ORIGINAL RESEARCH POT1 and Damage Response Malfunction Trigger Acquisition of Somatic Activating Mutations in the VEGF Pathway in Cardiac Angiosarcomas Oriol Calvete, PhD; Pablo Garcia-Pavia, MD; Fernando Domınguez, MD; Lluc Mosteiro, PhD; Lucıa Perez-Cabornero, PhD; Diego Cantalapiedra, PhD; Esther Zorio, MD; Teresa Ram  on y Cajal, MD; Maria G. Crespo-Leiro, MD; Alex Teule, PhD; Conxi Lazaro, PhD; Manuel M. Morente, MD; Miguel Urioste, MD; Javier Benitez, PhD Background-—Mutations in the POT1 gene explain abnormally long telomeres and multiple tumors including cardiac angiosarcomas (CAS). However, the link between long telomeres and tumorigenesis is poorly understood. Methods and Results-—Here, we have studied the somatic landscape of 3 different angiosarcoma patients with mutations in the POT1 gene to further investigate this tumorigenesis process. In addition, the genetic landscape of 7 CAS patients without mutations in the POT1 gene has been studied. Patients with CAS and nonfunctional POT1 did not repress ATR (ataxia telangiectasia RAD3-related)–dependent DNA damage signaling and showed a constitutive increase of cell cycle arrest and somatic activating mutations in the VEGF (vascular endothelial growth factor)/angiogenesis pathway (KDR gene). The same observation was made in POT1 mutation carriers with tumors different from CAS and also in CAS patients without mutations in the POT1 gene but with mutations in other genes involved in DNA damage signaling. Conclusions-—Inhibition of POT1 function and damage-response malfunction activated DNA damage signaling and increased cell cycle arrest as well as interfered with apoptosis, which would permit acquisition of somatic mutations in the VEGF/angiogenesis pathway that drives tumor formation. Therapies based on the inhibition of damage signaling in asymptomatic carriers may diminish defects on cell cycle arrest and thus prevent the apoptosis deregulation that leads to the acquisition of driver mutations. ( J Am Heart Assoc. 2019;8:e012875. DOI: 10.1161/JAHA.119.012875.) Key Words: cardiac angiosarcoma • cell cycle arrest • damage response • POT1 • VEGF/angiogenesis pathway T he Li-Fraumeni syndrome is an autosomal dominant syndrome representing a genetic predisposition to a wide spectrum of tumors and is typically linked to mutations TP53 gene. Recently, we studied different Li-Fraumeni-like families with multiple tumors including various cases of cardiac angiosarcoma (CAS), which is the most common and of the TP53 tumor suppressor gene.1 Li-Fraumeni-like families most aggressive type of primary malignant neoplasm of the have a similar clinical presentation, but Li-Fraumeni-like heart in adults.2 Patients affected with CAS are generally syndrome is less frequently associated with mutations in the diagnosed at advanced stages with very poor prognosis and From the Human Genetics Group (O.C., J.B.), Tumour Suppression Group (L.M.), Biobank Unit (M.M.M.), and Familial Cancer Clinical Unit (M.U.), Spanish National Cancer Research Center (CNIO), Madrid, Spain; Center for Biomedical Network Research on Rare Diseases (CIBERER), Madrid, Spain (O.C., J.B.); Department of Cardiology, Hospital Universitario Puerta de Hierro, Madrid, Spain (P.G.-P., F.D., M.G.C.-L.); Center for Biomedical Network Research on Cardiovascular Diseases (CIBERCV), Madrid, Spain (P.G.-P., F.D.); Facultad de Ciencias de la Salud, Universidad Francisco de Vitoria (UFV), Madrid, Spain (P.G.-P.); Spanish National Cardiovascular Research Center (CNIC), Madrid, Spain (F.D.); Medical Genetics Unit, Sistemas Genomicos, Parque Tecnologico de Valencia, Paterna, Spain (L.P.-C., D.C.); Department of Cardiology, Hospital Universitario y Politecnico La Fe, Valencia, Spain (E.Z.); Medical Oncology Service, Hospital Sant Pau, Barcelona, Spain (T.R.yC.); Department of Cardiology, Instituto de Investigacion Biomedica de A Coru~na (INIBIC), Complexo Hospitalario Universitario de A Coru~na (CHUfSiAC), A Coru~na, Spain (M.G.C.-L.); Hereditary Cancer Program-Medical Oncology Service (A.T.)  and Medical Oncology Service (C.L.), Catalan Institute of Oncology, ICO-IDIBELL and CIBERONC, Barcelona, Spain. Accompanying Tables S1 through S5 are available at https://www.ahajournals.org/doi/suppl/10.1161/JAHA.119.012875 Correspondence to: Oriol Calvete, PhD, Spanish National Cancer Research Center (CNIO), Melchor Fernandez Almagro, 3. 28029, Madrid, Spain. E-mail: ocalvete@cnio.es or Javier Benitez, PhD, Spanish National Cancer Research Center (CNIO), Melchor Fernandez Almagro, 3. 28029, Madrid, Spain. E-mail: jbenitez@cnio.es Received April 8, 2019; accepted July 22, 2019. ª 2019 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. DOI: 10.1161/JAHA.119.012875 Journal of the American Heart Association 1 Genetic Landscape of Cardiac Angiosarcomas Calvete et al ORIGINAL RESEARCH and damage.6 Other described POT1 mutations associated Clinical Perspective with risk of developing familial glioma and familial melanoma tumors also led to abnormally long and fragile What Is New? telomeres.7 However, the link between telomere instability • In this study we describe how mutations in the POT1 gene, and tumorigenesis is not well understood. In addition, which explain long telomeres, correlate with cell cycle arrest studies of the biological pathways involved in the progres- increase in angiosarcoma patients. sion of angiosarcomas are very scarce. Recently, next- • The same increase was observed in other cardiac angiosar- generation sequencing studies uncovered somatic alter- coma patients even without mutations in the POT1 gene but ations in the VEGF/PLCG1 pathway in cardiac angiosar- in the damage response signaling. • This malfunction would bypass the apoptosis mechanism coma16,17 and driver mutations in the PI3K/AKT/mTOR and and would trigger the acquisition of somatic activating MEK pathways in angiosarcomas other than cardiac,18-21 but mutations in the angiogenesis pathway. these studies did not distinguish between constitutive and somatic mutations and did not clarify the genetics and What Are the Clinical Implications? underlying mechanisms. In order to understand how telomere instability links the • Our results suggest that the use of angiogenesis inhibitors might regulate the tumor progression; however, targeting angiosarcoma process, in the present work we studied ATM/ATR (ataxia telangiectasia mutated/RAD3-related) somatic events in different angiosarcoma patients carrying activity would rescue the cell cycle control and would the POT1 p.Arg117Cys mutation described in Calvete et al6: 2 prevent the acquisition of somatic driver mutations in patients (F1 and F3) affected with CAS and 1 patient (F2) with patients affected with angiosarcoma tumors and asymp- 2 tumors, a breast AS and a papillary thyroid tumor. In tomatic patients carrying POT1 mutations. addition, we studied the genetic and molecular somatic landscape that drives tumor progression in 7 patients with sporadic CAS tumors (NT1-NT3 and T1 to T4) who did not short survival rates (5-year survival rate of 14%).3 The genetic carry mutations in the POT1 gene. landscape that determines the tumorigenic process of angiosarcomas (AS) is poorly understood and not well established.4,5 Previous studies by our group uncovered a Methods deleterious missense mutation in the POT1 gene (c.349C>T The data, methods used in the analysis, and materials used to [p.Arg117Cys], pathogenic, Li-Fraumeni-like syndrome/CAS, conduct the research are available to any researcher for autosomal dominant)6 causing AS in 4 families (3 in cardiac purposes of reproducing the results or replicating the tissue and 1 in breast).6 Germline mutations in the POT1 procedure. gene have also been related with the development of other familial cancer types.7-12 POT1 is a component of the so- called shelterin complex, which is involved in telomere Ethics Statement elongation in germline and stem cells (Figure 1A).13 In normal conditions the shelterin complex protects telomere cap ends Institutional Review Board approval was obtained; the ethics in somatic cells by preventing access of the telomerase to committee of the CNIO, the Institute of Health Carlos III, and chromosome ends.14 The shelterin complex also masks SaludMadrid (Autonomous Community of Madrid) approved single-stranded telomeres from the DNA damage response, this study (CS9679). Informed consent was received from thereby preventing the activation of ATM (ataxia telangiec- participants before inclusion in the study. tasia mutated) and ATR (ataxia telangiectasia RAD3-related) to avoid cell cycle arrest through POT1 and TPP1 (Fig- ure 1B).15 TPP1 (which is also called ACD gene) anchors the Patients telomere by POT1 and TRF1 (telomeric repeat binding factor Formalin-fixed paraffin-embedded (FFPE) tissue samples from 1) proteins. When telomeres are critically short, the shelterin 10 patients were selected. The 3 familial angiosarcoma complex does not prevent activation of the ATM and ATR individuals carrying the POT1 p.Arg117Cys mutation were response, which can drive the cell to senescence and selected from Calvete et al.6 Seven FFPE tissue samples from apoptosis (Figure 1C). individuals affected with sporadic CAS not carrying mutations Our previous studies demonstrated that cardiac angiosar- in the POT1 gene were also selected: 3 FFPE tissue sections coma patients carrying the POT1 p.Arg117Cys mutation contained normal (N) and tumor (T) tissue (NT1, NT2 and showed abnormally long telomeres due to the lack of NT3), and the other 4 FFPE sections contained only tumor repression of telomerase, which led to increased fragility tissue (T1 to T4). DOI: 10.1161/JAHA.119.012875 Journal of the American Heart Association 2 Genetic Landscape of Cardiac Angiosarcomas Calvete et al ORIGINAL RESEARCH A Elongation in germline cells/stem cells C Extremely short telomere ATM ATR TPP1 TRF POT1 Activation of DNA damage markers · γH2AX TPP1 · p53BP1 TRF POT1 ATM TP53 p21 Telomerase ATR CDK1/2 p27 B Telomere shortening in somatic cells D POT1 p.R117C carriers ATM ATM Activation of DNA damage markers ATR · γH2AX ATR · p53BP1 TP53 p21 TPP1 POT1 CDK1/2 p.R117C p27 TRF POT1 TPP1 TRF Telomerase Telomerase Figure 1. Telomere biology and damage signaling. A, Elongation in germline/stem cells. The shelterin complex mediates telomere elongation by recruiting telomerase. Shelterin also represses the DNA damage response by preventing the activation of ATM and ATR through TPP1 and POT1 proteins, respectively. TPP1 is anchored to the chromosome by POT1 and TRF (telomeric repeat binding factor 1), which is another component of the shelterin complex. B, Telomere shortening in somatic cells. Somatic divisions entail telomere shortening due to the inhibition of telomerase recruitment mediated by the POT1 protein. C, Extremely short telomere. The shelterin complex cannot bind critically short telomeres. DNA damage response ATM/ATR activates the TP53/p21 cascade to inhibit CDK1/2. DNA damage markers such as cH2AX and TP53BP1 bind the short telomeres. DNA damage signaling mediates senescence, cell cycle arrest, and apoptosis. D, POT1 p.Arg117Cys (p.R117C) mutation carriers. The POT1 p.R117C mutation prevents POT1 from binding to TPP1 and from forming the OB-fold to bind single-strand DNA, which prevents POT1 from repressing ATR signaling. Telomere damage response is activated in POT1 p.R117C mutation carriers. Samples negative staining. Staining was considered separately in normal (N) and tumor (T) tissues. IHC controls were performed Genomic DNA was extracted from FFPE tissue samples and by using normal tissue from biopsies of healthy individuals. from fixed tissue slides (microdissection) using the DNeasy Blood & Tissue Kit (Cat No. 69504, Qiagen, Hilden, Germany) following the manufacturer’s instructions. Histology of hema- Whole Exome Sequencing and Bioinformatics toxylin and eosin–stained sections of the tissues was Pipelines assessed by a pathologist (M.M.). Exomes from selected tissues were captured and enriched using the SureSelect Human All Exon Kit (78 Mb) (Agilent Technologies, Santa Clara, CA). Enriched samples were Immunohistochemistry paired-end sequenced on an Illumina Genome Analyzer II FFPE blocks were cut into 5-lm-thick sections for immuno- sequencing platform using 2 lanes per sample and generating histochemistry (IHC) studies. The cell cycle in normal tissue 101 base-pair long reads. FASTQ files of short reads were was tested with anti-p21 (WAF1) from Merck (Darmstadt, aligned using the BWA algorithm to the GRCh37/hg19 Germany; Ref MABE325), anti-p27 (57/Kip1) from BD Bio- reference genome22; 96.11% (ranging from 93.34% to sciences (Franklin Lakes, NJ; Ref 610242), and anti–phospho- 98.74%) of the reads aligned in the reference genomes histone cH2AX (Ser139) from Millipore (Burlington, MA; Ref (effectiveness). GATK-based variant calling was performed for 05-636) antibodies. Activation of the VEGF-angiogenesis aligned reads considering DP (Read Depth) values of >30 and pathway was assessed with 2 different antibodies against Quality-by-Depth scores for a variant confidence of >1.00. A phosphorylated (activated) proteins: anti–phospho-p44/42- total of 92.28% (ranging from 91.70% to 92.16%) of the MAPK (ERK1/2) and anti–phospho-S6 ribosomal protein variants that were well aligned and annotated passed the (Ser235/236) from Cell Signaling Technology (Danvers, MA; quality and coverage filters. Strict filtering for only well- Refs 9101 and 2211, respectively). Both the absence of defined variants by quality controls, and those not included in staining and excess nonspecific staining were considered as repeat regions were included to prevent false positives. Tumor DOI: 10.1161/JAHA.119.012875 Journal of the American Heart Association 3 Genetic Landscape of Cardiac Angiosarcomas Calvete et al ORIGINAL RESEARCH variants (<10% alternative variant allele frequency) and those supported with >7 reads and filtered by frequency (minor allele with low coverage (<69) were discarded. frequency <0.05). The copy number variation call was calculated Only quality-filtered variants affecting coding sequences of for the fold-change results for each gene. Variants were canonical transcripts (nonsynonymous, essential splice site, classified and analyzed later by the IBM Watson for Genomics frame shift or gain/loss of stops) were taken into account. platform, which searches electronic medical databases to find Variant type annotation, population statistics, disease-specific information that may be relevant to a particular genomic sequence databases, and in silico predictive algorithms were sequence (available at https://www.ibm.com/watson/). according to AMCG standards and guidelines.23 Variants with a minor allele frequency of <0.01 and <0.05 (dbSNP130, HapMap, or 1000 Genomes) were considered for stringent Results and relaxed filtering, respectively. Their potential damaging Angiosarcoma Patients Carrying the POT1 effect was assessed using the VEP24 script software package p.Arg117Cys Mutation (including Sift, Polyphen and Condel damage predictors). Stringent filtering only considered the variants annotated as Constitutional Effect in Normal Tissue of POT1 pathogenic by all damage predictors. Whole-exome sequenc- p.Arg117Cys Mutation Carriers ing data have been deposited in the ArrayExpress public In normal conditions the POT1 protein represses downstream database under accession number E-MTAB-7999 (available at activation of the DNA damage response at telomeres in https://www.ebi.ac.uk/fg/annotare/). somatic cells (Figure 1B).15 The POT1 p.Arg117Cys protein Variants found in DNA blood samples and variants found in shows a reduced capacity to bind telomeres and TPP16 and common between paired normal/tumor tissues were consid- may affect the regulation of the damage response. Damage ered as constitutional. Variants found only in tumor tissue response activation leads to cell cycle arrest, replicative were considered somatic variants. Constitutional variants senescence, and apoptosis (Figure 1C). In order to decipher were validated in DNA from blood and normal tissue samples, the putative effect of POT1 malfunction, patient tissues were and somatic variants were validated in DNA from tissue stained with anti-P-cH2AX (DNA damage marker) and anti-p21 samples (tumor) by Sanger sequencing. and anti-p27 antibodies (inhibitors of CDK1/2 to arrest the cell cycle). IHC studies were carried out in normal (N) tissues of patients from the families carrying the constitutional POT1 Pathway Enrichment Analyses p.Arg117Cys mutation studied in Calvete et al6: 2 patients Two different software packages were used for independent (F1 and F3) affected with CAS and 1 patient (F2) with 1 breast assessments of the gene set analyses. Data were analyzed AS and 1 papillary thyroid tumor. with Qiagen’s Ingenuity Pathway Analysis (IPA, Qiagen, Increased IHC staining with anti-p21, -p27, and -P-cH2AX Redwood City, CA www.qiagen.com/ingenuity) and Consen- antibodies was observed in N tissues of all studied patients in susPathDB (available at http://cpdb.molgen.mpg.de/).25 comparison with the corresponding N tissues from healthy Overrepresentation analysis of the gene set list was per- non–mutation carriers (Table 1 and Figure 2A). Therefore, the formed with a minimum overlap of 4 genes with the pathway reduced binding to telomeres and to TPP1 by the POT1 database set size and a P-value cutoff of 0.001. Induced p.Arg117Cys protein correlates with activation of damage network module analysis without intermediate nodes and response signaling mediated by p21 and p27 (Figure 1D). considering high-confidence binary protein and genetic/gene regulatory interactions were also evaluated. Somatic Events in POT1 p.Arg117Cys Mutation Carriers To evaluate possible somatic events in affected individuals Trusight Tumor 170 Panel and IBM Watson Study carrying the POT1 p.Arg117Cys mutation that might lead to Resequencing of tumor DNA from FFPE tissue samples was the formation of AS, the exomes of normal (N) and tumor (T) performed with TruSight Tumor 170 from Illumina (San Diego, tissues of the F1 and F2 individuals were sequenced. Variants CA), a novel sequencing platform that predicts the most found in T tissue but not in N tissue were considered to be probable variant causing the pathology and provides sugges- somatic (Table S1). In patient F1 (CAS), 46 filtered somatic tions for translating the data to the clinic. The sequencing of the variants were found in T but not in N cardiac tissue (Table 2) hybrid capture was run on the HiSeq 2500 System. The captured including an in-frame deletion in the KDR gene (p.Asn704del), gene content in the TruSight Tumor 170 Assay, data sheet, and which encodes VEGF receptor 2 (VEGFR2), which belongs to specifications are available at https://www.illumina.com. Low the VEGF-angiogenesis signaling pathway (Figure 3A and 3B). variant quality of <20 and low depth (<100 for variant calls and Regarding the F2 individual, only 13 filtered somatic <250 for reference call filters) were considered. Variants were variants were found in the T tissue of the breast AS DOI: 10.1161/JAHA.119.012875 Journal of the American Heart Association 4 Genetic Landscape of Cardiac Angiosarcomas Calvete et al ORIGINAL RESEARCH Table 1. Total Cases and Number of Variants Found in the Whole Exome Sequencing Individual Pathology Tissue Variant Calling Filtered Variants Somatic Variants* Constitutional Variants† POT1 p.Arg117Cys carriers F1 CAS T 1095 46 NA+ N F2 Papillary thyroid T 100 506 1294 5 NA+ N 100 371 1281 Breast AS T 93 496 1276 13 NA+ Without mutations in NT1 Sporadic CAS T 102 560 1266 62 1032 the POT1 gene N 111 686 1333 NT2 Sporadic CAS T 100 120 1231 36 1101 N 141 248 1239 NT3 Sporadic CAS T 97 233 704 37 1180 N 113 358 762 Individual Pathology Tissue Variant Calling Filtered Variants Stringent Filtering ‡ Only T tissue available T1 Sporadic CAS T 93 374 1181 315 T2 Sporadic CAS T 95 492 1274 403 T3 Sporadic CAS T 104 545 1328 443 T4 Sporadic CAS T 93 859 1233 361 CAS indicates cardiac angiosarcoma; N, normal tissue; NA+, not applicable (POT1 p.Arg117Cys carriers from Calvete et al [2015]); T, tumor tissue. *Variants found in tumor tissue that were not found in normal tissue. † Variants found in normal tissue that were not found in tumor tissue. ‡ Individuals with only T tissue sequenced. (Table S1). Two of them were variants annotated in the genes promotion (Figure 3C). In order to test the putative effects of PLCG1 (p.Leu752Val) and PIK3CA (p.Arg88Gln) belonging to the mutations found in the VEGF angiogenesis pathway, we the VEGF-angiogenesis pathway and previously described to studied the MAPK/ERK and AKT/PI3K molecular pathways by be involved in different angiosarcomas and primary breast IHC with antibodies against the activated (phosphorylated) cancer.26 Another 5 somatic variants were found in the forms of ERK and S6, respectively (Figure 3A and 3B). All papillary thyroid T tissue from the same individual (Table S1). angiosarcomas (CAS and the breast AS tumors) did not stain Three of these belonged to the VEGF-angiogenesis pathway with anti–P-ERK but were positively stained (>70%) with the (PIK3R6 [p.Arg59Lys], RASSF1 [p.Arg227His], and BRAF anti–P-S6 antibody. However, papillary thyroid tumor tissue [p.Val600Glu]). Overall, 32/52 (62%) of the somatic missenses stained with both antibodies (Table 1 and Figure 2B). were C:G>T:A changes (Table S1). Genes with mutations are In summary, somatic activating mutations of the VEGF- shown in the angiogenesis pathway depicted in Figure 3. angiogenesis pathway were found in all studied tumor tissues Finally, only cardiac tumor tissue from patient F3 (CAS) of POT1-mutated patients independently of the tumor type was sequenced (Table 2). A total of 297 filtered variants were (cardiac, breast, and thyroid). Somatic mutations in the KDR found in the tumor tissue of this patient (Table S2), including gene were found in both CAS patients (F1 and F3). In addition, another mutation in the KDR gene (p.Thr771Arg). Overall, 60% positive P-S6 staining was observed in all angiosarcomas with of the somatic variants found in patient F3 were annotated as somatic mutations in the KDR (CAS) and PI3K (breast AS) C:G>T:A changes (Table S2). genes (AKT/PI3K pathway). The papillary tumor (F2), which Thus, somatic mutations in the VEGF signaling pathway had a mutation in the MAPK/ERK signaling pathway (BRAF were found in the tumor tissues of all affected individuals gene), was stained with both anti–P-ERK and anti-PS6 carrying the POT1 p.Arg117Cys germline mutation. Interest- antibodies (Figures 2B and 3). ingly, both studied CAS patients had mutations in KDR, which activates VEGF signaling to regulate angiogenesis by the MAPK/ERK and AKT/PI3K pathways (Figure 3A and 3B). Sporadic Cardiac Angiosarcoma Patients Without MAPK/ERK regulates proliferation activity, while AKT/PI3K Mutations in the POT1 Gene promotes protein synthesis. Interestingly, both molecular To assess the whole genetic landscape of CAS tumors, activities regulate the cell cycle by inhibiting senescence another 7 patients with sporadic CAS tumors who were not DOI: 10.1161/JAHA.119.012875 Journal of the American Heart Association 5 Genetic Landscape of Cardiac Angiosarcomas Calvete et al ORIGINAL RESEARCH A N cardiac tissue N thyroid tissue B P-ERK P-S6 N cardiac tissue N cardiac tissue Wild type Wild type p21 mutation carriers (N tissue) NT2 (sporadic CAS) F2 POT1 p. R117C (thyroid) CAS (F1) CAS (F1) Damage Response POT1 p.R117C mutation carriers (T tissue) cardiactissue N cardiac tissue N thyroid tissue Breast AS (F2) Breast AS (F2) Wild type P-γH2AX NT2 (sporadic CAS) NT2 (sporadic CAS) F 2POT1 p. R117C (thyroid) Papillary thyroid (F2) Papillary thyroid (F2) mutation carriers (N tissue) Damage Response N cardiac tissue N thyroid tissue NT2 (sporadic CAS NT2 (sporadic CAS Damage Response mutation carriers (T tissue) Wild type p27 F2 POT1 p. R117C (thyroid) mutation carriers (N tissue) NT2 (sporadic CAS) CAS (T1) CAS (T1) Damage Response Figure 2. Immunohistochemical staining. A, Tissue stress was tested in normal tissue of carriers of the POT1 p.Arg117Cys (p.R117C) mutation and carriers of mutations in the damage response-signaling pathway (sporadic CAS) in comparison with the corresponding normal tissue without mutations (wild type). Above: Wild-type cardiac and thyroid tissues from healthy controls without mutations. Below: normal tissue of individual NT2 (sporadic CAS individual with constitutional mutations in the ATR, ATM and TP53BP genes) and normal tissue of individual F2 (papillary thyroid tumor with POT1 p.R117C mutation) as representative examples (see Table 2 for all studied individuals). Increased cell cycle arrest was observed in the normal tissue of both patients. Black arrowheads show some of the stained nuclei. Detailed fields (109) are also shown. Scale bar (in black): 100 lm. B, IHC staining with anti–P-ERK and anti–P-S6 antibodies in tumor tissues. Tumor tissues from carriers (F1 and F2) and noncarriers (T1 and NT2) of the POT1 p.R117C mutation compared with a normal tissue section (negative staining) are shown as examples (see Table 2 for all studied individuals). Three tumors from the 2 patients (F1 and F2) carrying the POT1 p.R117C mutation are shown: both angiosarcomas (CAS tumor tissue from F1 and breast AS from patient F2) only showed immunoreactivity with anti–P-S6 antibody, while the papillary thyroid tumor (patient F2) also showed immunoreactivity with anti–P-ERK antibody. Two staining patterns were observed in sporadic CAS patients without mutations in the POT1 gene: tissue from patient T1 only showed immunoreactivity with anti–P-S6, whereas tissue from patient NT2 (sporadic CAS) was stained with both anti–P-S6 and anti–P-ERK antibodies. Scale bar (in black): 100 lm. AS indicates angiosarcoma; CAS, cardiac angiosarcoma; N, normal tissue; T, tumor tissue. DOI: 10.1161/JAHA.119.012875 Journal of the American Heart Association 6 Genetic Landscape of Cardiac Angiosarcomas Calvete et al ORIGINAL RESEARCH carrying mutations in the POT1 gene were studied. Normal (N) (Figure 4). These genes belong to the damage response and tumor (T) cardiac tissues were available from 3 sporadic signaling pathway and may deregulate the cell cycle in the CAS individuals (NT1, NT2, and NT3), whereas only tumor same manner as previously observed for the POT1 mutation tissue was available from the other 4 CAS individuals (T1 to carriers. Thus, N cardiac tissue from the sporadic CAS T4). We found 1032, 1101, and 1180 constitutional variants individuals (NT series) was also stained with anti–P-cH2AX, in cardiac tissue for the NT1, NT2, and NT3 CAS individuals, anti-p21, and anti-p27 antibodies. Positive staining was also respectively; 62, 36, and 37 somatic variants were found for observed in N tissue of sporadic CAS individuals (Table 1), individuals NT1, NT2, and NT3, respectively (Table 2). which correlated with cell cycle deregulation as observed in Regarding the 4 tumor samples of which only T tissue was familial angiosarcomas (POT1 p.Arg117Cys mutation carriers). available, no distinction could be made between constitutional Therefore, the familial angiosarcomas (POT1 p.Arg117Cys and somatic variants. We found 1181, 1274, 1328, and 1233 carriers) and sporadic CAS patients behaved in a similar way variants in the T tissue of patients T1, T2, T3, and T4, regarding cell cycle arrest regulation. respectively (Table 2). To further delineate the genetic landscape of sporadic CAS Somatic Events in Tumor Tissue in Sporadic CAS tumors, the genes encompassing filtered variants were A second pathway enrichment analysis was performed with grouped into 2 different pathway enrichment analyses. The the gene set including the somatic variants found in T tissues first set included genes with constitutional (found in both N of sporadic CAS individuals (see above). This enrichment and T tissues from cases NT1, NT2, and NT3) and all genes analysis revealed that the most represented pathway was the with variants from the other 4 CAS individuals with only the “gf-hypoxia and angiogenesis” pathway (Biocarta: 16.7%) tumor tissue sequenced (constitutional or somatic) (2501 (P value 0.00557). The pathway with the major number of unique genes in total); the second set included the genes with affected genes was the “Signaling by VEGF” pathway somatic variants (only present in T tissue) from cases with N (Reactome, 24 genes) (P value 0.00101) (Table S3). Both and T tissue (NT1, NT2, and NT3) and again all genes with enrichment analyses corresponded with the VEGF-angiogen- variants from the other 4 CAS individuals with only the esis pathway. Genes with mutations are shown in the sequenced T tissue cases (T1 to T4) (1522 unique genes in angiogenesis pathway of Figure 3. Mutations in genes from total). these pathways were found in all 7 sporadic CAS individuals (Figure 4). Activation of the MAPK/ERK and AKT-PI3K path- Constitutional Events in Normal Tissue in Sporadic CAS ways was studied by IHC with anti–P-ERK and anti–P-S6 This study revealed that the most represented pathway and antibodies, respectively. the pathway with the major number of genes were the Tumors of all studied sporadic CAS individuals (NT and T “Sustainability of p53 pathway” (genes ATM, TP53, and series) stained positive with anti-PS6 antibodies, which RFWD2) (P value 0.003) and the “Repair modulation pathway” demonstrates that somatic mutations were activating the (genes ATR, ATM, TP53, RFWD2, SIRT7, BRCA2, CDK8, VEGF-angiogenesis pathway (Table 1). Especially intense UBE2D1, WRN, PMS2, BRIP1, TP53BP2, and APC2) (P value staining was also observed in the endothelial lining of blood 0.0022), respectively (Table S3). Mutations in genes from vessels. However, not all tissues from individuals affected these pathways were found in all 7 sporadic CAS individuals with sporadic CAS stained with anti-PERK antibody. Tissue of Table 2. Immunohistochemistry Staining Results for the Studied Cases N Tissue T Tissue Mutation Sample Pathology Tissue cH2AX p21 p27 Mutations in VEGF-Angiogenesis Pathway P-ERK P-S6* POT1 F1 CAS Cardiac (N+T) + + + KDR + p.Arg117Cys F2 Breast AS Breast T NA NA NA PI3K + Thyroid (N +T) + + + BRAF + + DR genes NT2 CAS Cardiac (N+T) + + + VEGF2/RAS-MAPK + + T1 CAS Cardiac (T) NA NA NA Akt-PI3K + T3 CAS Cardiac (T) NA NA NA RAS-MAPK/Akt-PI3K + + T4 CAS Cardiac (T) NA NA NA VEGFA/Akt-PI3K + + + indicates overexpression; , no expression; CAS, cardiac angiosarcoma; DR, damage response; N, normal; NA, tissue not available; T, tumor. *Positive staining in lining epithelium and tissue. DOI: 10.1161/JAHA.119.012875 Journal of the American Heart Association 7 Genetic Landscape of Cardiac Angiosarcomas Calvete et al ORIGINAL RESEARCH individual T1, who only had mutations in the AKT-PI3K therapies for the alterations common to the 2 CAS patients signaling pathway (Table 1), did not stain with anti–P-ERK. target TP53 to restore cell cycle regulation. The tumors with mutations in the 2 molecular signaling pathways (NT1, NT2, and T4) also stained positive with the 2 antibodies (anti–P-ERK and anti–P-S6) (Table 1). Interestingly, Discussion the individual with mutations only in the MAPK/ERK signaling Telomere Instability in POT1 Mutation Carriers pathway (T3) also stained positive with both antibodies Increases Cell Cycle Arrest in Constitutional (Table 1). Stained tissues from patients T1 and NT2 are Tissue and Increases Acquisition of Somatic shown in Figure 2B as representative examples. In summary, all studied individuals with CAS (either familial Mutations in the Angiogenesis Pathway or sporadic) had mutations (either constitutional or somatic) The single-strand DNA response in telomeres is inactivated in normal tissue affecting damage response signaling (Fig- by the shelterin complex (Figure 1A and 1B).15 We previously ure 4). IHC with anti-p21 and anti-p27 antibodies confirmed observed that the POT1 p.Arg117Cys mutation prevented the cell cycle arrest deregulation in N tissue that leads to POT1 protein from binding to TPP1 and forming the OB-fold constitutional cell cycle arrest and cessation of cell division to bind singles-strand DNA, which led to abnormally long (Figure 2A and Table 1). In addition, somatic activating telomeres.6 Here, we observed that abnormal telomere mutations in the VEGF-angiogenesis pathway were found in length found in our patients correlates with a genomic tumor tissue of familial and sporadic angiosarcomas, inde- instability scenario that, in consequence, activates DNA pendently of the presence of POT1 mutations (Figures 2B and damage-signaling (cH2AX-positive staining). Individuals car- 4; Table 1). rying the POT1 p.Arg117Cys mutation overexpressed the p21 and p27 proteins in constitutional tissue (Table 1), which correlates with prevention of the repression of ATR Sequencing Replication With the Truseq170 signaling and leads to cell cycle arrest (Figure 1D). This Panel and IBM Watson for Genomics Platform increased senescence in nontumor tissue would result in A sequencing replication was performed for 2 previously reduced cell cycling and cessation of cell division. A similar sequenced CAS patients without mutations in the POT1 gene. mechanism was proposed to explain how short telomeres The Truseq170 panel was run for tumor tissue of the T1 and can lead to vascular senescence and diminished proliferative T4 individuals and analyzed with the IBM Watson for capacity that involved exhaustion of cell pools in mice.27,28 Genomics platform (version 33.148), which is a novel In addition, tumor tissue of POT1 p.Arg117Cys mutation sequencing platform that predicts the most probable variant carriers was studied in order to evaluate the involvement of causing the pathology and provides suggestions for translat- constitutional cell cycle arrest in tumor progression. On ing the data to the clinic. The Watson for Genomics pipeline average, 61% of the somatic variants found in these patients revealed 15 variants of unknown significance, 2 alterations were C:G>T:A changes (Tables S1 and S2). At this time we without proposed therapies, and only 1 actionable alteration cannot rule out a correlation between this observed bias and for patient T1 (Table S4). Three variants of unknown a specific mutation signature in these tumors. Megquier significance, 3 alterations with no proposed therapies, and et al29 have established an association with somatic deam- another 3 actionable alterations were described for patient T4 ination of cytosine to thymine in a large series of angiosar- (Table S5). Interestingly, a not previously detected copy comas (different from cardiac), which is in agreement with number gain was found for the KIT gene in the tumor tissue of our observation. Somatic mutations in the VEGF-angiogen- patient T1. The gained region is involved in tumor cell esis pathway were found in tumor tissue of all individuals proliferation, angiogenesis, and metastatic disease. Only 1 (Figure 4). Interestingly, the F1 and F3 individuals (CAS) also actionable pathway was found in common for both CAS had a somatic mutation in the KDR gene, which is involved in patients. The variants found in the TP53 gene were highlighted VEGF/PLCG1 activation and was described altered in 2 as actionable alterations, as previously found in the WES recently studied sporadic CAS cases.16,17 In the F2 patient study (TP53 p.Arg175His and TP53 p.Val143Met for patients the recurrent somatic mutations PIK3CA p.Arg88Gln and T1 and T4, respectively). No Food and Drug Administration– BRAF p.Val600Glu were found in the breast AS and the approved therapies for angiosarcoma were recommended for papillary thyroid tumor, respectively. Twenty-five percent of these 2 patients, but 3 therapies with clinical trials were all breast cancers have somatic mutations in the PIK3CA observed in common for both patients: AZD1775 (NCT gene. Specifically, the PIK3CA p.Arg88Gln mutation was numbers NCT02576444 and NCT018227384), Transferrin described in a primary breast cancer.26 The somatic Receptor-Targeted Liposomal p53 (NCT02354547), and Mod- mutation BRAF p.Val600Glu is the most common genetic ified Vaccinia Virus Ankara Vaccine (NCT02432963). The 3 change in papillary thyroid cancers (35.8%).30 In addition, the DOI: 10.1161/JAHA.119.012875 Journal of the American Heart Association 8 Genetic Landscape of Cardiac Angiosarcomas Calvete et al ORIGINAL RESEARCH VEGFA KDR FGFR/EGFR/PDGFR A B (VEGFR2) POT1 SRC Telomere Damage PI3K SOS PLCG1 NRG2 AKT MYC RAS ITGAV FOXO IQGAP p27 RAF C TP53 p21 P-S6 mTOR ATM MAPK BRAF CDK1/2 ATR ERK P-ERK RICTOR CHK1/2 cdc25 Proliferaon Protein synthesis Cell cycle arrest & Senescence Figure 3. VEGF (vascular endothelial growth factor)-angiogenesis signaling and cell cycle regulation pathways. VEGF signaling is a growth factor pathway to stimulate vasculogenesis and angiogenesis. A, MAPK/ERK signaling regulates cell proliferation. B, AKT/PI3K signaling is related to protein synthesis and cell cycle signaling. Locations of anti–P-ERK and anti–P-S6 used in IHC studies are also shown in the pathway (white arrows). C, Damage-signaling pathway. AKT/PI3K signaling inhibits apoptosis, senescence, and cell cycle arrest through inhibition of FOXO, which in turn positively regulates p21 and p27 activity. BRAF p.Val600Glu and PIK3CA p.Arg88Gln mutations have Sporadic Cardiac Angiosarcomas Without POT1 been described as activators of the angiogenic response.31,32 Mutations The somatic mutation BRAF p.Val600Glu has also been reported in gliomas33 and melanomas,34 where constitutional Although in 4 of the 10 patients studied no discrimination mutations in the POT1 gene were also described.10-12 In between germline and somatic mutations could be made, summary, individuals with nonfunctional POT1 did not mutations in the pathways that were affected by the mutation repress damage signaling (ATR) and showed a constitutive in the POT1 gene at both the constitutional and the somatic increase of p21 and p27 expression, which is in agreement level were also found in these patients. with cell cycle arrest. In addition, somatic activating Studied CAS individuals without constitutional mutations in mutations in the angiogenesis pathway were acquired. the POT1 gene presented mutations in genes that were Interestingly, activating somatic mutations in the KDR gene involved in damage response signaling (ATM-ATR-TP53) were found in both studied patients affected with CAS and (Figure 4). IHC studies with anti-p21 and anti-p27 antibodies carrying a POT1 germline mutation (F1 and F3). This was confirmed the damage response activation in all sporadic CAS also observed in other tissues and tumors different from individuals (Table 1 and Figure 2). Therefore, our results cardiac angiosarcomas with mutations in the POT1 gene suggest a relation between telomere instability (familial CAS) (thyroid and breast from patient F2), demonstrating that not and altered damage signaling (sporadic CAS) on 1 hand, and only in cardiac tissue is there a strong correlation between increased cell cycle arrest leading to the cessation of cell constitutional senescence mediated by POT1 malfunction division on the other. Our observation is in agreement with and the acquisition of somatic mutations in the angiogen- previous studies in which overexpression of TP53 was esis pathway. detected by immunohistochemistry in 49% of angiosarcoma DOI: 10.1161/JAHA.119.012875 Journal of the American Heart Association 9 Genetic Landscape of Cardiac Angiosarcomas Calvete et al ORIGINAL RESEARCH Sporadic CAS N/T CAS T CAS Telomere POT1 instability ATR & BRCA1 Damage BRCA2 response ATM RAD51B RAD51C TP53 TP53RK TB53BP1 TB53BP2 APC2 CDK8 BRIP1 VEGF- KDR angiogenesis VEGFA FGF22 DOCK1 VAV3 SPTAN1 SRC SOS1 RASA2/4 RASGEF1A NRAS RASGEF1A ITGAV IQGAP1 MAP2K1 MAP2K3 PPP2R5E PTPRQ BRAF V600E PDPK1 PLCG1 PRKCQ NRG2 PIK3C2G PI3K RICTOR SHOX2 DUSP8 confirmed constitutional mutation confirmed somatic mutation confirmed somatic mutation (less stringent filtering) Mutation (only tumor tissue studied) Mutation (only tumor tissue studied) (less stringent filtering) DOI: 10.1161/JAHA.119.012875 Journal of the American Heart Association 10 Genetic Landscape of Cardiac Angiosarcomas Calvete et al ORIGINAL RESEARCH Figure 4. Genes with mutations in the studied CAS individuals. Genes are distributed considering the gene ontology among telomere instability and damage response or VEGF (vascular endothelial growth factor)-angiogenesis signaling pathways. Exomes from normal/tumor tissues of 3 patients (F1, F2, and F3) carrying the constitutional POT1 p.Arg117Cys mutation (orange) and 3 sporadic CAS individuals not carrying mutations in the POT1 gene (NT1, NT2, and NT3) were sequenced. Constitutional mutations refer to variants found in blood samples and found in common in both N and T tissues (red). Mutations found only in tumor tissue were considered somatic (purple). Only tumor tissue from another 4 sporadic CAS individuals (T1 to T4) was also sequenced. A distinction between first event and somatic variants could not be made for the mutations found in these individuals (blue). Less stringent frequency filtering corresponds to a minor allele frequency <0.05 (light purple) instead of <0.01 (light blue). AS indicates angiosarcoma; CAS, cardiac angiosarcoma; N, normal tissue; P. thyroid, papillary thyroid tumor; T, tumor tissue. patients.19 Somatic activating mutations in the VEGF- bypass the proliferation defect imposed by POT1 inhibition.36 angiogenesis pathway were also found in tumor tissue of all Therefore, under senescence conditions, activating somatic sporadic CAS individuals (Figures 2 and 4). Similar studies mutations in the angiogenesis pathway would acquire an performed in angiosarcomas also uncovered somatic driver important role to replenish the depleted tissue. However, the mutations in the VEGF/PLCG1 angiogenesis pathway.18,20,21 mutations found in the angiogenesis pathway would con- tribute to angiosarcoma formation and progression. Constitutional Cell Cycle Arrest May Fuel the Acquisition of Somatic Mutations in the Clinical Significance of the Identified Mechanism Angiogenesis Pathway in Angiosarcomas Activation of the VEGF-angiogenesis pathway was found in all Activation of damage signaling in both familial (POT1 muta- studied individuals with AS (familial and sporadic). However, tions carriers) and sporadic angiosarcomas (ATM-ATR-TP53 our IHC studies revealed 2 different staining patterns that mutation carriers) induced constitutional senescence and correlated with the location of the somatic mutations reduced cell division (Figure 2). Our results strongly suggest a (Figure 2). Tumors with mutations in the AKT-PI3K signaling correlation between constitutional cell cycle arrest and the pathway (F1, F2, F3, and T1) were only positively stained with acquired somatic mutations in the VEGF-angiogenesis path- the anti-P-S6 antibody (Table 1 and Figure 4). The individuals way that drive angiosarcoma formation. Moreover, increased with mutations only in the MAPK/ERK signaling pathway cell cycle arrest due to POT1 malfunction also uncovered (NT1, NT2 and the papillary thyroid tumor of F2) or in both somatic angiogenesis activation in tumors other than cardiac molecular signaling pathways (T3 and T4) were positively angiosarcomas. Damage response signaling and the VEGF- stained with both antibodies (Table 1). Therefore, different signaling pathway are mutually regulated (Figure 3). Recently, somatic alterations regarding increased angiogenesis may telomere biology and the PI3K pathway (angiogenesis) were occur in response to senescence. These results give impor- also shown to be functionally connected, and phosphorylation tant clues regarding the diagnosis and classification of activity of the PI3K/AKT pathway was demonstrated to affect angiosarcomas. telomere stability in vitro.35 In vitro studies with stem cells Our results also have an important clinical relevance from Pot1a knockout mice with increased telomere dysfunc- regarding treatment and translational research. Inhibition of tion also suggested a correlation with increased prolifera- angiogenesis may be useful to stop tumor progression.37 tion.36 Our results indicate that the observed cell cycle However, angiogenesis inhibition would mitigate the effect of deregulation may interfere with apoptosis. This bypass of the driving somatic mutations but would not revert cell cycle apoptosis would permit the acquisition of somatic mutations. arrest or the suggested bypass of apoptosis and would In addition, cells carrying somatic mutations in genes involved therefore not curtail the acquisition of new somatic mutations. in attenuating cell cycle arrest, which depletes progenitor Treatment with PI3K inhibitors of patient-derived xenografts stem cells in nontumor tissues, may undergo positive also showed increased telomeric DNA damage.35 Our results selection. A bypass of apoptosis was also suggested in suggest that further studies regarding ATM/ATR, damage studies in which POT1 was inactivated in vitro, and induced signaling and cell cycle inhibition activity might lead to genomic instability enabled cancer cells to acquire additional recovering cell cycle control and preventing the acquisition of mutations and conferred aggressive behavior. Attenuation of somatic mutations, including in asymptomatic patients carry- the damage response was suggested to allow tumor cells to ing POT1 mutations. Regarding this issue, the second DOI: 10.1161/JAHA.119.012875 Journal of the American Heart Association 11 Genetic Landscape of Cardiac Angiosarcomas Calvete et al ORIGINAL RESEARCH sequencing experiment with the IBM Watson platform also Sources of Funding pointed to the damage-signaling pathway as a therapeutic Benitez’s laboratory is partially funded by Centro de Investi- target. Actionable variants in the TP53 gene were highlighted gacion (CIBERER), Horizon2020 BRIDGES project, and by the in both studied CAS individuals (T1 and T4). Therefore, Spanish Ministry of Health supported by Federaci on Espa~ nola although somatic driver variants were found to occur in the de Enfermedades Raras (FEDER) funds (PI16/00440). Garcia- angiogenesis pathway, only damage response signaling was Pavia’s group is partially supported by the Instituto de Salud found actionable for both studied angiosarcomas (Tables S4 Carlos III (ISCIII) (grants CB16/11/00432 and PI14/0967) and S5). and by the Spanish Ministry of Economy and Competitiveness In summary, our current results demonstrate that inhibi- (grant SAF2015-71863-REDT). Garcia-Pavia’s and Crespo- tion of POT1 gene function and damage response malfunc- Leiro’s groups are supported by FEDER funds. Urioste’s tion both activate ATR-dependent DNA damage signaling, laboratory is funded by Spanish Ministry of Health supported which increases cell cycle arrest that would diminish cell by FEDER fund (PI14/00459). proliferation in constitutional tissue, and that triggers somatic activating mutations in the angiogenesis pathway in angiosarcomas. 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Cell Rep. 2016;15:2170–2184. the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association 37. Maj E, Papiernik D, Wietrzyk J. Antiangiogenic cancer treatment: the great for Molecular Pathology. Genet Med. 2015;17:405–424. discovery and greater complexity (review). Int J Oncol. 2016;49:1773–1784. DOI: 10.1161/JAHA.119.012875 Journal of the American Heart Association 13 SUPPLEMENTAL MATERIAL Table S1. Filtered somatic variants found in the WES of POT1 p.Arg117Cys carriers, one CAS and one breast AS with papillary thyroid cancer. Genes previously shown to be involved in the corresponding pathology are highlighted in grey. ALT allele Amino acid Pathology Chr Gene Position REF ALT dbSNP Cosmic GMAF ExAC fraction change CAS 1 TMEM52 1850627 CAGCGGCAGG C 0.224 L26del COSM1167507 - - 1 RCC1 28858834 G A 0.184 G169Asp - - - 0.432 rs20027726 1 CELSR2 109792751 T C L17P COSM1200708 - - 5 1 NOTCH2 120612234 G C 0.234 - - - - 0.232 rs20120485 1 NOTCH2NL 145248838 A G - - - 4 1 ARNT 150849103 C A 0.165 - rs10305649 0.0289 0.05 1 LOR 153233990 T G 0.212 Y189D - - - 2 NRXN1 50765449 C T 0.346 W735* - - - 0.432 COSM1632174, 2 ANKRD36 97820478 G A c.1260+3D rs79756591 - - COSM1632175 2 RABL2A 114386168 C T 0.218 - - - - 2 HS6ST1 129075877 G T 0.214 D87E - - - 0.168 rs20224738 2 HS6ST1 129075939 T A K67* COSM1129578 - - 7 2 HOXD11 176972061 G C 0.344 - - - - TCAGCAGCCT 0.188 2 SPATA3 231861032 AGCCCTGAAT T Q30_Q38del rs72362780 COSM1406147 - - CCACACCA 2 NGEF 233748132 C T 0.234 R549H - - - 3 FANCD2 10088266 G T 0.434 c.1137G>T+3D rs72492997 - - 4 KDR 55968550 CATT C 0.424 N704del - - - 4 ALB 74280882 G A 0.546 V397M - - - TGATA 0.288 4 DSPP 88537069 T S1085_insDSS - - - GCAGC 4 CENPE 104119549 G C 0.212 - - - - 4 SYNPO2 119951749 C T 0.188 R607* - - - 4 CBR4 169931326 CA C 0.434 - rs67305871 - - 6 ATAT1 30595639 G C 0.465 G79A - - - 6 HLA-DRB5 32497905 G A 0.365 R33* rs71549219 - - 6 HBS1L 135290447 T G 0.345 E609D - - - GGCTTCAGCT 0.208 COSM1329481, ACCGCTTGCA 7 ZAN 100385561 G F2344SfsTer10 rs72364644 COSM1329482, - - AGGCCGCATG COSM1329483 ACCTAT 7 TRBV5-5 142148969 A T 0.498 L101* - - - 10 ARMC4 28250610 C A 0.370 D425Y - - - 10 FGFR2 123325217 C T 0.430 c.111-1 - - - 0.708 rs20098033 COSM1603961, 11 MUC6 1030228 A C C334G - - 0 COSM1603962 11 CLCF1 67141148 AT A 0.278 - - - - 11 PDE2A 72301235 CGT C 0.234 Y252SfsTer74 - - - 12 DDX11 31237978 C T 0.188 R186W rs74087925 - - 12 PRKAG1 49397570 C A 0.234 V234F - - - 0.436 COSM144609, 12 KMT2D 49434325 G A R2410* - - COSM1299436 12 KMT2D 49444088 GGA G 0.298 L1094PfsTer20 - - - 14 ZFP36L1 69256806 T G 0.436 K154T - - - 16 TOX3 52484238 G A 0.186 Q31L - - - 16 PSMB10 67970188 G C 0.688 R58G COSM139019 - - 17 SHPK 3524660 CGGCGAT C 0.444 Il30_Al31del - - - 19 RHPN2 33490566 T C 0.212 Q384R - - - 19 RHPN2 33490585 G A 0.388 Q378* rs78615454 COSM1318333 - - 0.268 rs20037519 22 POTEH 16287784 C T W34* 0.006 - 0 22 SRRD 26879946 GGAGGCGGC G 0.160 Al33_A39del rs66831137 - - GCCCCGGGGG AGA 0.426 rs11441358 X SLC25A5 118604444 G C R236P - - 2 0.348 rs12191328 Breast AS 3 PIK3CA 178916876 G A R88Q 7 COSM746 - 4 TRIM2 154217059 G A 0.656 V461M - <0.0001 5 EIF4E1B 176070736 G A 0.655 - <0.0001 10 HPSE2 100503738 C T 0.258 R229H - 0.00473 0.288 - <0.0001 11 TENM4 78516508 G A R670W 11 CBL 119148966 T G 0.212 C396G COSM34074 - 12 VWF 6103147 T C 0.214 Y2160C - <0.0001 13 PARP4 25021323 A G 0.344 I1039T rs73172125 COSM147647 - 0.347 13 COL4A2 111160303 C T 0.256 A1539V - <0.0001 15 CSPG4 75982085 C T 0.156 E441K rs79463888 COSM1317754 - 0.15 20 BPIFB4 31680303 C A 0.128 P395T - 20 PLCG1 39795452 C G 0.434 L752V - 0.344 rs14756052 20 WFDC8 44180670 G A R241C 2 COSM1027261 - <0.0001 Papillary 3 RASSF1 50369082 C T 0.212 R227H - <0.0001 Thyroid 6 MDGA1 37606032 C T 0.234 V909I COSM1600157 - <0.0001 6 ARID1B 157528658 G A 0.168 R2168Q - <0.0001 0.324 rs11348802 7 BRAF 140453136 A T V600E 2 COSM476 - <0.0001 17 PIK3R6 8741894 C T 0.254 R59K Table S2. Filtered variants found in the WES of F3 patient affected with CAS (POT1 p.R117C carrier). Genomic Chr Gene Symbol Position REF ALT Fraction HGVSp Existing Variation GMAF chr1 KYAT3 88949093 A T 0.128 ENSP00000260508.4:p.Leu380Gln rs144984854 0.0006 chr2 ANKRD36 97211741 G A 0.436 ENSP00000391950.2:p.Val1157Met rs10194525 chr6 HLA-DRB1 32580249 G C 0.310 ENSP00000353099.5:p.Thr262Arg chr10 EEF1AKM 124774782 C T 0.168 ENSP00000357829.2:p.Ala98Thr rs199727427 0.0006 chr13 PARP4 24447185 A G 0.304 ENSP00000371419.3:p.Ile1039Thr chr17 MYH13 10306461 G A 0.344 ENSP00000404570.3:p.Arg1822Trp rs116935297 0.007 chr17 MAP2K3 21300875 G T 0.456 ENSP00000345083.4:p.Arg94Leu rs56067280 chr18 ANKRD30B 14843024 C G 0.478 ENSP00000351875.4:p.Pro918Ala rs180690700 chr19 COLGALT1 17560463 C G 0.268 ENSP00000252599.3:p.Leu163Val rs764429704 chr17 63491216 C T 0.675 ENSP00000464149.1:p.Thr342Met rs3730043 0.0034 chr1 VPS13D 12249234 A C 0.677 ENSP00000478104.1:p.Gln153His rs116415833 0.0018 chr1 FAM131C 16058547 G A 0.104 ENSP00000364814.4:p.Arg245Trp rs77667563 chr1 CROCC 16940041 G C 0.126 ENSP00000364691.4:p.Asp586His rs9435714 chr1 KDF1 26952098 A G 0.456 ENSP00000319179.5:p.Cys95Arg rs148853297 chr1 PPCS 42456962 A T 0.546 ENSP00000361642.3:p.Arg133Trp rs199807362 chr1 IPP 45716925 G A 0.344 ENSP00000379739.3:p.Arg427Cys rs142095376 0.0002 chr1 PODN 53078994 G A 0.778 ENSP00000308315.5:p.Arg543His rs61999355 0.003 chr1 FAM151A 54619889 C G 0.128 ENSP00000306888.2:p.Lys79Asn rs114883650 0.0144 chr1 RBMXL1 88983615 C G 0.376 ENSP00000446099.1:p.Gly71Ala rs111779380 chr1 TSPAN2 115072962 C G 0.201 ENSP00000358529.2:p.Gly39Arg rs147800870 0.0004 chr1 PDE4DIP 149005208 C T 0.567 ENSP00000358363.4:p.Arg1396Trp rs2798901 chr1 FLG2 152351277 G A 0.234 ENSP00000373370.4:p.Ser2170Phe rs201200591,COSM896166 chr1 CRNN 152409620 A G 0.198 ENSP00000271835.3:p.Ser488Pro chr1 GPA33 167068982 C A 0.304 ENSP00000356842.3:p.Val119Phe rs72689400 0.0058 chr1 KIF14 200592110 T G 0.567 ENSP00000356319.4:p.Lys928Thr rs150766596 chr1 PKP1 201322032 G A 0.436 ENSP00000263946.3:p.Glu489Lys rs748085816 rs142615706,COSM5042798,COSM50 42797,COSM5042796,COSM3360727, chr1 OBSCN 228350867 C G 0.128 ENSP00000455507.2:p.Arg7072Gly COSM3360726,COSM3360725 0.0012 chr1 RHOU 228743354 G A 0.676 ENSP00000355652.3:p.Val131Met chr1 EDARADD 236482309 C T 0.398 ENSP00000335076.4:p.Ser103Phe rs114632254,CM114961 0.013 chr1 OR2T3 248473745 G A 0.298 ENSP00000352604.2:p.Cys132Tyr rs148025314 chr1 OR2T34 248573866 G A 0.126 ENSP00000330904.2:p.Arg298Cys rs148590921 chr1 OR2T27 248650526 T C 1.000 ENSP00000342008.3:p.Tyr120Cys chr2 MBOAT2 8873294 C T 0.107 ENSP00000302177.3:p.Val233Ile rs34573615 0.0142 chr2 47087059 C A 0.172 ENSP00000408527.2:p.Met232Ile rs184422124 0.0008 chr2 ASB3 53765485 G C 0.243 ENSP00000263634.2:p.Leu30Val rs36020289 0.0056 chr2 TSPYL6 54255416 G A 0.778 ENSP00000417919.2:p.Arg246Cys rs17189743 0.0204 chr2 USP34 61278194 G A 0.166 ENSP00000381577.2:p.Pro1802Ser chr2 ANKRD36C 95944662 T A 0.214 ENSP00000403302.1:p.Asn486Tyr rs77220524 chr2 ASTL 96132701 C T 0.463 ENSP00000343674.2:p.Arg159His rs61735195 0.0066 chr2 NIFK 121732168 C T 0.184 ENSP00000285814.4:p.Glu94Lys rs751855520 chr2 LCT 135817693 T A 0.564 ENSP00000264162.2:p.Lys452Met chr2 LRP1B 140868259 G A 0.804 ENSP00000374135.3:p.Leu1392Phe rs200955532,COSM1007053 chr2 NMI 151275501 G A 0.756 ENSP00000243346.5:p.Thr206Met rs199573903 0.0002 chr2 SLC39A10 195716880 A G 0.278 ENSP00000386766.3:p.His647Arg rs755523552 chr2 TNS1 217882350 A C 0.674 ENSP00000171887.4:p.Ile311Met rs11680854 0.0104 chr2 ESPNL 238131340 A G 0.201 ENSP00000339115.3:p.Lys876Glu rs149541007 0.0062 chr3 FANCD2 10065867 G C 0.436 ENSP00000287647.3:p.Cys758Ser rs540805431 0.0002 chr3 TTC21A 39138609 G A 0.678 ENSP00000398211.2:p.Glu1291Lys rs80238762 0.0058 chr3 DNAH1 52360039 G A 0.322 ENSP00000401514.2:p.Val1511Met rs61734638 0.0084 chr3 ZNF717 75737092 C T 0.128 ENSP00000419377.1:p.Cys794Tyr rs139633377 chr3 ZNF717 75737101 G T 0.201 ENSP00000419377.1:p.Pro791His rs79138891 chr3 ZNF717 75737127 A C 0.128 ENSP00000419377.1:p.His782Gln rs79811623 chr3 ZNF717 75737622 T A 0.344 ENSP00000419377.1:p.Arg617Ser rs76175438 chr3 ZNF717 75737840 C T 0.166 ENSP00000419377.1:p.Glu545Lys rs76496075 chr3 ZNF717 75738575 G A 0.236 ENSP00000419377.1:p.Arg300Cys rs1962893 chr3 OR5K1 98469710 T C 0.768 ENSP00000373193.2:p.Leu45Ser rs200654905 0.003 chr3 BBX 107716685 C T 0.924 ENSP00000319974.8:p.Arg81Trp rs142400819 0.003 chr3 ARHGEF26 154225981 C G 0.440 ENSP00000348828.4:p.Asn687Lys chr3 GPR149 154428985 G C 0.778 ENSP00000374390.2:p.Pro211Ala rs774089990 chr3 MUC20 195726080 G C 0.234 ENSP00000414350.2:p.Asp493His chr3 MUC20 195729690 C G 0.128 ENSP00000414350.2:p.Ser671Cys chr3 MUC4 195762138 C A 1.000 ENSP00000417498.3:p.Ala4821Ser CM066583 chr3 MUC4 195789859 G A 0.184 ENSP00000417498.3:p.Thr574Ile chr4 IDUA 987896 C G 0.134 ENSP00000247933.4:p.His82Gln rs148775298 0.0012 chr4 HTT 3207344 G A 0.310 ENSP00000347184.5:p.Gly2047Arg rs181217572 0.0004 chr4 TLR1 38796634 G A 0.780 ENSP00000354932.2:p.Pro733Leu rs5743621 0.0126 chr4 KDR 55098758 G C 0.654 ENSP00000263923.4:p.Thr771Arg COSM94566,COSM4381082 chr4 UGT2B15 68670105 T G 0.388 ENSP00000341045.5:p.Ser172Arg rs200638397 0.0002 chr4 RUFY3 70806627 C G 0.124 ENSP00000370394.3:p.Leu611Val rs754675264 chr4 CCSER1 90308466 G C 0.432 ENSP00000425040.1:p.Ser61Thr rs184253075 0.0008 chr4 PDLIM5 94455353 A G 0.567 ENSP00000480359.1:p.Lys22Arg rs75841704 0.0078 chr4 UNC5C 95170263 C T 0.128 ENSP00000406022.1:p.Ala841Thr rs34585936 0.0184 chr4 QRFPR 121380442 A C 0.214 ENSP00000377948.2:p.Val69Gly rs34270076 0.0076 chr4 CBR4 169002197 C A 0.678 ENSP00000303525.3:p.Val137Phe chr4 CBR4 169002200 T A 0.455 ENSP00000303525.3:p.Ile136Phe chr4 TLR3 186084674 C A 0.134 ENSP00000296795.2:p.Ala839Asp rs201540925,COSM4584973 0.0002 chr5 SLC9A3 488354 C T 0.436 ENSP00000264938.3:p.Val213Ile rs143027124 chr5 C5orf49 7831902 T G 0.166 ENSP00000382708.2:p.Asn131Thr chr5 ADGRV1 90705420 G A 0.124 ENSP00000384582.2:p.Ala2803Thr rs111033530 0.0048 chr5 MARCH3 126878360 C T 0.356 ENSP00000309141.5:p.Arg143Gln rs138413676 chr5 LECT2 135951324 C T 0.304 ENSP00000274507.1:p.Gly63Glu rs149560293 0.0068 chr5 PCDHGA6 141374360 G A 0.256 ENSP00000429601.1:p.Glu93Lys rs200765071 chr5 PCDH12 141945665 C T 0.732 ENSP00000231484.3:p.Gly1091Ser rs779814208 chr5 SCGB3A2 147881560 G A 0.652 ENSP00000296694.4:p.Gly57Asp rs140599638 0.0004 chr5 SLC36A2 151343594 C A 0.224 ENSP00000334223.4:p.Gly87Val rs77010315,CM086960 0.005 chr5 GFPT2 180301596 T C 0.364 ENSP00000253778.8:p.Arg673Gly chr6 SOX4 21594616 C G 0.578 ENSP00000244745.1:p.Leu28Val rs140231408,COSM3697699 0.0066 chr6 BTN2A2 26392401 G A 0.576 ENSP00000349143.4:p.Ala336Thr rs142803339,COSM3745097 0.0092 CD994645,COSM5446135,COSM5446134,COSM459 chr6 HLA-A 29944253 G C 0.168 ENSP00000379873.1:p.Asp251His 3407 chr6 PPP1R10 30609877 C G 0.222 ENSP00000365694.2:p.Gly23Ala chr6 HLA-DRB1 32580779 C T 0.128 ENSP00000353099.5:p.Ala244Thr rs3830125 chr6 HLA-DRB1 32589669 G C 0.201 ENSP00000353099.5:p.Pro25Arg rs148093782 chr6 HLA-DQB2 32757848 T C 0.344 ENSP00000396330.2:p.Ser228Gly rs9276572 chr6 PPIL1 36871822 C G 0.134 ENSP00000362803.5:p.Cys36Ser rs12194408 0.0098 chr6 PTK7 43130655 G A 0.276 ENSP00000418754.1:p.Arg277His rs78949718 0.0028 chr6 POLR1C 43520104 C T 0.128 ENSP00000361465.3:p.Arg141Cys rs148385032 0.0014 chr6 DST 56552557 C T 0.128 ENSP00000307959.7:p.Gly3155Glu rs775546048 chr6 TTK 80035318 C T 0.166 ENSP00000358813.2:p.Leu609Phe rs540538876 0.0002 chr6 PNRC1 89081009 C T 0.124 ENSP00000336931.3:p.Pro39Ser rs2231267 0.0056 chr6 ARMC2 108953073 C T 0.278 ENSP00000376417.4:p.Thr546Met rs61741720 0.0132 chr6 VNN1 132683243 T C 0.128 ENSP00000356905.4:p.Tyr480Cys rs567530094 0.0002 chr6 SHPRH 145922832 G A 0.243 ENSP00000356475.2:p.Arg1184Cys rs148401398 0.0014 chr7 AMZ1 2709683 G T 0.376 ENSP00000308149.4:p.Cys272Phe rs148137749 0.0006 chr7 SDK1 3962743 G A 0.455 ENSP00000385899.2:p.Gly441Arg rs139796765 0.0058 chr7 TNRC18 5309293 C T 0.677 ENSP00000395538.1:p.Gly2822Arg COSM3881739,COSM3881740 chr7 PSPH 56021118 T C 0.295 ENSP00000378854.3:p.Asp32Gly chr7 GATAD1 92456449 C T 0.184 ENSP00000287957.3:p.Arg233Trp rs34768413 0.007 chr7 FBXO24 100590263 A C 0.128 ENSP00000416558.2:p.Glu114Asp rs545357401 0.0002 chr7 POT1 124863547 G A 0.457 ENSP00000350249.3:p.Arg117Cys rs780936436 chr7 CPA2 130289628 C A 0.335 ENSP00000222481.4:p.Leu381Met rs146796996 0.0012 chr7 TRBV20-1 142627134 A T 0.310 ENSP00000374917.3:p.Arg24Trp chr7 PRSS1 142750558 C G 0.436 ENSP00000308720.7:p.Ala15Gly rs200665515,COSM3723223 chr7 PRSS1 142750660 G T 0.567 ENSP00000308720.7:p.Gly49Val rs138464021 chr7 PRSS1 142750672 T A 0.668 ENSP00000308720.7:p.Ile53Asn rs149246646 chr7 PRSS1 142752506 G T 0.574 ENSP00000308720.7:p.Gly177Val chr7 SSPO 149802064 C T 0.256 ENSP00000485256.1:p.Ser2508Phe rs140977594 0.0056 chr7 KMT2C 152247922 C T 0.446 ENSP00000262189.6:p.Gly838Ser COSM4591270 chr7 KMT2C 152265180 C T 0.675 ENSP00000262189.6:p.Asp348Asn rs201834857,COSM228110 chr7 VIPR2 159031954 C T 0.788 ENSP00000262178.2:p.Cys362Tyr rs147214125 0.0004 chr8 SGCZ 14108196 G A 0.476 ENSP00000371512.1:p.Pro196Leu rs145213189 0.0004 chr8 PDGFRL 17642653 T A 0.324 ENSP00000444211.1:p.Ile327Asn rs35346456 0.0022 chr8 MTUS1 17653257 C G 0.222 ENSP00000262102.6:p.Glu1105Gln rs61733705 0.0066 chr8 MTUS1 17684434 T G 0.166 ENSP00000262102.6:p.Lys911Thr rs61748836 0.0046 chr8 CYP7A1 58497114 A G 0.436 ENSP00000301645.3:p.Leu133Pro chr8 CSPP1 67116017 C G 0.657 ENSP00000262210.5:p.Ser459Cys rs146431326 0.0012 chr8 NBN 89971232 G A 0.436 ENSP00000265433.3:p.Arg215Trp rs34767364,CM044022 0.001 chr8 PABPC1 100709584 G A 0.566 ENSP00000313007.5:p.Arg374Cys rs200409148 chr8 PABPC1 100709589 T C 0.534 ENSP00000313007.5:p.Glu372Gly rs201076736 chr8 PABPC1 100709611 C A 0.128 ENSP00000313007.5:p.Val365Leu rs202074479 chr8 PABPC1 100717815 G C 0.312 ENSP00000313007.5:p.Ala154Gly rs113614781 chr8 DEPTOR 119965343 G C 0.122 ENSP00000286234.5:p.Glu179Asp rs766410544 rs184113048,COSM1196723,COSM11 chr8 PHF20L1 132832298 G A 0.376 ENSP00000378784.2:p.Arg603Gln 96724 0.003 rs149221350,COSM3906436,COSM39 chr9 ADAMTSL 18684733 G T 0.184 ENSP00000369921.4:p.Ala503Ser 06437 0.0012 chr9 LINGO2 27949346 A C 0.345 ENSP00000369328.1:p.Ile442Met chr9 SHC3 89042122 G A 0.304 ENSP00000364995.4:p.Pro422Ser rs771704230,COSM24411 chr9 OR13C5 104599179 G A 0.431 ENSP00000363911.2:p.Pro79Ser rs7025570,COSM4163183 chr9 TRIM32 116698344 G A 0.128 ENSP00000408292.1:p.Arg201His chr9 LAMC3 131072789 C T 0.655 ENSP00000354360.4:p.Ser1124Phe rs113259170 0.0032 chr9 GBGT1 133153893 C T 0.756 ENSP00000361110.3:p.Arg243His rs143563851,COSM4163479 0.001 chr10 CUBN 16890385 G A 0.678 ENSP00000367064.4:p.Ala2914Val rs45551835,COSM32707 0.0064 chr10 NPY4R 46462341 C A 0.234 ENSP00000363431.1:p.Ala99Ser chr10 GPRIN2 46549695 C A 0.112 ENSP00000363436.1:p.Val348Leu rs4926046 chr10 GPRIN2 46550133 C A 0.324 ENSP00000363436.1:p.Gly202Trp rs11204658 chr10 GPRIN2 46550598 C T 0.436 ENSP00000363436.1:p.Val47Met chr10 GPRIN2 46550622 G C 0.234 ENSP00000363436.1:p.Leu39Val chr10 LHPP 124517198 G A 0.536 ENSP00000357835.5:p.Asp215Asn rs140823928 0.0038 chr10 CTBP2 124994530 G A 0.598 ENSP00000311825.6:p.Ser780Phe rs374871777,COSM2021369,COSM2021368 chr10 CTBP2 124994542 C T 0.504 ENSP00000311825.6:p.Ser776Asn rs78155918 chr10 CTBP2 124994563 A T 0.544 ENSP00000311825.6:p.Leu769Gln rs150320719 chr10 CTBP2 124994577 C A 0.657 ENSP00000311825.6:p.Gln764His rs80025996,COSM5764564,COSM5764563 chr10 CTBP2 125002983 T G 0.346 ENSP00000311825.6:p.Asp652Ala rs796433756 chr10 CTBP2 125003006 T C 0.310 ENSP00000311825.6:p.Ile644Met rs112239066 chr10 CTBP2 125003010 C T 0.215 ENSP00000311825.6:p.Arg643Gln rs760489730,COSM5620576,COSM5620575 chr10 CTBP2 125003036 C A 0.166 ENSP00000311825.6:p.Lys634Asn rs201760950,COSM4441587,COSM4441586 chr10 CTBP2 125003065 T A 0.184 ENSP00000311825.6:p.Ile625Phe rs75794788,COSM4144456,COSM4144455 chr10 CTBP2 125003091 G A 0.345 ENSP00000311825.6:p.Ala616Val rs3198935 chr10 CTBP2 125003357 G A 0.128 ENSP00000311825.6:p.Thr605Met rs768573864,COSM2021395,COSM2021394 rs1058301,COSM5763526,COSM5763525,COSM4675 chr10 CTBP2 125003368 G C 0.267 ENSP00000311825.6:p.Asp601Glu 296,COSM4675295 chr10 CTBP2 125003410 C G 0.215 ENSP00000311825.6:p.Glu587Asp rs74705267,COSM4144460,COSM4144459 chr10 CTBP2 125003448 G C 0.166 ENSP00000311825.6:p.Leu575Val rs3198920 chr10 CTBP2 125003450 G A 0.376 ENSP00000311825.6:p.Pro574Leu rs796256730,COSM5620578,COSM5620577 chr10 CTBP2 125003460 G T 0.435 ENSP00000311825.6:p.His571Asn rs796388243 chr10 CTBP2 125003466 G T 0.243 ENSP00000311825.6:p.Pro569Thr chr10 CTBP2 125003468 C A 0.213 ENSP00000311825.6:p.Gly568Val rs368195398 chr10 CTBP2 125003470 G T 0.178 ENSP00000311825.6:p.Asn567Lys rs797010536 chr11 MUC6 1016961 G A 0.166 ENSP00000406861.2:p.Thr1947Ile rs773824783 chr11 MUC6 1017069 G A 0.801 ENSP00000406861.2:p.Thr1911Met rs80333708 chr11 MUC6 1017135 G A 0.726 ENSP00000406861.2:p.Thr1889Ile rs747429892 chr11 MUC6 1017183 G T 0.405 ENSP00000406861.2:p.Pro1873Gln rs34844844 0.0004 chr11 MUC6 1017337 T C 0.435 ENSP00000406861.2:p.Thr1822Ala rs76686156 chr11 MUC6 1018042 A G 0.235 ENSP00000406861.2:p.Ser1587Pro rs200364398 chr11 MUC6 1018473 A T 0.387 ENSP00000406861.2:p.Leu1443His rs112923701 chr11 OR51J1 5403408 C T 0.468 ENSP00000332473.1:p.Ser271Phe rs183738872 0.0008 chr11 SAA2-SAA4 18245916 A G 0.234 ENSP00000485552.1:p.Val75Ala rs71469388 chr11 LRP4 46895950 G A 0.435 ENSP00000367888.1:p.Arg373Trp rs118009068 0.0106 chr11 FOLH1 49186715 G A 0.665 ENSP00000256999.2:p.Arg190Trp rs75111588 chr11 OR4C46 54603244 C T 0.657 ENSP00000329056.1:p.Cys252Tyr rs11246608 chr11 OR4C46 54603280 G A 0.879 ENSP00000329056.1:p.Ser240Phe rs11246607 chr11 OR9G1 56700722 A G 0.342 ENSP00000309012.1:p.Tyr112Cys rs4990194 0.0056 chr11 OR9G1 56700892 C T 0.210 ENSP00000309012.1:p.Arg169Cys rs11228733 0.0008 chr11 OR9G1 56701223 T A 0.128 ENSP00000309012.1:p.Val279Glu rs79251113 chr11 ESRRA 64315848 T C 0.184 ENSP00000384851.1:p.Leu385Pro rs201072913 chr11 ESRRA 64315856 C T 0.376 ENSP00000384851.1:p.Leu388Phe rs79204587 chr11 ESRRA 64315859 C T 0.435 ENSP00000384851.1:p.Arg389Cys rs80310817 chr11 ACTN3 66554587 C T 0.231 ENSP00000422007.1:p.Pro217Leu rs370740496 chr11 NUMA1 72014043 G A 0.435 ENSP00000377298.3:p.Arg1154Trp rs61740456 0.001 chr11 USP35 78210302 G A 0.768 ENSP00000431876.1:p.Arg816His rs75370284 0.0128 chr11 TRIM49C 90041372 C A 0.465 ENSP00000388299.1:p.Thr394Asn rs75119043,COSM3998623,COSM3998622 chr11 MAML2 96093510 C T 0.376 ENSP00000434552.1:p.Gly174Asp rs61749254 0.0042 chr11 SORL1 121496918 G A 0.346 ENSP00000260197.6:p.Glu270Lys rs117260922 0.0078 chr11 OR8B2 124382854 G A 0.567 ENSP00000364152.2:p.Leu164Phe rs886202 chr12 WNK1 827047 T A 0.234 ENSP00000341292.5:p.Leu313Gln chr12 FOXM1 2858912 G A 0.128 ENSP00000342307.2:p.Pro711Leu rs28919870 0.0044 chr12 CRACR2A 3696811 A C 0.345 ENSP00000409382.2:p.Asp63Glu rs144314920 0.0004 chr12 KLRC2 10435931 C G 0.376 ENSP00000371327.2:p.Arg19Pro rs75545535 chr12 10435931 C G 0.346 ENSP00000437563.1:p.Arg19Pro rs75545535 chr12 TAS2R19 11021703 A G 0.205 ENSP00000375091.2:p.Phe290Ser chr12 TAS2R19 11021868 A C 0.346 ENSP00000375091.2:p.Leu235Arg rs763119571 chr12 TAS2R31 11030522 G A 0.145 ENSP00000375093.2:p.Pro272Ser rs763119976 chr12 TAS2R31 11031043 A G 0.754 ENSP00000375093.2:p.Leu98Pro rs73049067 chr12 TAS2R31 11031194 G C 0.304 ENSP00000375093.2:p.Leu48Val rs760444623 chr12 TAS2R30 11134103 G C 0.166 ENSP00000444736.1:p.Leu48Val rs113026132 chr12 PTPRO 15503912 C G 0.456 ENSP00000281171.4:p.Asn370Lys rs61754411 0.009 chr12 CCDC65 48918345 A G 0.345 ENSP00000312706.4:p.Gln227Arg rs149640178 0.0004 chr12 TROAP 49331358 G A 0.545 ENSP00000257909.3:p.Arg748Gln rs200487146,COSM4921428 0.0006 rs62638191,CM991095,RISN_RDH5:c chr12 RDH5 55724028 G T 0.787 ENSP00000257895.5:p.Gly238Trp .712G>T 0.0004 chr12 MDM1 68332236 G C 0.456 ENSP00000302537.7:p.Arg4Gly rs199537908 chr12 LRRIQ1 85153046 A C 0.476 ENSP00000376910.2:p.Asp1481Ala rs191782536 0.0018 chr12 DNAH10 123916480 C A 0.675 ENSP00000489675.1:p.Asp3464Glu rs61745785 0.011 chr13 MPHOSPH 19642121 G T 0.778 ENSP00000355388.4:p.Val74Phe chr13 PARP4 24447125 T C 0.368 ENSP00000371419.3:p.Gln1059Arg rs77269056 chr13 PABPC3 25096638 C T 0.215 ENSP00000281589.3:p.Thr147Ile rs78432860 chr13 PABPC3 25096659 C G 0.567 ENSP00000281589.3:p.Ala154Gly rs75760907 chr13 PABPC3 25096889 A G 0.786 ENSP00000281589.3:p.Lys231Glu rs78826513 chr13 PABPC3 25096951 G T 0.745 ENSP00000281589.3:p.Met251Ile rs75281454 chr13 PABPC3 25097154 C T 0.564 ENSP00000281589.3:p.Thr319Ile rs80261016 chr13 PABPC3 25097291 G T 0.678 ENSP00000281589.3:p.Val365Leu rs77466429 chr13 NEK5 52086336 G A 0.124 ENSP00000347767.4:p.Arg474Cys rs35465612 0.0042 chr13 SLC10A2 103049340 G A 0.434 ENSP00000245312.3:p.Pro290Ser rs56398830,CM952446 0.0074 chr14 WDR89 63599649 A T 0.376 ENSP00000378399.2:p.Asp98Glu chr14 WDR89 63599677 C T 0.266 ENSP00000378399.2:p.Cys89Tyr COSM5764373 chr14 WDR89 63599684 A G 0.234 ENSP00000378399.2:p.Ser87Pro chr14 SYNE2 64130105 C A 0.340 ENSP00000350719.3:p.Pro4733Thr rs75568433 0.0028 chr14 VIPAS39 77453359 C T 0.675 ENSP00000452181.1:p.Val46Met rs148360332 0.0006 chr14 IGHV4-31 106349457 G T 0.564 ENSP00000395656.2:p.Pro61Gln rs77489245 chr15 NUTM1 34355873 C A 0.567 ENSP00000444896.1:p.Ser622Tyr chr15 TYRO3 41573327 G T 0.254 ENSP00000263798.3:p.Val669Leu rs62001448 chr15 SPATA5L1 45403247 C G 1.000 ENSP00000305494.6:p.Ser273Cys rs143453038,COSM4509821 0.01 chr15 SPATA5L1 45403421 G T 0.654 ENSP00000305494.6:p.Gly331Val chr15 SNX1 64136360 G A 0.435 ENSP00000261889.5:p.Asp466Asn rs1802376 0.0074 chr15 MYO9A 71968075 T C 0.675 ENSP00000348349.5:p.His632Arg rs552840125 0.0002 chr16 RPL3L 1947003 C T 0.532 ENSP00000268661.7:p.Val262Met rs113956264 0.0212 chr16 ACSM2A 20465673 G A 0.345 ENSP00000459451.1:p.Val112Met chr16 ACSM3 20775918 T C 0.546 ENSP00000289416.5:p.Leu100Pro rs5713 0.004 chr16 OR16-13 33827483 G T 0.180 ENSP00000474814.1:p.Ala59Ser rs4077614 chr16 CES1 55828971 C A 0.184 ENSP00000353720.3:p.Gly19Val rs3826190 chr16 ZNF319 57996752 C A 0.184 ENSP00000299237.2:p.Arg505Leu rs149758697,COSM1191791 0.0002 chr16 AARS 70252728 T A 0.215 ENSP00000261772.7:p.Lys967Met rs35744709 0.0036 chr16 CLEC18B 74409592 C T 0.304 ENSP00000341051.5:p.Asp430Asn chr16 ZNF469 88437795 G C 0.128 ENSP00000402343.1:p.Arg3414Thr rs199528724,COSM1380396 0.0018 chr17 ITGAE 3757758 G A 0.166 ENSP00000263087.4:p.Thr323Met rs71366574 0.0164 chr17 CTC1 8232058 G A 0.678 ENSP00000313759.8:p.Arg744Cys chr17 MYH13 10309647 C G 0.456 ENSP00000404570.3:p.Asp1614His rs35069886 0.0068 chr17 MAP2K3 21300880 C T 0.765 ENSP00000345083.4:p.Arg96Trp chr17 MAP2K3 21304522 C T 0.678 ENSP00000345083.4:p.Thr222Met rs58609466 chr17 KCNJ12 21415757 G A 0.234 ENSP00000463778.1:p.Glu139Lys rs76265595 chr17 KCNJ12 21415775 G A 0.345 ENSP00000463778.1:p.Gly145Ser rs75029097 0.0002 chr17 KCNJ12 21416124 G A 0.212 ENSP00000463778.1:p.Arg261His rs77270326 chr17 KCNJ12 21416127 T G 0.256 ENSP00000463778.1:p.Ile262Ser rs76684759 chr17 KCNJ12 21416474 G A 0.345 ENSP00000463778.1:p.Glu378Lys rs78547883 chr17 SPAG5 28578086 T G 0.348 ENSP00000323300.5:p.Asn1145Thr rs138772502 0.0014 chr17 KRTAP29-1 41302224 A T 0.678 ENSP00000375148.1:p.Cys210Ser chr17 KRT33B 41364890 C T 0.486 ENSP00000251646.3:p.Arg329His chr17 KRT33B 41369498 C T 0.567 ENSP00000251646.3:p.Glu85Lys chr17 ETV4 43528697 G A 0.278 ENSP00000321835.4:p.Ala426Val rs117364926 0.001 chr17 KANSL1 46171833 T G 0.215 ENSP00000387393.3:p.Lys104Thr chr17 EPN3 50536971 C T 0.676 ENSP00000268933.3:p.Arg139Trp rs371737517 0.0002 chr17 ACE 63491216 C T 0.234 ENSP00000290866.4:p.Thr916Met rs3730043 0.0034 chr17 CSH2 63872181 T C 0.178 ENSP00000376623.2:p.Glu200Gly chr18 PIEZO2 10671669 C T 0.134 ENSP00000421377.3:p.Arg2706Gln rs189453524 0.0004 chr18 OSBPL1A 24167436 A G 0.265 ENSP00000320291.3:p.Ser810Pro rs35693789 0.0094 chr19 PODNL1 13934406 C T 0.234 ENSP00000345175.4:p.Val174Met rs781238470 chr19 PALM3 14053817 C T 0.376 ENSP00000344996.3:p.Asp604Asn rs75841596,COSM1390897 0.0116 chr19 WTIP 34482629 C T 0.184 ENSP00000466953.2:p.Arg219Trp chr19 ZNF599 34769537 C T 0.180 ENSP00000333802.6:p.Asp13Asn rs117610843 0.0058 chr19 CYP2A6 40848628 A T 0.256 ENSP00000301141.4:p.Leu160His rs1801272,CM980517 0.0092 chr19 PPP1R37 45144962 C T 0.324 ENSP00000221462.3:p.Leu366Phe rs146723120 0.003 chr19 NOP53 47745650 G C 0.134 ENSP00000246802.4:p.Asp31His rs78530808 0.0158 chr19 NTN5 48664274 A G 0.102 ENSP00000270235.3:p.Ile280Thr rs142533877 0.0056 chr19 LILRB2 54279040 G A 0.304 ENSP00000375629.4:p.Leu243Phe COSM1326184 chr19 LILRA2 54574799 C A 0.356 ENSP00000251377.3:p.Leu141Ile chr19 LILRA2 54575019 A G 0.405 ENSP00000251377.3:p.Glu214Gly chr19 KIR2DL1 54773491 C A 0.546 ENSP00000336769.5:p.His77Asn chr19 KIR2DL1 54773534 C A 0.625 ENSP00000336769.5:p.Thr91Lys COSM5712917,COSM5545196,COSM3404605 chr19 KIR2DL4 54804874 A G 0.645 ENSP00000339634.5:p.Tyr53Cys chr19 KIR3DL1 54819832 G T 0.654 ENSP00000375608.4:p.Gly159Trp chr19 KIR3DL2 54865893 G C 0.564 ENSP00000325525.3:p.Trp363Cys chr20 PYGB 25276685 T C 0.678 ENSP00000216962.3:p.Tyr234His chr20 ANKRD60 58218668 G A 0.564 ENSP00000396747.1:p.Pro289Ser rs41275658 0.002 chr20 LAMA5 62317394 C T 0.304 ENSP00000252999.3:p.Ala2488Thr rs148169370 0.0048 chr22 IGLV5-45 22376251 C T 0.234 ENSP00000374831.2:p.Leu39Phe chr22 GSTTP1 23998546 C A 0.215 ENSP00000492273.1:p.Arg241Met chr22 GSTTP1 23998547 T A 0.435 ENSP00000492273.1:p.Arg241Trp chr22 SUSD2 24183189 G T 0.180 ENSP00000351075.3:p.Gly70Val rs79232038 chr22 KIAA1671 25029315 A G 0.376 ENSP00000351207.3:p.Lys439Arg chr22 CYP2D6 42127526 C T 0.128 ENSP00000353820.5:p.Arg365His rs1058172 chr22 CYP2D6 42130692 G A 0.166 ENSP00000353820.5:p.Pro34Ser CM900081 chr22 TUBGCP6 50221169 C T 0.924 ENSP00000248846.5:p.Gly1064Arg rs149231425 0.0132 chr22 PLXNB2 50280899 A G 0.184 ENSP00000409171.1:p.Tyr1280His COSM4444878 chrX PAGE1 49689423 T C 1.000 ENSP00000365320.3:p.Glu138Gly rs151117508 0.0079 0.567 rs111638770,COSM5461083,COSM5461082,COSM38 chrX FAM104B 55146247 G C ENSP00000397188.2:p.Pro63Arg 8435 chrX MED12 71119383 C G 0.215 ENSP00000363193.3:p.Thr37Arg chrX RTL9 110450737 G C 1.000 ENSP00000419786.2:p.Gln40His rs150383653,COSM4721270 0.0003 chrX SLC25A5 119469766 G A 0.345 ENSP00000360671.4:p.Gly73Ser rs143413528 chrX SLC25A5 119469779 A C 0.204 ENSP00000360671.4:p.Asn77Thr rs148294496 chrX SLC25A5 119469784 A T 0.184 ENSP00000360671.4:p.Ile79Phe rs141428607 chrX SLC25A5 119470481 G C 0.444 ENSP00000360671.4:p.Arg236Pro rs114413582 Table S3. Mutations found in the WES for chromosome/telomere instability and the VEGF-angiogenesis pathway in sporadic CAS sequenced individuals (non-POT1 mutation carriers). Genomic Reference Alternative ALT Allelic Amino acid Pathway Sample Gene position allele allele fraction change dbsnp MAF NT1* TP53BP2 223991119 G T 0.213 Q229K rs34683843 0.0467 NT2* TP53BP1 43762077 TGGGATAGG TGG 0.205 PI454- ATR 142215368 GGAAGTAA - 0.215 Mutations NT3* 0.166 ATM 108200982 T A L2450* involved in damage BRCA2 32912956 C A 0.166 H1488Q response and T1 BRCA1 41245120 T A 0.128 N810Y rs28897682 repair BRCA1 41246481 T C 0.257 Q356R rs1799950 0.0279 TP53 7578503 C T 0.451 V143M TP53RK 45315786 C T 0.255 R123Q rs34983477 0.0279 APC2 1457002 G C 0.376 G323R rs143870588 0.0119 T2 TP53BP2 223998162 G A 0.201 P115S T3 BRCA1 41245471 C T 0.312 D693N rs4986850 0.0398 RAD51C 56798128 A G 0.255 T287A rs28363317 0.0055 TTTAGGAAAT 0.423 ATM 108165638 TAACCATTTT CTCT - -1593 CDK8 26911760 C T 0.310 S62L BRIP1 59861662 C A 0.567 V533L T4 TP53 7576876 C G 0.346 D324H TP53 7578406 C T 0.134 R175H rs28934578 RAD51B 68331765 A T 0.126 M121L rs150928231 RAD51B 68352648 T G 0.104 L172W rs34094401 0.0188 ATM 108123551 C T 0.104 P604S rs2227922 0.0032 APC2 1468408 C A 0.376 A1703E rs200444154 NT2*; T3 ATM 108175462 G A 0-202 D1853N rs1801516 0.0788 Mutations NT1** KDR 55979558 C T 0.184 V297I rs2305948 0.1310 involved in RASA2 141326602 T C 0.422 rs295322 0.1140 Angiogenesis DOCK1 128810642 C T 0.273 rs9418773 0.1230 RICTOR 38952300 G A 0.179 S1042L IQGAP1 90996087 G A 0.189 E415K rs200988874 0.0005 SRC 36012590 A G 0.304 S12G PIK3C2G 18691253 C T 0.435 rs12315010 0.0389 NRAS 115256530 G T 0.134 Q61K rs121913254 NT2* PDPK1 2607915 CT C 0.310 -79 RASA4 102246366 G A 0.124 R123W rs139960113 0.0020 NT3* SHOX2 157823580 TACA T 0.556 p.77_78del T1 NRG2 139231255 C T 0.345 R577H rs75431091 0.0229 SPTAN1 131367372 A G 0.204 N1260S FGF22 640065 C T 0.256 S47F PIK3C2G 18443809 C A 0.107 A261E rs7133666 0.0417 T2 RASA2 141299289 G A 0.172 S557N MAP2K1 66736993 G T 0.243 IQGAP1 91025227 T G 0.435 L1122R VAV3 108145045 C T 0.172 E732K DUSP8 1579018 T C 0.234 rs113161544 0.0640 PIK3C2G 18473929 C T 0.432 Q391* T3 RASA2 141328901 G A 0.134 G839R PLCG1 39792063 A G 0.345 S279G rs2228246 0.0952 RICTOR 38960053 C T 0.180 V627I DUSP8 1579506 A G 0.172 RASA4 102235804 G A 0.102 T340I RASA4 102246393 C T 0.165 E114K ITGAV 187540655 G A 0.184 T4 PTPRQ 81004265 A C 0.436 E1589D PIK3C2G 18656259 C T 0.174 H1021Y rs201569993 0.0006 PRKCQ 6498721 G A 0.102 A521V T1; T3; T4 SPTAN1 131377915 G A 0.304 R1718H T3; NT1* MAP2K1 66777404 C T 0.174 A257V T4; NT2* VEGFA 43738526 C G 0.184 A28G rs201786558 * Confirmed constitutional ** Confirmed somatic. Table S4. Actionable variants found in the sequencing experiment with IBM Watson for Genomics platform for T1 patient. Gene Actionable variant Variant evidence (Pubmed) FDA-approved drug for Other molecules with clinical evidence (NCT) Angiosarcoma MSH6 p.Glu1088fs 12019211 Pembrolizunab Nivolumab (NCT0366819) TP53 p.Arg175His 8510927; 17401432 - Adavosertib (NCT01827384) Transferrin (NCT02354547) ARID1A p.Pro21del 23097632 - ** ATR p.Ile774fs 22960745 - ** NOTCH p.Pro2415del 15472975*; 25564152* - ** NCT: Number of Clinical Trial * Similar variant within the same domain. ** Compelling preclinical evidence and/or case study reports support the biomarker as being predictive of response to this drug Table S5. Actionable variants found in the sequencing experiment with IBM Watson for Genomics platform for T4 patient. Gene Actionable variant Variant evidence (Pubmed) FDA-approved drug for Other molecules with clinical evidence (NCT) Angiosarcoma CDK2A p.Arg99Pro 20340136; 19260062 - Palbociclib (NCT03239015) TP53 p.Val143Met 23469205 - Adavosertib (NCT01827384) Transferrin (NCT02354547) KIT Amplification 21523721; 17189383; 16166280 Pazopanib (NCT01462630)* Pazopanib (NCT03628131) Imatinib (NCT02461849) Nilotinib (NC02379416) NCT: Number of Clinical Trial * Phase II study