 106 39 S NYG ODA DNA WGS, RNA, NM SNORA81 3 Gain 207 39 S NYG ODA DNA WGS, RNA, NM SPARC 5 Gain 10015 39 S NYG ODA DNA WGS, RNA, NM SPECC1L 22 Gain 159 39 S NYG ODA DNA WGS, RNA, NM TNFAIP3 6 Gain 191 39 S NYG ODA DNA WGS, RNA, NM

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Supplemental Table S3. Compiled sequencing results from lung metastasis (13 months).

TMB Data Gene Chr Variant type HGVS DNA HGVS protein CNV TPM (Muts) MS source Analysis Data type

BCR Loss NYG NYG DNA WGS, RNA, NM CARS Loss NYG NYG DNA WGS, RNA, NM CHEK2 Gain NYG NYG DNA WGS, RNA, NM CLTCL1 Loss NYG NYG DNA WGS, RNA, NM CR1 1 Substitution - Missense c.4189A>G p.Lys1397Glu NYG NYG DNA WGS, RNA, NM CREB3L1 Loss NYG NYG DNA WGS, RNA, NM CREB3L1 Gain NYG NYG DNA WGS, RNA, NM EP300 Loss NYG NYG DNA WGS, RNA, NM EWSR1 Loss NYG NYG DNA WGS, RNA, NM EWSR1 Gain NYG NYG DNA WGS, RNA, NM EWSR1-CREB3L1 Fusion NYG NYG DNA WGS, RNA, NM EXT2 Loss NYG NYG DNA WGS, RNA, NM FAM208A 3 Substitution - Missense c.3232G>T p.Val1078Leu NYG NYG DNA WGS, RNA, NM FANCF Loss NYG NYG DNA WGS, RNA, NM GON4L 1 Substitution - Missense c.4615C>G p.Gln1539Glu NYG NYG DNA WGS, RNA, NM HCAR3 12 Substitution - Missense c.1127C>G p.Ala376Gly NYG NYG DNA WGS, RNA, NM HRAS Loss NYG NYG DNA WGS, RNA, NM IGL Loss NYG NYG DNA WGS, RNA, NM LILRB4 19 Substitution - Missense c.1232G>A p.Gly411Glu NYG NYG DNA WGS, RNA, NM LMO1 Loss NYG NYG DNA WGS, RNA, NM LMO2 Loss NYG NYG DNA WGS, RNA, NM MKL1 Loss NYG NYG DNA WGS, RNA, NM MYH9 Loss NYG NYG DNA WGS, RNA, NM MYOD1 Loss NYG NYG DNA WGS, RNA, NM NF2 Loss NYG NYG DNA WGS, RNA, NM NUP98 Loss NYG NYG DNA WGS, RNA, NM PATZ1 Loss NYG NYG DNA WGS, RNA, NM 48

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PDGFB Loss NYG NYG DNA WGS, RNA, NM PILRA 7 Substitution - Missense c.316A>G p.Lys106Glu NYG NYG DNA WGS, RNA, NM SEPT5 Loss NYG NYG DNA WGS, RNA, NM SIGLEC10 19 Substitution - Missense c.430C>A p.Gln144Lys NYG NYG DNA WGS, RNA, NM WT1 Loss NYG NYG DNA WGS, RNA, NM AC092807.3-DDAH1 Fusion 30 S NYG ODA DNA WGS, RNA, NM AC139769.2-AC139769.1 Fusion 30 S NYG ODA DNA WGS, RNA, NM AGAP6-LINC00843 Fusion 30 S NYG ODA DNA WGS, RNA, NM AKR1E2-LINC01697 Fusion 30 S NYG ODA DNA WGS, RNA, NM B4GALT1-MRPS18A Fusion 30 S NYG ODA DNA WGS, RNA, NM CFAP44-AC112128.1 Fusion 30 S NYG ODA DNA WGS, RNA, NM CSNK1E-SLC16A5, ARMC7 Fusion 30 S NYG ODA DNA WGS, RNA, NM DPP6-ACTR3B Fusion 30 S NYG ODA DNA WGS, RNA, NM DYRK1A-TNKS Fusion 30 S NYG ODA DNA WGS, RNA, NM ESCO1-ROCK1 Fusion 30 S NYG ODA DNA WGS, RNA, NM EWSR1-CREB3L1 Fusion 30 S NYG ODA DNA WGS, RNA, NM FRG1DP-DUX4L35, FRG1EP Fusion 30 S NYG ODA DNA WGS, RNA, NM GALR1-GALR1, AC124254.1 Fusion 30 S NYG ODA DNA WGS, RNA, NM GBP7-GBP4 Fusion 30 S NYG ODA DNA WGS, RNA, NM HCG4-AL671883.2 Fusion 30 S NYG ODA DNA WGS, RNA, NM KLHL5-AP000350.2 Fusion 30 S NYG ODA DNA WGS, RNA, NM NRP1-PARD3 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA2-PCDHGA10 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA2-PCDHGA11 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA2-PCDHGA12 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA2-PCDHGA8 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA2-PCDHGA9 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA2-PCDHGB5 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA2-PCDHGB6 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA2-PCDHGB7 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA2-PCDHGC3 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA2-PCDHGC4 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA2-PCDHGC5 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA3-PCDHGA10 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA3-PCDHGA11 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA3-PCDHGA12 Fusion 30 S NYG ODA DNA WGS, RNA, NM 49

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PCDHGA3-PCDHGA8 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA3-PCDHGA9 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA3-PCDHGB5 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA3-PCDHGB6 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA3-PCDHGB7 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA3-PCDHGC3 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA3-PCDHGC4 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA3-PCDHGC5 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA4-PCDHGA10 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA4-PCDHGA11 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA4-PCDHGA12 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA4-PCDHGA8 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA4-PCDHGA9 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA4-PCDHGB5 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA4-PCDHGB6 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA4-PCDHGB7 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA4-PCDHGC3 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA4-PCDHGC4 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA4-PCDHGC5 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA5-PCDHGA10 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA5-PCDHGA11 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA5-PCDHGA12 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA5-PCDHGA8 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA5-PCDHGA9 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA5-PCDHGB5 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA5-PCDHGB6 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA5-PCDHGB7 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA5-PCDHGC3 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA5-PCDHGC4 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA5-PCDHGC5 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA6-PCDHGA10 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA6-PCDHGA11 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA6-PCDHGA12 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA6-PCDHGA8 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA6-PCDHGA9 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA6-PCDHGB5 Fusion 30 S NYG ODA DNA WGS, RNA, NM 50

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PCDHGA6-PCDHGB6 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA6-PCDHGB7 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA6-PCDHGC3 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA6-PCDHGC4 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGA6-PCDHGC5 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGB1-PCDHGA10 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGB1-PCDHGA11 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGB1-PCDHGA12 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGB1-PCDHGA8 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGB1-PCDHGA9 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGB1-PCDHGB5 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGB1-PCDHGB6 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGB1-PCDHGB7 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGB1-PCDHGC3 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGB1-PCDHGC4 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGB1-PCDHGC5 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGB2-PCDHGA10 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGB2-PCDHGA11 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGB2-PCDHGA12 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGB2-PCDHGA8 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGB2-PCDHGB5 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGB2-PCDHGB6 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGB2-PCDHGB7 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGB2-PCDHGC3 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGB2-PCDHGC4 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGB2-PCDHGC5 Fusion 30 S NYG ODA DNA WGS, RNA, NM PCDHGB5-PCDHGA9 Fusion 30 S NYG ODA DNA WGS, RNA, NM PPP1R21-STON1 Fusion 30 S NYG ODA DNA WGS, RNA, NM PRS10P7-NAV1 Fusion 30 S NYG ODA DNA WGS, RNA, NM RAB11FIP3-CAPN15 Fusion 30 S NYG ODA DNA WGS, RNA, NM RF00019, CD86-CD86 Fusion 30 S NYG ODA DNA WGS, RNA, NM SIPA1L2, RNU-1211P- SIPA1L2 Fusion 30 S NYG ODA DNA WGS, RNA, NM TGM3-SIRPB1 Fusion 30 S NYG ODA DNA WGS, RNA, NM TTC3-ZNF532 Fusion 30 S NYG ODA DNA WGS, RNA, NM XBP1 22 Gain 122 30 S NYG ODA DNA WGS, RNA, NM 51

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YY1AP1-ASH1L Fusion 30 S NYG ODA DNA WGS, RNA, NM ZNF267-ZNF720 Fusion 30 S NYG ODA DNA WGS, RNA, NM ZNF676-ZNF814 Fusion 30 S NYG ODA DNA WGS, RNA, NM COL1A1 8854 TH ODA RNA COL1A2 12404 TH ODA RNA COL3A1 13816 TH ODA RNA CREB3L1 98 TH ODA RNA EWSR1 289 TH ODA RNA EWSR1-CREB3L1 Fusion TH ODA RNA H19 9313 TH ODA RNA MT-ATP6 9385 TH ODA RNA MT-ATP8 16586 TH ODA RNA MT-CO1 12016 TH ODA RNA MT-CO2 9900 TH ODA RNA MT-CO3 14459 TH ODA RNA SPARC 30899 TH ODA RNA CDK1 high TH TH RNA EWSR1-CREB3L1 Fusion TH TH RNA NOTCH1 Splice site (subclonal) TH TH RNA NOTCH3 high TH TH RNA TMEM20A Y134fs*1 TH TH RNA

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Supplemental Table S4. Variant table for compiled genes.

dbSNP/dbVar Genotype Gene Chr HGVS DNA HGVS protein Variant type Effect Biotype ID Genotype depth Sample type

DUSP9 Primary kidney Bx EWSR1-CREB3L1 Fusion Primary kidney Bx IRF1 V175A Primary kidney Bx MLL2 P2210L Primary kidney Bx Splice site NOTCH1 (subclonal) Primary kidney Bx NTRK1 P171S Primary kidney Bx PLCG2 K1019E Primary kidney Bx PRDM1 K532Q Primary kidney Bx ROS1 R2269 Primary kidney Bx TMEM30A Y134fs*1 Primary kidney Bx ATRX R1803H Primary nephrectomy EWSR1-CREB3L1 Fusion Primary nephrectomy IRF1 V175A Primary nephrectomy KMT2C (MLL3) L804V Primary nephrectomy MLL2 P2210L Primary nephrectomy NTRK1 P171S Primary nephrectomy PLCG2 K1019E Primary nephrectomy PRDM1 K532Q Primary nephrectomy ROS1 R2269 Primary nephrectomy SPEN R620Q Primary nephrectomy AHCTF1 1 c.1277+2T>C Splice Donor Site Splice donor Protein coding 0/1 53,44 Primary nephrectomy AQR 15 c.1523C>T p.Ala508Val Missense Substitution Protein coding COSM4054187 0/1 101,20 Primary nephrectomy ATP2B3 X Primary nephrectomy BCR 22 Primary nephrectomy BMPR1A 10 Primary nephrectomy CARS Primary nephrectomy CHD7 8 c.5588C>A p.PRO1863Gln Missense Substitution Protein coding 0/1 43,30 Primary nephrectomy CLTCL1 Primary nephrectomy CREB3L1 11 Primary nephrectomy 53

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DND1 5 c.1000G>A p.Glu334Lys Missense Substitution Primary nephrectomy EP300 22 Primary nephrectomy EWSR1 22 Primary nephrectomy EWSR1-CREB3L1 c.902G>A p.Gly301Glu Fusion Primary nephrectomy EXT2 11 Primary nephrectomy FANCF 11 Primary nephrectomy FAS Primary nephrectomy FGFR2 Primary nephrectomy FOXP1 Primary nephrectomy GON4L 1 c.4615C>G p.Gln1539Glu Missense Substitution Primary nephrectomy GPR32 19 c.994A>C p.Thr332Pro Missense Substitution Primary nephrectomy HCAR3 12 c.1127C>G p.Ala376Gly Missense Substitution Primary nephrectomy HRAS 11 Primary nephrectomy IGL Primary nephrectomy KIAA1598 Primary nephrectomy LILRB1 19 c.955G>A p.Ala319Thr Missense Substitution Primary nephrectomy LMO1 11 Primary nephrectomy LMO2 11 Primary nephrectomy MCAM 11 c.1079G>A p.Ser360Asn Missense Substitution Protein coding 0/1 67,9 Primary nephrectomy MKL1 22 Missense Substitution Protein coding 0/1 12,39 Primary nephrectomy MKL1 Primary nephrectomy MYH9 22 Primary nephrectomy MYOD1 11 Primary nephrectomy NBPF9 1 c.755A>G p.Asn252Ser Missense Substitution Protein coding rs1324516211 0/1 63,4 Primary nephrectomy

NF2 22 Primary nephrectomy NFKB2 10 Primary nephrectomy NT5C2 10 Primary nephrectomy NUP98 11 Primary nephrectomy NUTM2A 10 Primary nephrectomy PATZ1 22 Primary nephrectomy PDGFB 22 Primary nephrectomy PTEN 10 Primary nephrectomy SEPT5 Primary nephrectomy SIGLEC10 19 c.430C>A p.Gln144Lys Missense Substitution Primary nephrectomy SMARCB1 22 Primary nephrectomy SUFU 10 Primary nephrectomy 54

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TCF7L2 10 Primary nephrectomy TLX1 10 Primary nephrectomy TMEM30A 6 c.401dupA p.Tyr134fs Frameshift Insertion Protein coding 0/1 Primary nephrectomy UGT2B11 4 c.1442A>G p.His481Arg Missense Substitution Primary nephrectomy VTI1A 10 Primary nephrectomy WT1 11 Primary nephrectomy ANG 14 Primary nephrectomy ATP6V1G1 9 Primary nephrectomy CALM1 14 Primary nephrectomy DDX5 17 Primary nephrectomy EIF4A2 3 Primary nephrectomy EIF5 14 Primary nephrectomy FNBP4 11 Primary nephrectomy IRF2BP2 1 Primary nephrectomy KHDC4 1 Primary nephrectomy MALAT1 11 Primary nephrectomy MCL1 1 Primary nephrectomy MIR1248 3 Primary nephrectomy PLK2 5 Primary nephrectomy PNN 14 Primary nephrectomy PNRC1 6 Primary nephrectomy RN7SL2 14 Primary nephrectomy RNU11-2P 17 Primary nephrectomy RNU4-1 12 Primary nephrectomy RNU4-2 12 Primary nephrectomy RNU5A-1 15 Primary nephrectomy RNU5B-1 15 Primary nephrectomy RPL35A 3 Primary nephrectomy SBDS 7 Primary nephrectomy SNORA31 13 Primary nephrectomy SNORA63 3 Primary nephrectomy SNORA70G 12 Primary nephrectomy SNORA81 3 Primary nephrectomy SPARC 5 Primary nephrectomy SPECC1L 22 Primary nephrectomy TNFAIP3 6 Primary nephrectomy 55

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BCR 22 Lung relapse met CARS Lung relapse met CHEK2 22 Lung relapse met CLTCL1 22 Lung relapse met CR1 1 c.4189A>G p.Lys1397Glu Missense Substitution Protein coding 0/1 93,21 Lung relapse met CREB3L1 11 Lung relapse met CREB3L1 11 Lung relapse met EP300 22 Lung relapse met EWSR1 22 Lung relapse met EWSR1 22 Lung relapse met EWSR1-CREB3L1 Fusion Lung relapse met EXT2 11 Lung relapse met FAM208A 3 c.3232G>T p.Val1078Leu Missense Substitution Protein coding 0/1 90,19 Lung relapse met FANCF 11 Lung relapse met GON4L 1 c.4615C>G p.Gln1539Glu Missense Substitution Lung relapse met HCAR3 12 c.1127C>G p.Ala376Gly Missense Substitution Lung relapse met HRAS 11 Lung relapse met IGL Lung relapse met LILRB4 19 c.1232G>A p.Gly411Glu Missense Substitution Lung relapse met LMO1 11 Lung relapse met LMO2 11 Lung relapse met MKL1 Lung relapse met MYH9 22 Lung relapse met MYOD1 11 Lung relapse met NF2 22 Lung relapse met NUP98 11 Lung relapse met PATZ1 22 Lung relapse met PDGFB 22 Lung relapse met PILRA 7 c.316A>G p.Lys106Glu Missense Substitution Lung relapse met SEPT5 Lung relapse met SIGLEC10 19 c.430C>A p.Gln144Lys Missense Substitution Lung relapse met WT1 11 Lung relapse met AC092807.3-DDAH1 Fusion Lung relapse met AC139769.2-AC139769.1 Fusion Lung relapse met AGAP6-LINC00843 Fusion Lung relapse met AKR1E2-LINC01697 Fusion Lung relapse met 56

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B4GALT1-MRPS18A Fusion Lung relapse met CFAP44-AC112128.1 Fusion Lung relapse met CSNK1E-SLC16A5, ARMC7 Fusion Lung relapse met DPP6-ACTR3B Fusion Lung relapse met DYRK1A-TNKS Fusion Lung relapse met ESCO1-ROCK1 Fusion Lung relapse met EWSR1-CREB3L1 Fusion Lung relapse met FRG1DP-DUX4L35, FRG1EP Fusion Lung relapse met GALR1-GALR1, AC124254.1 Fusion Lung relapse met GBP7-GBP4 Fusion Lung relapse met HCG4-AL671883.2 Fusion Lung relapse met KLHL5-AP000350.2 Fusion Lung relapse met NRP1-PARD3 Fusion Lung relapse met PCDHGA2-PCDHGA10 Fusion Lung relapse met PCDHGA2-PCDHGA11 Fusion Lung relapse met PCDHGA2-PCDHGA12 Fusion Lung relapse met PCDHGA2-PCDHGA8 Fusion Lung relapse met PCDHGA2-PCDHGA9 Fusion Lung relapse met PCDHGA2-PCDHGB5 Fusion Lung relapse met PCDHGA2-PCDHGB6 Fusion Lung relapse met PCDHGA2-PCDHGB7 Fusion Lung relapse met PCDHGA2-PCDHGC3 Fusion Lung relapse met PCDHGA2-PCDHGC4 Fusion Lung relapse met PCDHGA2-PCDHGC5 Fusion Lung relapse met PCDHGA3-PCDHGA10 Fusion Lung relapse met PCDHGA3-PCDHGA11 Fusion Lung relapse met PCDHGA3-PCDHGA12 Fusion Lung relapse met PCDHGA3-PCDHGA8 Fusion Lung relapse met PCDHGA3-PCDHGA9 Fusion Lung relapse met PCDHGA3-PCDHGB5 Fusion Lung relapse met PCDHGA3-PCDHGB6 Fusion Lung relapse met PCDHGA3-PCDHGB7 Fusion Lung relapse met PCDHGA3-PCDHGC3 Fusion Lung relapse met PCDHGA3-PCDHGC4 Fusion Lung relapse met PCDHGA3-PCDHGC5 Fusion Lung relapse met 57

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PCDHGA4-PCDHGA10 Fusion Lung relapse met PCDHGA4-PCDHGA11 Fusion Lung relapse met PCDHGA4-PCDHGA12 Fusion Lung relapse met PCDHGA4-PCDHGA8 Fusion Lung relapse met PCDHGA4-PCDHGA9 Fusion Lung relapse met PCDHGA4-PCDHGB5 Fusion Lung relapse met PCDHGA4-PCDHGB6 Fusion Lung relapse met PCDHGA4-PCDHGB7 Fusion Lung relapse met PCDHGA4-PCDHGC3 Fusion Lung relapse met PCDHGA4-PCDHGC4 Fusion Lung relapse met PCDHGA4-PCDHGC5 Fusion Lung relapse met PCDHGA5-PCDHGA10 Fusion Lung relapse met PCDHGA5-PCDHGA11 Fusion Lung relapse met PCDHGA5-PCDHGA12 Fusion Lung relapse met PCDHGA5-PCDHGA8 Fusion Lung relapse met PCDHGA5-PCDHGA9 Fusion Lung relapse met PCDHGA5-PCDHGB5 Fusion Lung relapse met PCDHGA5-PCDHGB6 Fusion Lung relapse met PCDHGA5-PCDHGB7 Fusion Lung relapse met PCDHGA5-PCDHGC3 Fusion Lung relapse met PCDHGA5-PCDHGC4 Fusion Lung relapse met PCDHGA5-PCDHGC5 Fusion Lung relapse met PCDHGA6-PCDHGA10 Fusion Lung relapse met PCDHGA6-PCDHGA11 Fusion Lung relapse met PCDHGA6-PCDHGA12 Fusion Lung relapse met PCDHGA6-PCDHGA8 Fusion Lung relapse met PCDHGA6-PCDHGA9 Fusion Lung relapse met PCDHGA6-PCDHGB5 Fusion Lung relapse met PCDHGA6-PCDHGB6 Fusion Lung relapse met PCDHGA6-PCDHGB7 Fusion Lung relapse met PCDHGA6-PCDHGC3 Fusion Lung relapse met PCDHGA6-PCDHGC4 Fusion Lung relapse met PCDHGA6-PCDHGC5 Fusion Lung relapse met PCDHGB1-PCDHGA10 Fusion Lung relapse met PCDHGB1-PCDHGA11 Fusion Lung relapse met PCDHGB1-PCDHGA12 Fusion Lung relapse met 58

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PCDHGB1-PCDHGA8 Fusion Lung relapse met PCDHGB1-PCDHGA9 Fusion Lung relapse met PCDHGB1-PCDHGB5 Fusion Lung relapse met PCDHGB1-PCDHGB6 Fusion Lung relapse met PCDHGB1-PCDHGB7 Fusion Lung relapse met PCDHGB1-PCDHGC3 Fusion Lung relapse met PCDHGB1-PCDHGC4 Fusion Lung relapse met PCDHGB1-PCDHGC5 Fusion Lung relapse met PCDHGB2-PCDHGA10 Fusion Lung relapse met PCDHGB2-PCDHGA11 Fusion Lung relapse met PCDHGB2-PCDHGA12 Fusion Lung relapse met PCDHGB2-PCDHGA8 Fusion Lung relapse met PCDHGB2-PCDHGB5 Fusion Lung relapse met PCDHGB2-PCDHGB6 Fusion Lung relapse met PCDHGB2-PCDHGB7 Fusion Lung relapse met PCDHGB2-PCDHGC3 Fusion Lung relapse met PCDHGB2-PCDHGC4 Fusion Lung relapse met PCDHGB2-PCDHGC5 Fusion Lung relapse met PCDHGB5-PCDHGA9 Fusion Lung relapse met PPP1R21-STON1 Fusion Lung relapse met PRS10P7-NAV1 Fusion Lung relapse met RAB11FIP3-CAPN15 Fusion Lung relapse met RF00019, CD86-CD86 Fusion Lung relapse met SIPA1L2, RNU-1211P- SIPA1L2 Fusion Lung relapse met TGM3-SIRPB1 Fusion Lung relapse met TTC3-ZNF532 Fusion Lung relapse met XBP1 22 Lung relapse met YY1AP1-ASH1L Fusion Lung relapse met ZNF267-ZNF720 Fusion Lung relapse met ZNF676-ZNF814 Fusion Lung relapse met COL1A1 Lung relapse met COL1A2 Lung relapse met COL3A1 Lung relapse met CREB3L1 Lung relapse met EWSR1 Lung relapse met 59

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EWSR1-CREB3L1 Fusion Lung relapse met H19 Lung relapse met MT-ATP6 Lung relapse met MT-ATP8 Lung relapse met MT-CO1 Lung relapse met MT-CO2 Lung relapse met MT-CO3 Lung relapse met SPARC Lung relapse met CDK1 Lung relapse met EWSR1-CREB3L1 Fusion Lung relapse met Splice site NOTCH1 (subclonal) Lung relapse met NOTCH3 Lung relapse met TMEM20A Y134fs*1 Lung relapse met

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Supplemental Table S5. Sequencing coverage table for compiled genes.

Gene HGVS protein Raw VAF Total depth Post Dx (Months) Sample type DUSP9 0 Primary kidney Bx EWSR1-CREB3L1 0 Primary kidney Bx IRF1 V175A 0 Primary kidney Bx MLL2 P2210L 0 Primary kidney Bx NOTCH1 0 Primary kidney Bx NTRK1 P171S 0 Primary kidney Bx PLCG2 K1019E 0 Primary kidney Bx PRDM1 K532Q 0 Primary kidney Bx ROS1 R2269 0 Primary kidney Bx TMEM30A 0 Primary kidney Bx ATRX R1803H 5 Primary nephrectomy EWSR1-CREB3L1 5 Primary nephrectomy IRF1 V175A 5 Primary nephrectomy KMT2C (MLL3) L804V 5 Primary nephrectomy MLL2 P2210L 5 Primary nephrectomy NTRK1 P171S 5 Primary nephrectomy PLCG2 K1019E 5 Primary nephrectomy PRDM1 K532Q 5 Primary nephrectomy ROS1 R2269 5 Primary nephrectomy SPEN R620Q 5 Primary nephrectomy AHCTF1 54.64% 146 5 Primary nephrectomy AQR p.Ala508Val 83.47% 175 5 Primary nephrectomy ATP2B3 5 Primary nephrectomy BCR 5 Primary nephrectomy BMPR1A 5 Primary nephrectomy CARS 5 Primary nephrectomy CHD7 p.PRO1863Gln 58.90% 123 5 Primary nephrectomy CLTCL1 5 Primary nephrectomy CREB3L1 5 Primary nephrectomy DND1 p.Glu334Lys 5 Primary nephrectomy EP300 5 Primary nephrectomy EWSR1 5 Primary nephrectomy EWSR1-CREB3L1 p.Gly301Glu 5 Primary nephrectomy EXT2 5 Primary nephrectomy FANCF 5 Primary nephrectomy FAS 5 Primary nephrectomy FGFR2 5 Primary nephrectomy FOXP1 5 Primary nephrectomy GON4L p.Gln1539Glu 5 Primary nephrectomy GPR32 p.Thr332Pro 5 Primary nephrectomy HCAR3 p.Ala376Gly 5 Primary nephrectomy HRAS 5 Primary nephrectomy IGL 5 Primary nephrectomy KIAA1598 5 Primary nephrectomy LILRB1 p.Ala319Thr 5 Primary nephrectomy

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LMO1 5 Primary nephrectomy LMO2 5 Primary nephrectomy MCAM p.Ser360Asn 88.16% 123 5 Primary nephrectomy MKL1 p.Gly301Glu 23.53% 111 5 Primary nephrectomy MKL1 5 Primary nephrectomy MYH9 5 Primary nephrectomy MYOD1 5 Primary nephrectomy NBPF9 p.Asn252Ser 94.03% 104 5 Primary nephrectomy NF2 5 Primary nephrectomy NFKB2 5 Primary nephrectomy NT5C2 5 Primary nephrectomy NUP98 5 Primary nephrectomy NUTM2A 5 Primary nephrectomy PATZ1 5 Primary nephrectomy PDGFB 5 Primary nephrectomy PTEN 5 Primary nephrectomy SEPT5 5 Primary nephrectomy SIGLEC10 p.Gln144Lys 5 Primary nephrectomy SMARCB1 5 Primary nephrectomy SUFU 5 Primary nephrectomy TCF7L2 5 Primary nephrectomy TLX1 5 Primary nephrectomy TMEM30A p.Tyr134fs 56.45% 111 5 Primary nephrectomy UGT2B11 p.His481Arg 5 Primary nephrectomy VTI1A 5 Primary nephrectomy WT1 5 Primary nephrectomy ANG 5 Primary nephrectomy ATP6V1G1 5 Primary nephrectomy CALM1 5 Primary nephrectomy DDX5 5 Primary nephrectomy EIF4A2 5 Primary nephrectomy EIF5 5 Primary nephrectomy FNBP4 5 Primary nephrectomy IRF2BP2 5 Primary nephrectomy KHDC4 5 Primary nephrectomy MALAT1 5 Primary nephrectomy MCL1 5 Primary nephrectomy MIR1248 5 Primary nephrectomy PLK2 5 Primary nephrectomy PNN 5 Primary nephrectomy PNRC1 5 Primary nephrectomy RN7SL2 5 Primary nephrectomy RNU11-2P 5 Primary nephrectomy RNU4-1 5 Primary nephrectomy RNU4-2 5 Primary nephrectomy RNU5A-1 5 Primary nephrectomy RNU5B-1 5 Primary nephrectomy RPL35A 5 Primary nephrectomy SBDS 5 Primary nephrectomy SNORA31 5 Primary nephrectomy 62

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SNORA63 5 Primary nephrectomy SNORA70G 5 Primary nephrectomy SNORA81 5 Primary nephrectomy SPARC 5 Primary nephrectomy SPECC1L 5 Primary nephrectomy TNFAIP3 5 Primary nephrectomy BCR 13 Lung relapse met CARS 13 Lung relapse met CHEK2 13 Lung relapse met CLTCL1 13 Lung relapse met CR1 p.Lys1397Glu 81.58% 172 13 Lung relapse met CREB3L1 13 Lung relapse met CREB3L1 13 Lung relapse met EP300 13 Lung relapse met EWSR1 13 Lung relapse met EWSR1 13 Lung relapse met EWSR1-CREB3L1 13 Lung relapse met EXT2 13 Lung relapse met FAM208A p.Val1078Leu 82.57% 171 13 Lung relapse met FANCF 13 Lung relapse met GON4L p.Gln1539Glu 13 Lung relapse met HCAR3 p.Ala376Gly 13 Lung relapse met HRAS 13 Lung relapse met IGL 13 Lung relapse met LILRB4 p.Gly411Glu 13 Lung relapse met LMO1 13 Lung relapse met LMO2 13 Lung relapse met MKL1 13 Lung relapse met MYH9 13 Lung relapse met MYOD1 13 Lung relapse met NF2 13 Lung relapse met NUP98 13 Lung relapse met PATZ1 13 Lung relapse met PDGFB 13 Lung relapse met PILRA p.Lys106Glu 13 Lung relapse met SEPT5 13 Lung relapse met SIGLEC10 p.Gln144Lys 13 Lung relapse met WT1 13 Lung relapse met AC092807.3-DDAH1 13 Lung relapse met AC139769.2-AC139769.1 13 Lung relapse met AGAP6-LINC00843 13 Lung relapse met AKR1E2-LINC01697 13 Lung relapse met B4GALT1-MRPS18A 13 Lung relapse met CFAP44-AC112128.1 13 Lung relapse met CSNK1E-SLC16A5, ARMC7 13 Lung relapse met DPP6-ACTR3B 13 Lung relapse met DYRK1A-TNKS 13 Lung relapse met ESCO1-ROCK1 13 Lung relapse met EWSR1-CREB3L1 13 Lung relapse met FRG1DP-DUX4L35, FRG1EP 13 Lung relapse met 63

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GALR1-GALR1, AC124254.1 13 Lung relapse met GBP7-GBP4 13 Lung relapse met HCG4-AL671883.2 13 Lung relapse met KLHL5-AP000350.2 13 Lung relapse met NRP1-PARD3 13 Lung relapse met PCDHGA2-PCDHGA10 13 Lung relapse met PCDHGA2-PCDHGA11 13 Lung relapse met PCDHGA2-PCDHGA12 13 Lung relapse met PCDHGA2-PCDHGA8 13 Lung relapse met PCDHGA2-PCDHGA9 13 Lung relapse met PCDHGA2-PCDHGB5 13 Lung relapse met PCDHGA2-PCDHGB6 13 Lung relapse met PCDHGA2-PCDHGB7 13 Lung relapse met PCDHGA2-PCDHGC3 13 Lung relapse met PCDHGA2-PCDHGC4 13 Lung relapse met PCDHGA2-PCDHGC5 13 Lung relapse met PCDHGA3-PCDHGA10 13 Lung relapse met PCDHGA3-PCDHGA11 13 Lung relapse met PCDHGA3-PCDHGA12 13 Lung relapse met PCDHGA3-PCDHGA8 13 Lung relapse met PCDHGA3-PCDHGA9 13 Lung relapse met PCDHGA3-PCDHGB5 13 Lung relapse met PCDHGA3-PCDHGB6 13 Lung relapse met PCDHGA3-PCDHGB7 13 Lung relapse met PCDHGA3-PCDHGC3 13 Lung relapse met PCDHGA3-PCDHGC4 13 Lung relapse met PCDHGA3-PCDHGC5 13 Lung relapse met PCDHGA4-PCDHGA10 13 Lung relapse met PCDHGA4-PCDHGA11 13 Lung relapse met PCDHGA4-PCDHGA12 13 Lung relapse met PCDHGA4-PCDHGA8 13 Lung relapse met PCDHGA4-PCDHGA9 13 Lung relapse met PCDHGA4-PCDHGB5 13 Lung relapse met PCDHGA4-PCDHGB6 13 Lung relapse met PCDHGA4-PCDHGB7 13 Lung relapse met PCDHGA4-PCDHGC3 13 Lung relapse met PCDHGA4-PCDHGC4 13 Lung relapse met PCDHGA4-PCDHGC5 13 Lung relapse met PCDHGA5-PCDHGA10 13 Lung relapse met PCDHGA5-PCDHGA11 13 Lung relapse met PCDHGA5-PCDHGA12 13 Lung relapse met PCDHGA5-PCDHGA8 13 Lung relapse met PCDHGA5-PCDHGA9 13 Lung relapse met PCDHGA5-PCDHGB5 13 Lung relapse met PCDHGA5-PCDHGB6 13 Lung relapse met PCDHGA5-PCDHGB7 13 Lung relapse met PCDHGA5-PCDHGC3 13 Lung relapse met PCDHGA5-PCDHGC4 13 Lung relapse met PCDHGA5-PCDHGC5 13 Lung relapse met PCDHGA6-PCDHGA10 13 Lung relapse met 64

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PCDHGA6-PCDHGA11 13 Lung relapse met PCDHGA6-PCDHGA12 13 Lung relapse met PCDHGA6-PCDHGA8 13 Lung relapse met PCDHGA6-PCDHGA9 13 Lung relapse met PCDHGA6-PCDHGB5 13 Lung relapse met PCDHGA6-PCDHGB6 13 Lung relapse met PCDHGA6-PCDHGB7 13 Lung relapse met PCDHGA6-PCDHGC3 13 Lung relapse met PCDHGA6-PCDHGC4 13 Lung relapse met PCDHGA6-PCDHGC5 13 Lung relapse met PCDHGB1-PCDHGA10 13 Lung relapse met PCDHGB1-PCDHGA11 13 Lung relapse met PCDHGB1-PCDHGA12 13 Lung relapse met PCDHGB1-PCDHGA8 13 Lung relapse met PCDHGB1-PCDHGA9 13 Lung relapse met PCDHGB1-PCDHGB5 13 Lung relapse met PCDHGB1-PCDHGB6 13 Lung relapse met PCDHGB1-PCDHGB7 13 Lung relapse met PCDHGB1-PCDHGC3 13 Lung relapse met PCDHGB1-PCDHGC4 13 Lung relapse met PCDHGB1-PCDHGC5 13 Lung relapse met PCDHGB2-PCDHGA10 13 Lung relapse met PCDHGB2-PCDHGA11 13 Lung relapse met PCDHGB2-PCDHGA12 13 Lung relapse met PCDHGB2-PCDHGA8 13 Lung relapse met PCDHGB2-PCDHGB5 13 Lung relapse met PCDHGB2-PCDHGB6 13 Lung relapse met PCDHGB2-PCDHGB7 13 Lung relapse met PCDHGB2-PCDHGC3 13 Lung relapse met PCDHGB2-PCDHGC4 13 Lung relapse met PCDHGB2-PCDHGC5 13 Lung relapse met PCDHGB5-PCDHGA9 13 Lung relapse met PPP1R21-STON1 13 Lung relapse met PRS10P7-NAV1 13 Lung relapse met RAB11FIP3-CAPN15 13 Lung relapse met RF00019, CD86-CD86 13 Lung relapse met SIPA1L2, RNU-1211P-SIPA1L2 13 Lung relapse met TGM3-SIRPB1 13 Lung relapse met TTC3-ZNF532 13 Lung relapse met XBP1 13 Lung relapse met YY1AP1-ASH1L 13 Lung relapse met ZNF267-ZNF720 13 Lung relapse met ZNF676-ZNF814 13 Lung relapse met COL1A1 13 Lung relapse met COL1A2 13 Lung relapse met COL3A1 13 Lung relapse met CREB3L1 13 Lung relapse met EWSR1 13 Lung relapse met EWSR1-CREB3L1 13 Lung relapse met H19 13 Lung relapse met 65

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MT-ATP6 13 Lung relapse met MT-ATP8 13 Lung relapse met MT-CO1 13 Lung relapse met MT-CO2 13 Lung relapse met MT-CO3 13 Lung relapse met SPARC 13 Lung relapse met CDK1 13 Lung relapse met EWSR1-CREB3L1 13 Lung relapse met NOTCH1 13 Lung relapse met NOTCH3 13 Lung relapse met TMEM20A 13 Lung relapse met

Supplemental Table S6. Detailed fusion reads from lung metastasis (NYG data).

StarFusion FusionCatcher Arriba gene1 gene2 JunctionReadCount SpanningFragCount Spanning_pairs split_reads1,2 discordant mates RF00019(14433),CD86(15629) CD86 () () () 3,33 36 CFAP44 AC112128.1 () () () 1,0 2 PCDHGA5 PCDHGA8 () () () 5,3 1 PCDHGA5 PCDHGC5 () () () 5,3 1 PCDHGA5 PCDHGA10 () () () 5,3 1 PCDHGA5 PCDHGC4 () () () 5,3 1 PCDHGA5 PCDHGA11 () () () 5,3 1 PCDHGA5 PCDHGB6 () () () 5,3 1 PCDHGA5 PCDHGB5 () () () 5,3 1 PCDHGA5 PCDHGA12 () () () 5,3 1 PCDHGA5 PCDHGB7 () () () 5,3 1 PCDHGA5 PCDHGA9 () () () 5,3 1 PCDHGA5 PCDHGC3 () () () 5,3 1 PCDHGA2 PCDHGA8 () () () 1,0 6 PCDHGA2 PCDHGC5 () () () 1,0 6 PCDHGA2 PCDHGA10 () () () 1,0 6 PCDHGA2 PCDHGC4 () () () 1,0 6 PCDHGA2 PCDHGA11 () () () 1,0 6 PCDHGA2 PCDHGB6 () () () 1,0 6 PCDHGA2 PCDHGB5 () () () 1,0 6 PCDHGA2 PCDHGA12 () () () 1,0 6 PCDHGA2 PCDHGB7 () () () 1,0 6 PCDHGA2 PCDHGA9 () () () 1,0 6 PCDHGA2 PCDHGC3 () () () 1,0 6 66

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PCDHGB2 PCDHGA8 () () () 4,0 3 PCDHGB2 PCDHGC5 () () () 4,0 3 PCDHGB2 PCDHGA10 () () () 4,0 3 PCDHGB2 PCDHGC4 () () () 4,0 3 PCDHGB2 PCDHGA11 () () () 4,0 3 PCDHGB2 PCDHGB6 () () () 4,0 3 PCDHGB2 PCDHGB5 () () () 4,0 3 PCDHGB2 PCDHGA12 () () () 4,0 3 PCDHGB2 PCDHGB7 () () () 4,0 3 PCDHGB2 PCDHGA9 () () () 4,0 3 PCDHGB2 PCDHGC3 () () () 4,0 3 PCDHGA6 PCDHGA8 () () () 3,1 1 PCDHGA6 PCDHGC5 () () () 3,1 1 PCDHGA6 PCDHGA10 () () () 3,1 1 PCDHGA6 PCDHGC4 () () () 3,1 1 PCDHGA4 PCDHGA8 () () () 2,0 3 PCDHGA6 PCDHGA11 () () () 3,1 1 PCDHGA6 PCDHGB6 () () () 3,1 1 PCDHGA6 PCDHGB5 () () () 3,1 1 PCDHGA4 PCDHGC5 () () () 2,0 3 PCDHGA4 PCDHGA10 () () () 2,0 3 PCDHGA6 PCDHGA12 () () () 3,1 1 PCDHGA6 PCDHGB7 () () () 3,1 1 PCDHGA6 PCDHGA9 () () () 3,1 1 PCDHGA4 PCDHGC4 () () () 2,0 3 PCDHGA4 PCDHGA11 () () () 2,0 3 PCDHGA4 PCDHGB6 () () () 2,0 3 PCDHGA6 PCDHGC3 () () () 3,1 1 PCDHGA4 PCDHGB5 () () () 2,0 3 PCDHGA4 PCDHGA12 () () () 2,0 3 PCDHGA4 PCDHGB7 () () () 2,0 3 PCDHGA4 PCDHGA9 () () () 2,0 3 PCDHGA4 PCDHGC3 () () () 2,0 3 PCDHGA3 PCDHGA8 () () () 2,1 1 PCDHGA3 PCDHGC5 () () () 2,1 1 PCDHGA3 PCDHGA10 () () () 2,1 1 PCDHGA3 PCDHGC4 () () () 2,1 1 PCDHGA3 PCDHGA11 () () () 2,1 1 PCDHGA3 PCDHGB6 () () () 2,1 1 PCDHGA3 PCDHGB5 () () () 2,1 1 PCDHGA3 PCDHGA12 () () () 2,1 1 PCDHGA3 PCDHGB7 () () () 2,1 1 PCDHGA3 PCDHGA9 () () () 2,1 1 PCDHGA3 PCDHGC3 () () () 2,1 1 67

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PCDHGB1 PCDHGA8 () () () 3,1 0 PCDHGB1 PCDHGC5 () () () 3,1 0 PCDHGB1 PCDHGA10 () () () 3,1 0 PCDHGB1 PCDHGC4 () () () 3,1 0 PCDHGB1 PCDHGA11 () () () 3,1 0 PCDHGB1 PCDHGB6 () () () 3,1 0 PCDHGB1 PCDHGB5 () () () 3,1 0 PCDHGB1 PCDHGA12 () () () 3,1 0 PCDHGB1 PCDHGB7 () () () 3,1 0 PCDHGB1 PCDHGA9 () () () 3,1 0 PCDHGB1 PCDHGC3 () () () 3,1 0 AC139769.2 AC139769.1 () () () 1,0 2 EWSR1 CREB3L1 (0) (28) () 0,0 32 RPS10P7 NAV1 () () () 0,8 2 GBP7 GBP4 () () () 0,0 9 AC092807.3 DDAH1 () () () 0,2 6 ZNF267 ZNF720 () () (2) 0,0 6 B4GALT1 MRPS18A () () () 0,0 4 PPP1R21 STON1 () () (3) 0,0 4 TGM3 SIRPB1 () () () 3,0 0 GALR1 GALR1(52249),AC124254.1(91474) () () () 2,0 1 DYRK1A TNKS () () () 0,0 3 FRG1DP DUX4L35(10378),FRG1EP(20463) () () () 0,0 3 ZNF676 ZNF814 (0) (3) () 0,0 2 CSNK1E SLC16A5(418),ARMC7(3372) () () () 0,0 2 SIPA1L2(61702),RNU6-1211P(76944) SIPA1L2 () () () 0,0 2 TTC3 ZNF532 (1) (1) () 1,0 1 NRP1 PARD3 (1) (1) (1) 0,1 1 ESCO1 ROCK1 () () () 1,0 0 AGAP6 LINC00843 (1) (1) () 1,0 0 HCG4 AL671883.2 3 30 () (,) () NAIP OCLN () () 43 (,) () NPEPPS TBC1D3 () () 36 (,) () HACL1 COLQ () () 15 (0,0) (14) SAMD5 SASH1 () () 14 (0,0) (7) SIDT2 TAGLN () () 14 (0,0) (0) YY1AP1 ASH1L () () 12 (0,4) (1) SMG1 NPIPB5 () () 11 (,) () AJAP1 AL353572.2 () () 9 (,) () KLHL5 AP000350.2 () () 6 (0,0) (1) SSC5D DDX3X () () 6 (,) () CACNA1B CHST11 () () 5 (,) () AKR1E2 LINC01697 () () 4 (,) () NKD2 AL353572.2 () () 4 (,) () 68

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RAB11FIP3 CAPN15 () () 4 (0,0) (0) YY1 BCL11A () () 4 (,) () ELN HIPK3 () () 3 (,) () FBXO25 FAM157B () () 3 (,) () MALRD1 PLXDC2 () () 3 (0,0) (1) SERF1A SMN1 () () 2 (,) () DPP6 ACTR3B () () 1 (,) ()

Supplemental Table S7. Detailed fusion reads from primary kidney (NYG data).

StarFusion FusionCatcher Arriba gene1 gene2 JunctionReadCount SpanningFragCount Spanning_pairs split_reads1,2 discordant mates DMD(70601),AL050305.1(902) DMD () () () 0,12 17 DMD DMD(70733),AL050305.1(770) () () () 1,0 0 UNC13C UNC13C(211072),AC025272.1(212261) () () () 1,0 13 AL591501.1 DMD () () () 0,3 6 AL050305.1(311785),AL591501.1 DMD () () () 0,1 25 PCDHGA6 PCDHGC5 () () () 1,2 2 PCDHGA6 PCDHGC4 () () () 1,2 2 PCDHGA6 PCDHGC3 () () () 1,2 2 PCDHGA6 PCDHGA8 () () () 1,2 2 PCDHGA6 PCDHGA11 () () () 1,2 2 PCDHGA6 PCDHGA10 () () () 1,2 2 PCDHGA6 PCDHGB6 () () () 1,2 2 PCDHGA6 PCDHGB7 () () () 1,2 2 PCDHGA6 PCDHGA12 () () () 1,2 2 PCDHGA6 PCDHGB5 () () () 1,2 2 PCDHGA6 PCDHGA9 () () () 1,2 2 PCDHGA4 PCDHGC5 () () () 2,2 0 PCDHGA4 PCDHGC4 () () () 2,2 0 PCDHGA4 PCDHGC3 () () () 2,2 0 PCDHGA4 PCDHGA8 () () () 2,2 0 69

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PCDHGA4 PCDHGA11 () () () 2,2 0 PCDHGA4 PCDHGA10 () () () 2,2 0 PCDHGA4 PCDHGB6 () () () 2,2 0 PCDHGA4 PCDHGB7 () () () 2,2 0 PCDHGA4 PCDHGA12 () () () 2,2 0 PCDHGA4 PCDHGB5 () () () 2,2 0 PCDHGA4 PCDHGA9 () () () 2,2 0 SKA3 DDX10 () () () 0,1 3 PCDHGA3 PCDHGC5 () () () 1,0 2 PCDHGA3 PCDHGC4 () () () 1,0 2 PCDHGA3 PCDHGC3 () () () 1,0 2 PCDHGA3 PCDHGA8 () () () 1,0 2 PCDHGB2 PCDHGC5 () () () 0,1 2 PCDHGB2 PCDHGC4 () () () 0,1 2 PCDHGA3 PCDHGA11 () () () 1,0 2 PCDHGA3 PCDHGA10 () () () 1,0 2 PCDHGA3 PCDHGB6 () () () 1,0 2 PCDHGA3 PCDHGB7 () () () 1,0 2 PCDHGA3 PCDHGA12 () () () 1,0 2 PCDHGB2 PCDHGC3 () () () 0,1 2 PCDHGA3 PCDHGB5 () () () 1,0 2 PCDHGA3 PCDHGA9 () () () 1,0 2 PCDHGB2 PCDHGA11 () () () 0,1 2 PCDHGB2 PCDHGA10 () () () 0,1 2 PCDHGB2 PCDHGB6 () () () 0,1 2 PCDHGB2 PCDHGB7 () () () 0,1 2 PCDHGB2 PCDHGA12 () () () 0,1 2 PCDHGB2 PCDHGB5 () () () 0,1 2 PCDHGB2 PCDHGA9 () () () 0,1 2 EWSR1 CREB3L1 (0) (16) () 0,0 22 GALR1 GALR1(52249),AC124254.1(91474) () () () 1,1 6 LEPROT LEPR () () () 0,1 6 B4GALT1 MRPS18A () () () 0,0 6 RPS10P7 NAV1 () () () 0,6 0 SPATA13 SPATA13 () () () 2,2 1 BMP2 BMP2(40211),AL096799.1(249123) () () () 0,0 4 70

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PPP1R21 STON1 () () (4) 0,0 4 CHSY1 AC019254.2(34301),AC019254.1(16683) () () () 0,0 4 ZNF416 ZNF550 () () (1) 0,0 3 IFT88 NSD3 () () () 0,0 3 PCDHGB2 PCDHGA8 () () () 1,0 2 ZFYVE16 FAM151B () () (2) 0,0 3 PCDHA5 PCDHAC2 () () () 1,1 0 PCDHA5 PCDHAC1 () () () 1,1 0 PCDHA5 PCDHA13 () () () 1,1 0 ZNF28 ZNF468 () () (2) 0,0 2 SKAP2 PDP1 (2) (0) () 2,0 0 SALL3 NFATC1 () () () 1,1 0 SFT2D1 RPS6KA2 (1) (0) () 1,0 0 NPEPPS TBC1D3 () () 105 (,) () NAIP OCLN () () 64 (,) () AJAP1 AL353572.2 (0) (1) 30 (0,0) (0) SMG1 NPIPB5 () () 30 (0,0) (0) AL591378.1 DMD () () 21 (,) () YY1AP1 ASH1L () () 17 (1,0) (0) HACL1 COLQ () () 16 (0,0) (15) TOMM40 AL589743.5 () () 16 (,) () C6ORF47 BAG6 () () 14 (,) () PDCD6IP AC091951.3 () () 12 (,) () PRKAA1 TTC33 () () 10 (0,0) (0) SIDT2 TAGLN () () 9 (0,0) (0) AJAP1 KDM6B (6) (0) 8 (0,0) (0) KDM6B AJAP1 (0) (1) 8 (0,0) (0) CACNA1B CHST11 () () 7 (,) () KLHL5 AP000350.2 () () 7 (0,0) (3) DPP6 ACTR3B () () 6 (,) () SAMD5 SASH1 () () 6 (0,0) (4) CBX4 FOXD2 () () 5 (,) () FBXO25 FAM157B () () 5 (,) () PDCD6IP HERC2 () () 5 (,) () ZMYM5 PSPC1 () () 5 (0,0) (4) GEN1 SMG1 () () 4 (,) () 71

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MTG1 SCART1 () () 4 (0,0) (3) YY1 BCL11A () () 4 (,) () AC008013.1 KDM2A (1) (0) 3 (0,1) (0) DDX17 OLFM1 () () 3 (,) () FNBP4 ALG13 (1) (1) 3 (0,0) (0) ICOSLG PLD1 () () 3 (,) () LINC00641 MDN1 () () 3 (,) () MANBAL SRC () () 3 (0,0) (4) R3HDM1 ALG13 (1) (0) 3 (0,0) (0)

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Supplemental Table S8. EWSR1-CREB3L1 fusion sequence and PCR primers.

ATGGCGTC CACGGATTAC AGTACCTATA GCCAAGCTGC AGCGCAGCAG GGCTACAGTG CTTACACCGC CCAGCCCACT CAAGGATATG CACAGACCAC CCAGGCATAT GGGCAACAAA GCTATGGAAC CTATGGACAG CCCACTGATG TCAGCTATAC CCAGGCTCAG ACCACTGCAA CCTATGGGCA GACCGCCTAT GCAACTTCTT ATGGACAGCC TCCCACTGTA GAAGGGACCA GTACAGGTTA TACTACTCCA ACTGCCCCCC AGGCATACAG CCAGCCTGTC CAGGGGTATG GCACTGGTGC TTATGATACC ACCACTGCTA CAGTCACCAC CACCCAGGCC TCCTATGCAG CTCAGTCTGC ATATGGCACT CAGCCTGCTT ATCCAGCCTA TGGGCAGCAG CCAGCAGCCA CTGCACCTAC AAGACCGCAG GATGGAAACA AGCCCACTGA GACTAGTCAA CCTCAATCTA GCACAGGGGG TTACAACCAG CCCAGCCTAG GATATGGACA GAGTAACTAC AGTTATCCCC AGGTACCTGG GAGCTACCCC ATGCAGCCAG TCACTGCACC TCCATCCTAC CCTCCTACCA GCTATTCCTC TACACAGCCG ACTAGTTATG ATCAGAGCAG TTACTCTCAG CAGAACACCT ATGGGCAACC GAGCAGCTAT GGACAGCAGA GTAGCTATGG TCAACAAAGC AGCTATGGGC AGCAGCCTCC CACTAGTTAC CCACCCCAAA CTGGATCCTA CAGCCAAGCT CCAAGTCAAT ATAGCCAACA GAGCAGCAGC TACGGGCAGC AGAGTTCATT CCGACAGGAC CACCCCAGTA GCATGGGTGT TTATGGGCAG GAGTCTGGAG GATTTTCCGG ACCAGGAGAG AACCGGAGCA TGAGTGGCCC TGATAACCGG GGCAGGGGAA GAGGGGGATT TGATCGTGGA GGCATGAGCA GAGGTGGGCG GGGAGGAGGA CGCGGTGGAA TGGGCGCTGG AGAGCGAGGT GGCTTCAATA AGCCTGGTGG ACCCATGGAT GAAGGACCAG ATCTTGATCT AGGCCCACCT GTAGATCCAG ATGAAGACTC TGACAACAGT GCAATTTATG TACAAGGATT AAATGACAGT GTGACTCTAG ATGATCTGGC AGACTTCTTT AAGCAGTGTG GGGTTGTTAA GAAATTACAG GGGACATCAG GGCCACTGCT CCTGACAGAG GAGGAGAAGC GGACCCTGAT TGCTGAGGGC TACCCCATCC CCACAAAACT CCCCCTCACC AAAGCCGAGG AGAAGGCCTT GAAGAGAGTC CGGAGGAAAA TCAAGAACAA GATCTCAGCC CAGGAGAGCC GTCGTAAGAA GAAGGAGTAT GTGGAGTGTC TAGAAAAGAA GGTGGAGACA TTTACATCTG AGAACAATGA ACTGTGGAAG AAGGTGGAGA CCCTGGAGAA TGCCAACAGG ACCCTGCTCC AGCAGCTGCA GAAACTCCAG ACTCTGGTCA CCAACAAGAT CTCCAGACCT TACAAGATGG CCGCCACCCA GACTGGGACC TGCCTCATGG TGGCAGCCTT GTGCTTTGTT CTGGTGCTGG GCTCCCTCGT GCCCTGCCTT CCCGAGTTCT CCTCCGGCTC CCAGACTGTG AAGGAAGACC CCCTGGCCGC AGACGGCGTC TACACGGCCA GCCAGATGCC CTCCCGAAGC CTCCTATTCT ACGATGACGG GGCAGGCTTA TGGGAAGATG GCCGCAGCAC CCTGCTGCCC ATGGAGCCCC CAGATGGCTG GGAAATCAAC CCCGGGGGGC CGGCAGAGCA GCGGCCCCGG GACCACCTGC AGCATGATCA CCTGGACAGC ACCCACGAGA CCACCAAGTA CCTGAGTGAG GCCTGGCCTA AAGACGGTGG AAACGGCACC AGCCCCGACT TCTCCCACTC CAAGGAGTGG TTCCACGACA GGGATCTGGG CCCCAACACC ACCATCAAAC TCTCCTAG

Primer Sequence Tm (°C) GC% EWSR1-CREB3L1 fus breakpoint (forward) 5'-TCTTTAAGCAGTGTGGGGTTG-3' 63.9 47.6 EWSR1-CREB3L1 fus breakpoint (reverse) 5'-TTTCCTCCGGACTCTCTTCA-3' 63.9 50 EWSR1-CREB3L1 fus portion (forward) 5'-CAGCCCACTGATGTCAGCTA-3' 64.1 55 EWSR1-CREB3L1 fus portion (reverse) 5'-CTGTCGTGGAACCACTCCTT-3' 64.2 55 EWSR1-CREB3L1 fus entire (forward) 5'-CCCAGCCTAGGATATGGACA-3' 63.7 55 EWSR1-CREB3L1 fus entire (reverse) 5'-AGGGGGTCTTCCTTCACAGT-3' 63.8 55

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Metastatic Pediatric Sclerosing Epithelioid Fibrosarcoma

Andy D Woods, Reshma Purohit, Laura Mitchell, et al.

Cold Spring Harb Mol Case Stud published online August 6, 2021 Access the most recent version at doi:10.1101/mcs.a006093

Published online August 6, 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 onlineAugust 6, 2021in advance of the full issue.

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