Telomerase Reverse Transcriptase

Author Manuscript Published OnlineFirst on April 21, 2020; DOI: 10.1158/1078-0432.CCR-19-3573 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Telomerase reverse transcriptase promoter mutations identify a genomically defined and 2 highly aggressive human pleural mesothelioma subgroup 3 Christine Pirker1, Agnes Bilecz2, Michael Grusch1, Thomas Mohr1, Barbara Heidenreich3, 4 Viktoria Laszlo4,5, Paul Stockhammer4, Daniela Lötsch1, Johannes Gojo1, 6, Lisa Gabler1, Sabine 5 Spiegl-Kreinecker7, Balazs Döme4, 5, 8, Ariane Steindl4, Thomas Klikovits4, Mir Alireza Hoda4, 6 Marko Jakopovic9, Miroslav Samarzija9, Katja Mohorcic10, Izidor Kern10, Barbara Kiesel11, Luka 7 Brcic12, Felicitas Oberndorfer13, Leonhard Müllauer13, Walter Klepetko4, Wolfgang M. 8 Schmidt14, Rajiv Kumar3, Balazs Hegedus2, 4, 15,*, Walter Berger1, * 9 10 Affiliations of authors: 11 1Institute of Cancer Research and Comprehensive Cancer Center, Department of Medicine I, 12 Medical University of Vienna, Austria. 13 22nd Institute of Pathology, Semmelweis University, Budapest, Hungary. 14 3Division of Molecular Genetic Epidemiology, German Cancer Research Center, Heidelberg, 15 Germany. 16 4Division of Thoracic Surgery, Department of Surgery, Comprehensive Cancer Center Vienna, 17 Medical University Vienna, Austria. 18 5Department of Tumor Biology, National Koranyi Institute of Pulmonology, Semmelweis 19 University, Budapest, Hungary 20 6Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Austria. 21 7Department of Neurosurgery, Neuromed Campus, Kepler University Hospital, Johannes 22 Kepler University, Linz, Austria. 23 8Department of Thoracic Surgery, Semmelweis University and National Institute of Oncology, 24 Budapest, Hungary. 25 9Department for Respiratory Diseases Jordanovac, University Hospital Center, University of 26 Zagreb, Croatia. 27 10University Clinic of Respiratory and Allergic Diseases, Golnik, Slovenia. 28 11Department of Neurosurgery, Medical University of Vienna, Austria. 29 12Medical University of Graz, Diagnostic and Research Institute of Pathology, Graz, Austria. 30 13Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria 31 14Center of Anatomy and Cell Biology, Neuromuscular Research Department, Medical 32 University of Vienna, Austria.

Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 21, 2020; DOI: 10.1158/1078-0432.CCR-19-3573 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 15Department of Thoracic Surgery, Ruhrlandklinik, University Duisburg-Essen, Essen, 2 Germany. 3 4 Short title: TERT promoter mutations in pleural mesothelioma 5 6 Key words: Telomerase, cancer, mesothelioma, survival, genomic changes 7 8 *Corresponding authors: 9 Walter Berger, Institute of Cancer Research and Comprehensive Cancer Center, Medical 10 University Vienna, Borschkegasse 8a, 1090 Vienna, Austria (e-mail: 11 [email protected]). 12 Balazs Hegedus, Department of Thoracic Surgery, Ruhrlandklinik, University Duisburg-Essen, 13 45239, Essen, Germany (e-mail: [email protected]). 14 15 Conflict of interests: The authors declare no potential conflicts of interest. 16 17 Translational relevance:

18 Acquisition of immortality as a hallmark of malignant transformation is achieved by 19 telomerase reactivation in the majority of human cancers. Point mutations in the telomerase 20 reverse transcriptase (TERT) gene promoter were recently discovered as one fundamental 21 driver of this process correlating with worse prognosis in several cancer types. The potential 22 role of these mutations in human malignant pleural mesothelioma (MPM) aggressiveness 23 was unknown far. Here we show that presence of TERT promoter mutations identifies a 24 distinct MPM patient subgroup with extremely dismal prognosis. The respective MPM cell 25 explants with TERT promoter mutations are characterized by a profoundly enhanced in vitro 26 immortalization potential, reduced chromosomal instability and a specific mutation/deletion 27 pattern compared to the wild-type counterparts. Consequently, our MPM-based results 28 indicate a unique way of malignant transformation of TERT promoter-mutated tumors which 29 has broad implications also for multiple other cancer types harboring this non-coding 30 genomic alteration. 31

1 2 Abstract 3 Purpose: Human malignant pleural mesothelioma (MPM) is characterized by dismal

4 prognosis. Consequently, dissection of molecular mechanisms driving malignancy is of key

5 importance. Here we investigate, whether activating mutations in the telomerase reverse

6 transcriptase (TERT) gene promoter are present in MPM and associated with disease

7 progression, cell immortalization, and genomic alteration patterns.

8 Experimental design: TERT promoters were sequenced in 182 MPM samples and compared

9 to clinicopathological characteristics. Surgical specimens from 45 MPM patients were tested

10 for in vitro immortalization. The respective MPM cell models (N=22) were analyzed by array

11 comparative genomic hybridization, gene expression profiling, exome sequencing, as well as

12 TRAP, telomere length and luciferase promoter assays.

13 Results: TERT promoter mutations were detected in 19/182 (10.4%) MPM cases and

14 significantly associated with advanced disease and non-epithelioid histology. Mutations

15 independently predicted shorter overall survival in both histological MPM subtypes.

16 Moreover, 9/9 (100%) mutated but only 13/36 (36.1%) wild-type samples formed

17 immortalized cell lines. TERT promoter mutations were associated with enforced promoter

18 activity and TERT mRNA expression, while neither telomerase activity nor telomere lengths

19 were significantly altered. TERT promoter-mutated MPM cases exhibited distinctly reduced

20 chromosomal alterations and specific mutation patterns. While BAP1 mutations/deletions

21 were exclusive with TERT promoter mutations, homozygous deletions at the RBFOX1 and the

22 GSTT1 loci were clearly enriched in mutated cases.

23 Conclusions: TERT promoter mutations independently predict a dismal course of disease in

24 human MPM. The altered genomic aberration pattern indicates that TERT promoter

1 mutations identify a novel, highly aggressive MPM subtype presumably based on a specific

2 malignant transformation process.

4 Introduction

5 Malignant pleural mesothelioma (MPM) is a rare, highly aggressive tumor arising from

6 mesothelial cells lining the pleural cavity closely linked to asbestos exposure (1,2). Additional

7 risk factors include certain genetic predispositions like germline BRCA1-associated protein 1

8 (BAP1) gene mutations. Three histological subtypes – epithelioid, sarcomatoid and a mixed

9 biphasic form – have been defined with the sarcomatoid type having the worst prognosis.

10 Current treatment options include surgery, radio-, and chemotherapy, however, median

11 overall survival is only about 6-12 months and the 5 year survival rate below 5% (1).

12 Genetically, MPM is characterized primarily by loss of key tumor suppressor and

13 transcription regulator genes (1,3) including BAP1, cyclin-dependent kinase inhibitor 2A

14 (CDKN2A), neurofibromin 2 (NF2, merlin), tumor suppressor protein 53 (TP53), large tumor

15 suppressor homolog 2 (LATS2), and RNA binding fox-1 homolog 1 (RBFOX1, A2BP1) (2,4).

16 Gains/amplifications of driver oncogenes occur less frequently, further complicating the

17 search for defined therapeutic targets (5). Despite increasing knowledge concerning

18 molecular factors underlying MPM development (6), effective therapeutic options are still

19 limited and clinically applicable prognostic markers urgently needed (7,8).

20 Virtually all MPM are characterized by reactivation of telomerase allowing telomere

21 stabilization and immortalization (9). Besides gene dose changes, epigenetic mechanisms

22 and the impact of deregulated oncogenic transcription factors (10), activating mutations in

23 the TERT promoter are a major cause for telomerase rejuvenation in several cancer types

24 (11). These mutations occur at -124 (-124C>T), -146 (-146C>T), or -57 (-57A>C) base pairs

1 upstream of the TERT gene coding region on chromosome 5p15.33 (also designated C228T,

2 C250T, and A161C in many reports). All three TERT promoter mutations create de novo

3 binding sites for E twenty-six-specific (ETS) transcription factors (12). Besides creating similar

4 ETS binding sites, TERT promoter mutations have been shown to affect transcription

5 differently and act through different mechanisms (13,14). These non-coding mutations occur

6 at highly variable frequencies in different cancer types and are widely missing e.g. in breast,

7 colon and lung cancer (10). Interestingly, TERT promoter mutations have been shown to

8 associate with an unfavorable patient outcome in several cancer types (12,15). More

9 recently, increased TERT gene transcription and even telomerase activity in promoter

10 mutated tumors have been observed in most studies (16,17). However, the exact

11 mechanisms driving worse prognosis associated with TERT promoter mutations are so far

12 unclear.

13 By analyzing two independent MPM patient cohorts, we aimed to dissect the association of

14 TERT promoter mutation status with clinical parameters and patient prognosis. Moreover, by

15 using an extended panel of MPM primo-cell cultures from surgical specimens, we aimed to

16 unravel cellular and molecular changes associated with activating TERT promoter mutations

17 in MPM. Besides a higher incidence in MPM with non-epithelioid histology, we found strong

18 prognostic power of TERT promoter mutations in both histological subgroups. Enhanced

19 aggressiveness of TERT promoter mutation-positive tumors was reflected at the level of

20 tumor cell biology by their higher propensity for in vitro immortalization. Besides moderately

21 but significantly enhanced TERT gene expression, TERT promoter mutated MPM cell models

22 were characterized by reduced levels of chromosomal instability and lack of BAP1

23 alterations.

1 Materials and Methods

2 Patient samples

3 In total 182 MPM patients were analyzed. The Austrian cohort consisted of 83 patients from

4 the Department of Thoracic Surgery, Medical University of Vienna. The Croatian/Slovenian

5 cohort included 76 Slovenian patients from the Department for Pulmonology, University

6 Clinic Golnik, and 23 Croatian patients from the Department for Respiratory Diseases

7 Jordanovac, University of Zagreb. The study was approved by the Ethics Committees at the

8 Medical Universitiy of Vienna (#904/2009) and the University Hospital Center Zagreb

9 (#02/21AG). The Institutional Review Board of the University Clinic Golnik granted a waiver

10 for the retrospective analyses. The studies were performed in accordance with the

11 Declaration of Helsinki and all prospectively included patients gave informed written

12 consent. More details are given in the Supplementary Materials and Methods.

14 TERT promoter analysis

15 DNA was extracted from FFPE samples, frozen tissue or MPM cell pellets using the respective

16 kits from Qiagen (Qiagen, Hilden, Germany). Mutational status of the TERT core promoter

17 region from position -27 to -286 from ATG start site was determined by PCR and Sanger

18 sequencing as described previously, and data were analyzed by Geneious Pro 5.6.5 software

19 with reference to the sequences from the NCBI gene database (chr5: 1,295,071–1,295,521,

20 hg19 GRCH37) (18,19).

22 Establishment of in vitro MPM cell models

23 Establishment of MPM cell cultures from surgical samples and cell line authentication by STR

24 analyses were performed as published previously (20,21). Cell cultures were regularly

1 checked for Mycoplasma contamination using the Mycoplasma PCR detection kit G238

2 (Applied Biological Materials, Inc). For experimental procedures, cell models ranging

3 between passage 4 and 20 were used. The length of time between thawing and

4 experimental use did not exceed 2 months.

6 Array CGH

7 Array CGH analysis was performed as described (22) using 4x44K whole genome

8 oligonucleotide-based microarrays (Agilent). Data visualization and evaluation is described in

9 the Supplementary Materials and Methods. Data are openly available at ArrayExpress

10 (https://www.ebi.ac.uk/arrayexpress/) under the accession number E-MTAB-8987.

12 Gene expression profiling

13 Whole genome gene expression arrays were performed using 4x44K microarrays from

14 Agilent as described (22). Feature extraction and data analysis were carried out using the

15 Feature Extraction (version 11.5.1.1.) and GeneSpring (version 13.0) softwares, respectively.

16 Data are openly available at ArrayExpress (https://www.ebi.ac.uk/arrayexpress/) under the

17 accession number E-MTAB-8986.

19 Whole exome sequencing and Ion Torrent sequencing

20 Exome sequencing of MPM cell lines was performed using the Nextera DNA Exome Kit

21 (Illumina TruSeq Rapid Capture Exome 45Mb) on a HiSeq 4000 (2x75 bp paired-end) at the

22 Medical University of Vienna Biomedical Sequencing Facility.

1 For confirmation of the detected mutations in MPM tumor tissue, Ion Torrent sequencing

2 was performed. Details for both methods are provided in the Supplementary Materials and

3 Methods.

5 Quantitative reverse transcription PCR (qRT-PCR) for TERT mRNA determination

6 qRT-PCR for TERT mRNA expression in MPM cell models was performed as published (15)

7 and as described in the Supplementary Materials and Methods.

9 PCR for analysis of genomic status of GSTT1 and RBFOX1

10 Genomic DNA of healthy tissues, MPM tumor tissues and cell lines were analyzed for GSST1

11 or RBFOX1 deletions as described in detail in the Supplementary Materials and Methods.

13 Analysis of parameters associated with telomerase activity

14 Protein activity of telomerase of all MPM cell models was assessed by Telomerase Repeat

15 Amplification Protocol (TRAP assay). In parallel, telomere lengths of the cell lines were

16 analyzed by qPCR and results calculated relative to the ALT-positive osteosarcoma model SA-

17 OS. Both methods are described in detail in the Supplementary Materials and Methods.

19 TERT promoter activity

20 TERT promoter activity was analyzed by luciferase reporter assays in a TERT promoter wild-

21 type and a TERT promoter mutated background as published recently (15). Knock-down of

22 GABPA by siRNA (Dharamcon) was performed using ON-TARGETplus SMARTpool siRNA and

23 Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientifc) for 48h. The respective non-

24 targeting pool served as negative control.

2 Chemosensitivity analysis

3 The impact of the telomerase inhibitor MST-312 (Sigma) (23) and the ETS factor inhibitor YK-

4 4-279 (Selleck Chemicals) (24) was determined by MTT-based viability assay as described

5 previously (15).

7 Protein isolation, Western blot analysis and immunohistochemistry

8 Analysis of total protein extracts of MPM cell models by Western blot as well as BAP1

9 staining by immunohistochemistry is described in Supplementary Materials and Methods.

10 Antibodies are given there as well.

11 Statistics

12 SPSS and GraphPad Prism software packages were used for statistical analyses. Overall

13 survival was defined as time between MPM diagnosis and death or, in censored patients,

14 diagnosis and last follow-up date. Analysis of survival data and all other statistical analyses

15 are described in the Supplementary Materials and Methods.

17 Results

18 TERT promoter mutations associate with non-epithelioid histology

19 The TERT promoter region containing the respective activating mutation sites was

20 sequenced in 182 MPM samples derived from two independent cohorts: one consisting of 83

21 Austrian and the other of 99 Croatian/Slovenian MPM patients. TERT promoter mutations

22 were detected in 19/182 (10.4%) MPM cases (selected sequencing charts are shown in

23 Supplementary Fig. S1). Patient characteristics of the entire patient collection are given in

24 Table 1 while those of the Austrian and the Croatian/Slovenian cohorts are separately

1 described in Supplementary Tables 1 and 2, respectively. TERT promoter mutations were

2 found at significantly higher frequency in patients with non-epithelioid histology both in the

3 entire patient collective (5.5% versus 22.2% in epithelioid versus non-epithelioid histology),

4 but also the two separate cohorts (6.8% versus 29.2% in the Austrian and 4.4% versus 16.7%

5 in the Croatian/Slovenian cohort). Moreover, all patients with TERT promoter mutated

6 tumors and evaluable IMIG stage derived from both cohorts presented at a late disease

7 stage (Table 1). Accordingly, within the Austrian cohort these patients were less frequently

8 treated in a multimodal therapeutic setting (Supplementary Table 1). With regard to the

9 mutation type, 13/19 mutated MPM harbored the -124C>T (68.4%), 2/19 the -146C>T

10 (10.5%), and 4/19 (21%) the -57A>C genotype. Strikingly, the distribution was different

11 between the histological subgroups. Comparable to many other tumor types (12,25), the -

12 124C>T mutation was strongly prevalent in the non-epithelioid cases (10/12 mutated

13 tumors). However, in case of the epithelioid histology, the prevalence of the -124C>T

14 mutation was equal with that of the rare -57A>C mutation (3/7 mutated cases each).

16 TERT promoter mutations are independent predictors of dismal overall survival in MPM

17 To assess the prognostic value of TERT promoter mutations we performed Kaplan-Meier

18 survival analyses (Fig. 1). Median overall survival in the entire patient cohort was 262 versus

19 469 days in TERT promoter mutated as compared to wild-type cases (p<0.0001) (Fig. 1A).

20 Hence, this genomic marker was of higher prognostic value for an aggressive course of

21 disease as compared to the histological subtype, a known marker of MPM prognosis (353

22 versus 459 days for non-epithelioid as compared to epithelioid cases; p=0.01) (Fig. 1B). TERT

23 promoter mutations significantly enriched in the non-epithelioid histology (Table 1,

24 Supplementary Tables 1 and 2) and only insignificantly enriched in tumors with pleomorphic

1 nuclear features (3/11; 27.3%). With regard to the quality as prognostic marker, the Austrian

2 and Croatian/Slovenian patient collectives were also evaluated separately as test and

3 validation cohorts. In the Austrian test cohort, we found a significantly shorter overall

4 survival in patients harboring TERT promoter mutations (262 versus 524 days, p=0.0012) (Fig.

5 1C, left panel). A comparable and significant difference was observed for the

6 Croatian/Slovenian validation cohort (104 versus 465 days, p=0.0024) (Fig. 1C, right panel).

7 This confirms independence of the prognostic quality with regard to regional patient origin

8 even despite differences in country-specific therapy settings. Importantly - even though

9 being more frequent in non-epithelioid tumors - TERT promoter mutations were significantly

10 associated with shorter overall survival in both the epithelioid (Fig. 1D, left; 340 versus 510

11 days, p=0.003) and the non-epithelioid (Fig. 1D, right; 199 versus 412 days, p=0.023)

12 subgroup. In order to test whether presence of a TERT promoter mutation is an independent

13 prognostic factor, we performed univariate as compared to multivariate Cox regression

14 analyses of the entire patient cohort (Supplementary Table 3 and Table 2, respectively). In

15 univariate analysis Karnofsky performance status, histology, IMIG stage, EORTC score and

16 TERT promoter status were significantly associated with patient survival. In the multivariate

17 setting, TERT promoter mutations and non-epithelioid histology remained significant

18 indicators of shorter overall survival (Table 2). Also, when including the EORTC performance

19 score, available for 140 patients, in multivariate analysis, TERT promoter mutation status

20 remained a significant predictor of poor prognosis (Supplementary Table 4). These data

21 prove that TERT promoter mutations are a robust, independent prognostic factor in human

22 MPM.

24 In vitro cell immortalization correlates with TERT promoter mutation

1 The strong and histology-independent negative prognostic impact of TERT promoter

2 mutations suggests a major role of these non-coding, genomic alterations in the aggressive

3 phenotype of human MPM. To clarify whether this is a consequence of altered cancer cell

4 biology and dissect the underlying molecular mechanisms, we set up primo-cell cultures

5 from 45 patient samples of the Austrian cohort (termed “original panel” in Supplementary

6 Table 5). Immortalized cell lines were successfully generated from 22 (48.9%) patients. Cell

7 line characteristics are summarized in Supplementary Table 5. The TERT promoter mutation

8 status of the cell lines reflected the ones of the original tumors in all cases available for

9 parallel analysis (N=10) with exception of Meso71. This MPM cell line was derived from a

10 recurrence after chemotherapy that harbored a -57A>C mutation neither detectable in the

11 patient`s blood nor in tumor tissue of the primary lesion before chemotherapy. 5/9 TERT

12 promoter-mutated cell models harbored also wild-type alleles in accordance with the

13 described mono-allelic TERT expression from the mutated alleles (26), while 4/9 showed

14 homozygous mutations, indicating LOH of the wild-type allele. Cell line generation was

15 achieved in all nine (100%) mutated but only 13/36 (36.1%) wild-type cases, demonstrating a

16 significant association of in vitro immortalization with TERT promoter status (Fig. 2B) not

17 seen with histology or disease stage (Fig. 2C, D). Successful cell line propagation in vitro,

18 however, obviously represents another strong negative prognostic factor itself. The survival

19 differences between patients with cell line-forming and -non-forming tumors remained

20 statistically significant (Fig. 2A; median overall survival 268.5 versus 607 days, p=0.0008),

21 even after exclusion of the nine TERT promoter mutant patients (not shown; median overall

22 survival 313.7 versus 577.3 days, p=0.014). Interestingly, also presence of pleomorphic

23 features was associated with successful cell line establishment (6/7; 85.7%) (Fig. 2E). The

24 relation between TERT promoter status, cell line formation capacity, histology, pleomorphic

1 features, and stage at time of cell culture are opposed to overall survival data on single

2 patient level in Fig. 2E. Moreover, C-reactive protein (CRP) levels - another marker of poor

3 MPM prognosis (27) - were significantly higher in patients with TERT promoter mutated as

4 compared to wild-type tumors (7.3±2.2 mg/dl versus 3.8±0.6 mg/dl; p<0.05) (Supplementary

5 Fig. S2). Together, these observations suggest that the worse prognosis of MPM patients

6 with TERT promoter mutations is at least in part attributable to a more aggressive cancer cell

7 biology.

9 Association between TERT promoter mutations and telomere-related parameters

10 Re-activation of TERT expression has been repeatedly found in virtually all MPM tumor

11 samples analyzed (9,28). Hence, we tested whether TERT promoter mutations are associated

12 with altered TERT gene expression, telomerase activity and telomere lengths. All MPM cell

13 models expressed detectable levels of TERT mRNA and telomerase activity. Despite a

14 significant correlation between these two parameters (linear regression; r=0.44, p=0.04),

15 only the mRNA transcript level but not telomerase activity was significantly higher in the

16 TERT promoter mutated cell lines (p=0.006; Supplementary Fig. S3A; p=0.07; Supplementary

17 Fig. S3B, respectively). Accordingly, TERT promoter status was not associated with altered

18 telomere length, although all rare cases with very long telomeres were TERT promoter wild-

19 type (Supplementary Fig. S3C). To estimate if TERT promoter mutations indeed drive

20 enhanced TERT mRNA expression, luciferase experiments with wild-type and mutated

21 promoter constructs were performed in MPM cell models with endogenous wild-type

22 (VMC48) or -124 C>T-mutated (VMC20) TERT promoter background. Presence of either

23 activating mutation -124C>T or -146C>T - significantly increased promoter activity both in

24 the wild-type and the -124C>T -mutated background. Interestingly, the effect was more

1 robust using the -124C>T promoter construct in the -124C>T-mutated MPM background

2 while both mutations were equally effective in the wild-type model (Supplementary Fig.

3 S4A). Knock-down of the ETS factor GABPA, a key factor for activation of the mutated TERT

4 promoter (29), resulted in massive downregulation of TERT promoter activity when using the

5 two mutated but not the wild-type constructs. The effect was, however, significantly

6 stronger for the -124C>T but not the -146C>T construct when transfected into the MPM cell

7 model with mutated (VMC20, -124C>T) as compared to the wild-type background (VMC48)

8 (Supplementary Fig. S4B). This suggests that cellular parameters like the transcription factor

9 expression pattern might be adapted for ideal utilization of the endogenous mutated TERT

10 promoter sequence. Accordingly, expression of several ETS transcription factor genes at the

11 mRNA level, including GABPA, ETS1 and FLI1, tended to be higher in mutated as compared to

12 wild-type models (Supplementary Fig. S4C).

13 To investigate, whether the TERT promoter status might predict sensitivity against

14 telomerase inhibition, nine MPM cell models each with either wild-type or mutated TERT

15 promoter were analyzed for responsiveness to the telomerase inhibitor MST-312 (23) and

16 the ETS factor inhibitor YK-4-279 (24). The mean IC50 values for the MPM models with

17 mutant TERT promoter were moderately but significantly lower as compared to the wild-

18 type subgroup in case of MST-312, while for YK-4-279 only an insignificant trend in this

19 direction was detected (Supplementary Fig. S5).

21 TERT promoter mutations are associated with a more stable chromosomal phenotype

22 As mentioned above, the enhanced TERT mRNA expression in TERT promoter mutant

23 mesothelioma cell models did not fully translate into significantly augmented telomerase

24 activity (compare Supplementary Fig. S3). Moreover, TERT mRNA expression positively but

1 only weakly correlated with shorter patient survival in TCGA database in silico analysis

2 (Supplementary Fig. S6). Clearly, this effect was minor as compared to the predictive power

3 of the TERT promoter mutations in our patient cohorts (compare Fig. 1). Therefore, we

4 hypothesized that the level of TERT expression and telomerase activity at the time of surgery

5 might not represent the sole factor driving aggressiveness in TERT promoter mutated MPM.

6 Array CGH analyses performed for the original MPM cell line panel (N=22, Supplementary

7 Table 5) revealed that, unexpectedly, TERT promoter wild-type cell lines (N=13) exhibited

8 distinctly and significantly (p=0.016) enhanced chromosomal alterations (35.3% genome

9 affected by gains/losses) as compared to the panel of mutated models (N=9; 16.6% genome

10 affected). While the amount of chromosomal alterations was rather comparable regarding

11 mutated models, a clearly higher variability was detected within wild-type models (Fig. 3A,

12 B). No differences were detected with regard to chromosome 5p harboring the TERT gene

13 locus, where both groups showed gains in more than 50% of cases (Fig. 3B). Interestingly,

14 the higher variability of the wild-type cell models involved genomic gains as well as losses

15 (Fig. 3A). Moreover, differences were not randomly distributed but concerned distinct

16 genomic regions including chromosomes 1, 5q, 9p, 7, 14, and 20 (Fig. 3B, upper panels).

17 Three affected chromosomes (chromosomes 1, 9 and 14) are enlarged in Fig. 3B (lower

18 panels). Quantification of aberrations at the single probe-based level for chromosome 1, 9p,

19 and 14 is given in Fig. 3C. While at chromosome 1 the gains observed for the TERT promoter

20 wild-type models were widely missing in the mutated cell lines, much stronger losses were

21 found on chromosome 14 in the mutated cells. The characteristic loss at chromosome 9p

22 including the CDKN2A locus was narrow and focused in case of the TERT promoter mutant

23 cells but highly variable and much broader in the wild-type panel, affecting frequently

1 multiple adjacent genes. This suggests a higher chromosomal stability of MPM cells

2 harboring TERT promoter mutations.

4 Exclusiveness of TERT promoter mutations and BAP1 alterations

5 An extended MPM cell line panel (N=26; Supplementary Table 5) was analyzed by exome

6 sequencing (sequencing depth >100x) to explore a potential relationship of TERT promoter

7 mutations with known genomic alterations of MPM including mutations/loss of BAP1 and

8 NF2 (Fig. 4; for mutations of BAP1, CDKN2A, and NF2 compare Supplementary Table 6).

9 Generally, although a tendency towards a lower mutational burden in TERT promoter

10 mutated samples was detected, this difference did not reach statistical significance (see

11 Supplementary Fig. S7 for the coding variants). The general mutational pattern in MPM cell

12 lines was equally detected in the original tumor tissues using Ion Torrent sequencing (Fig.

13 4B). Interestingly, neither loss/mutations of the CDKN2A locus nor of NF2 or TP53 showed

14 any association with TERT promoter status or MPM histology (Fig. 4A, B). In contrast, BAP1

15 mutations (5/26; 19.2%) or deletions (3/26; 11.5%) were exclusively found in MPM cell

16 models lacking TERT promoter mutations (8/17; 47.1%) (Fig. 4A, B). BAP1 protein expression

17 by Western blot was completely absent in seven cell extracts, of which four harbored a BAP1

18 mutation and three a gene deletion, and only one sample with a BAP1 point mutation

19 (Meso221) showed detectable protein levels (Supplementary Fig. S8A). Accordingly, both

20 BAP1 mRNA and protein expression levels were lower in the TERT promoter wild-type

21 models (Supplementary Fig. S8B, upper panels) and inversely correlated with overall survival

22 (Supplementary Fig. S8B, lower panels). Cell line data widely correlated with BAP1

23 expression in the corresponding tumor specimens detected by immunohistochemistry (Fig.

24 4B and Supplementary Fig. S9). Loss of nuclear BAP1 in tumor samples (N=75, Austrian

1 cohort) was clearly associated with epithelioid histology (Supplementary Tables 7 and 8) and

2 wild-type TERT promoter status (Supplementary Table 9).

3 While for the frequencies of other mutations/deletions described for MPM before (3) no

4 significant associations with the TERT promoter status were found, two distinct deletions

5 concerning the splicing regulator RBFOX1 and the glutathione S transferase GSTT1 were

6 strongly related to both TERT promoter status as well as tumor histology (Fig. 4A, B).

7 Deletion of the GSTT1 locus is a well-known germline polymorphism which has been shown

8 to increase the risk for severe fibrotic changes upon asbestos exposure (30). In contrast,

9 RBFOX1 deletions at 16p13.3 represent a cancer phenomenon and have been reported for

10 example in glioblastoma, where RBFOX1 is crucial for terminal cell differentiation (31).

11 Concerning our mesothelioma cases, RBFOX1 deletions were solely found in the malignant

12 but never the corresponding non-malignant tissues. In case of GSTT1 deletions, only 2/10

13 affected patients harbored also a germline deletion (Fig. 4B). RBFOX1 deletions occurred

14 either in TERT promoter mutated (7/9) or non-epithelioid (3/17) samples but were

15 completely absent in TERT promoter wild-type, epithelioid cell lines, an association also seen

16 for the GSTT1 deletion.

18 Discussion

19 In this translational research study we describe a strong and independent negative

20 prognostic impact of activating TERT promoter mutations in human MPM. Besides a clear-

21 cut enrichment in non-epithelioid specimens, the prognostic quality remained significant

22 also in patients harboring tumors of epithelioid histology, despite the relatively low number

23 of affected cases. This negative impact on overall survival was equally found in two

24 independent patient cohorts from Austria and Croatia/Slovenia despite differences in stages

1 of disease and treatment modalities. This implicates that TERT promoter mutations might

2 represent a novel marker for worse prognosis of human MPM, as recently suggested for

3 several other cancer types including gliomas (32). Our data are in accordance with a very

4 recent report concerning the Inserm series, also identifying TERT promoter mutations in a

5 comparable percentage of MPM cases. Mutations were significantly associated with non-

6 epithelioid histology and poor patient prognosis mainly within a three gene mutation

7 signature (28,33).

8 Concerning the TERT promoter mutation types in MPM, preferentially the -124C>T mutation,

9 predominant in most cancers (10), was detected. However, in contrast to the French study

10 (33), we also found the less frequent -146C>T mutation in two cases of our MPM cohort.

11 More prevalent was, surprisingly, the very rare -57A>C mutation, with three affected cases

12 concerning the rarely mutated epithelioid histology. Originally, -57A>C has been described as

13 a causal high-penetrance germline mutation in a melanoma-prone family (11) and only

14 occasionally as a somatic event in melanoma and bladder cancer (34,35). Thus, relatively

15 high prevalence of this mutation type might be specific for epithelioid MPM.

16 To investigate the impact of TERT promoter mutations on MPM cell characteristics, we set

17 out to establish permanent cell cultures from part of the Austrian MPM sample collection.

18 Interestingly, TERT promoter mutations were strongly enriched in samples with in vitro

19 immortalization potential. Corresponding associations of TERT promoter mutations and

20 more efficient in vitro immortalization have been reported for mixed brain tumors (36),

21 thyroid cancer (37), and by our group for meningioma (15). In addition, our data

22 demonstrate that effective in vitro cell immortalization itself might represent a marker for

23 high MPM aggressiveness, even after exclusion of the TERT promoter mutated cases. One

24 underlying factor might be the comparably high prevalence of samples with pleomorphic

1 nuclear features, known to be associated with unfavorable prognosis (38), in the TERT

2 promoter wild-type cell line-forming (3/13; 23%) as compared to non-cell line-forming (1/23;

3 4.3%) cases.

4 An association of TERT promoter mutations with worse clinical outcome has been observed

5 in multiple cancer types (12). However, the underlying factors are not entirely clear so far.

6 Besides telomerase-mediated replicative immortality, also non-canonical functions of TERT

7 (10) in cancer cell motility, stemness and therapy resistance have been suggested as

8 underlying mechanisms (39). In our study, MPM cell models with TERT promoter mutations

9 were significantly more responsive to telomerase inhibition than wild-type cases, suggesting

10 an enhanced dependency on telomerase activity in this MPM subgroup. Additionally, ETS

11 factor expression profiles and luciferase reporter data indicated that TERT promoter

12 mutated MPM cells might be ideally adapted for optimal promoter activation, for example

13 by binding of the ETS factor GABPA (29).

14 However, based on our data, it is questionable whether enhanced TERT promoter activity at

15 the time of diagnosis is the sole mechanism underlying the dismal prognosis of patients

16 harboring TERT promoter mutated tumors. Generally, all MPM are telomerase-positive and

17 several wild-type MPM models in the present study displayed comparable TERT mRNA

18 expression and telomerase activity levels as the mutated ones. Additionally, the enhanced

19 TERT mRNA expression in TERT promoter mutated cases did not translate into pronounced

20 augmentation of telomerase activity and no correlation with telomere lengths was observed.

21 In search for additional oncogenic driver mechanisms, we analyzed whether defined

22 chromosomal alteration or mutation patterns might be associated with TERT promoter

23 mutations in human MPM cell cultures. Generally, our array CGH and whole exome

24 sequencing data are well in agreement with previous studies, with losses at chromosomes

1 4q, 9p, 13, 14, and 22, gains in chromosomes 5p and 7p as well as mutations/deletions of

2 CDKN2A, NF2, BAP1 and TP53 as predominant genomic alterations in human MPM

3 (2,3,40,41). Interestingly, we found distinct differences in the general pattern of

4 chromosomal alterations and at defined gene loci associated with TERT promoter mutations.

5 Most strikingly, TERT promoter mutated MPM samples showed a distinctly lower level of

6 chromosomal instability. These results were rather unexpected, as Ivanov et al. reported

7 association of enhanced chromosomal instability with poor survival of 22 MPM patients (40).

8 The underlying mechanisms are unknown so far, but it has to be considered that the TERT

9 promoter mutated MPM subgroup is rather minor and as such might have been missed in

10 smaller patient collectives. Moreover, our MPM cell model approach clearly selected for

11 TERT promoter mutated MPM cases, significantly prone to immortalize with higher efficacy.

12 In addition, the differences in chromosomal aberration patterns suggest that TERT promoter

13 mutations might be associated with an alternative malignant transformation process.

14 Accordingly, it has been shown that TERT promoter mutations time-dependently supported

15 immortalization especially by stabilizing short telomeres at a critical length, hence modifying

16 telomere shortening-associated crisis (42). It might be hypothesized that early acquisition of

17 a TERT promoter mutation in a small subgroup of MPM may circumvent critical telomere

18 shortening during malignant transformation and hence avoid massive chromosomal

19 rearrangements. Accordingly, a study about clonal evolution of glioblastoma cells revealed

20 TERT promoter mutations as an early event during malignant progression shared by all

21 tumor cell subclones (43).

22 While several well-known MPM alterations (CDKN2A mutations/deletions, NF2 and TP53

23 mutations) did not associate with TERT promoter status, BAP1 mutations/deletions were

24 exclusively found in TERT promoter wild-type MPM models. This association was confirmed

1 in a large series of surgical specimens by immunohistochemical BAP1 detection. Accordingly,

2 BAP1 mRNA expression was lower in the TERT promoter wild-type cells, and samples

3 completely missing BAP1 protein expression were only found within this genotype.

4 Interestingly, the deubiquitylase BAP1 has been shown to prevent chromosomal instability

5 by several mechanisms (44). These include cell death induction following DNA damage (45),

6 de-ubiquitination of γ-tubulin (46), the centrosome protein MCRS1 (47), and the chromatin

7 remodeling molecule Ino80 in several human cancer cell models including mesothelioma

8 (48). Thus, exclusiveness of BAP1 loss with TERT promoter mutations might be another

9 explanation for the lower chromosomal instability observed in the latter subgroup.

10 Accordingly, BAP1 loss might support telomerase activation by other mechanisms than

11 promoter mutations based on accelerated chromosomal instability. In agreement with our

12 results, mutual exclusivity between BAP1 and TERT promoter mutations was observed within

13 the clinical Inserm MPM series (33), while - in a MPM cell culture collection from the same

14 research group - 3/12 cell models harbored both alterations (28). The reasons for this

15 discrepancy are currently unclear. Interestingly, in urothelial carcinoma the very frequently

16 occurring TERT promoter mutations did not show any association with the BAP1 mutation

17 status (49), while in liver cancer with biliary phenotype, as in our study in MPM cell models,

18 exclusiveness of these two mutations was found (50). It might be hypothesized, that the

19 better prognosis of BAP1 mutated mesothelioma cases (2) might be, at least partly, based on

20 the lack or reduced frequency of BAP1 alterations in the small but very aggressive subgroup

21 of TERT promoter mutated MPM patients.

22 Two gene deletions, namely RBFOX1 (also designated A2BP1) and GSTT1, were significantly

23 enriched in TERT promoter mutated MPM cell models. Moreover, the only two cell models

24 with tumor necrosis factor (TNF) receptor–associated factor 7 (TRAF7) mutations (compare

1 Fig. 4), previously detected in about 2% of MPM (3), both harbored the -57 A>C mutation

2 and were of epithelioid histology. Together, this data might reflect an altered propensity in

3 the TERT promoter mutated MPM subset to undergo gene deletions and to associate with

4 specific genomic alterations.

5 Summarizing, we report that activating TERT promoter mutations identify a specific

6 subgroup of aggressive human MPM characterized by reduced chromosomal instability and

7 lack of BAP1 mutations. TERT promoter mutations represent an independent prognostic

8 marker for dismal patient survival. After validation in prospective studies, this marker might

9 support better estimation of mesothelioma patient prognosis.

11 Acknowledgements:

12 Research support funding: This work was supported by the Austrian Science Fund (FWF,

13 I2872 to B. Hegedus, T906-B28 to D. Lötsch, and I3522-B33 to V. Laszlo), the Hungarian

14 National Research, Development and Innovation Office (K109626, K108465, KNN121510 and

15 SNN114490 to B. Döme), the Vienna Fund for Innovative Interdisciplinary Cancer Research

16 (to B. Döme), the Medical Scientific Fund of the Mayor of the City of Vienna (17028 to T.

17 Klikovits), and the EFOP (3.6.3-VEKOP-16-2017-00009 Fund to A. Bilecz).

18 We thank Petra Vician, Gerald Timelthaler, Jennifer Hsu, Mirjana Stojanovic, Barbara Dekan,

19 Andreas Wagner, and Violetta Piurko for competent technical assistance and cell culture

20 establishment.

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1 Table 1. Clinicopathological characteristics of all MPM patients grouped by TERT promoter status.

Total TERTpwt TERTpmut P-value (N= 182) (N= 163) (N= 19)

male 143 125 18 Gender 0.070 female 39 38 1 Age (years) mean ± SD 64.18 ± 0.8 64.0 ± 0.8 65.3 ± 2.6 0.614 Karnofsky PS PS 80 140 128 12 0.313 (NA = 15) PS <80 27 23 4 Epithelioid 127 120 7

Histology Non-epithelioid 54 42 12 <0.001a (NA = 1) - biphasic 44 38 6 - sarcomatoid 9 3 6 - NA = 1 IMIG stage I / II 54 54 0 0.002 (NA = 42) III / IV 86 64 12 EORTC Prognostic 1.27 40 38 2 0.232 Score >1.27 100 87 13 (NA = 42) 2 NA, not available; SD, standard deviation; TERTp, TERT promoter. 3 a P-value calculated from epithelioid versus biphasic versus sarcomatoid 4 5 6 Table 2. Multivariate Cox regression analysis adjusted for clinical factors influencing 7 overall survival.

HR 95% CI P-value

Gender male 0.923 0.580-1.470 0.737 female

Age (years) <70 1.013 0.665-1.544 0.952 70

Histology epithelioid non-epithelioid 0.563 0.366-0.867 0.009

IMIG stage early (I/II) late (III/IV) 0.673 0.447-1.013 0.058

TERTp status TERTpwt 0.427 0.220-0.826 0.011 TERTpmut

8 CI, confidence interval; HR, hazard ratio; TERTp, TERT promoter.

4 Figure Legends

5 Figure 1: Impact of TERT promoter status and tumor histology on MPM patient overall

6 survival. Kaplan-Meier survival curves are given for the entire MPM patient cohort (N=182)

7 subgrouped with regard to (A) the TERT promoter status into wild-type (wt) versus mutated

8 (mut) cases and (B) MPM histology into epithelioid (epi) versus non-epithelioid (non-epi)

9 samples. Survival curves according to the TERT promoter status are shown (C) separately for

10 the Austrian (left) and the Croatian/Slovenian (right) cohorts and (D) for the epithelioid (left)

11 and non-epithelioid MPM histology (right). Statistical analyses were performed using log-

12 rank (Mantel-Cox) test; p-values are indicated.

14 Figure 2: TERT promoter status and in vitro MPM cell immortalization. (A) Association

15 between the ability for in vitro cell line formation and MPM patient overall survival is

16 depicted by Kaplan-Meier survival curves. Statistical analyses were performed using log-rank

17 (Mantel-Cox) test; p-values are indicated. Impact of (B) TERT promoter status, (C) MPM

18 histology, and (D) tumor stage at time of cell culture establishment on immortalized cell line

19 formation is shown. Statistical analyses were performed by Fisher`s exact test. *** =

20 p<0.001; ns = not significant. (E) Relation between patient overall survival and TERT

21 promoter status (wild-type = wt; mutated = mut), cell line formation ability (yes; no), MPM

22 histology (epithelioid = ep; non-epithelioid = non-ep; pleomorphic nuclear features = black

23 hashtags), C-reactive protein (CRP) levels before surgery, and tumor stage at time of cell

24 culture establishment (early; late) is depicted. Patients censored for survival analysis due to

1 death within 30 days following surgery or loss to follow-up are indicated by black asterisks.

2 Unknown = uk; not determined = n.d.

4 Figure 3: Array CGH analysis showing the association of TERT promoter mutations and

5 chromosomal instability in human MPM. (A) Number of altered (left), gained (middle), and

6 lost (right) probes in the TERT promoter wild-type (wt; N=13) and TERT promoter mutated

7 (mut; N=9) MPM cell models were calculated from the “interval-based text report” using the

8 Agilent Genomic workbench software (see Material and Methods). Single samples and mean

9 ± SEM are depicted. Differences were tested for significance by two-tailed Student`s t-test

10 with Welch`s correction; p-values are indicated. (B) Graphical penetrance analysis indicating

11 percentage of cell models with genome-wide gains and losses for the TERT promoter wild-

12 type (wt) and mutated (mut) MPM cell models (upper two panel). Gains are depicted in dark

13 gray above the 0 line and losses in light gray below. A more detailed view of chromosomes 1,

14 9, and 14 is given in the lower two panels. (C) The numbers of probes gained at chromosome

15 1 and lost on chromosome 9p and chromosome 14 are shown. Data were calculated from

16 the “interval based text report” as in (A). Single samples and mean ± SEM are depicted.

17 Differences were tested for significance by two-tailed Student`s t-test with Welch`s

18 correction; p-values are indicated.

20 Figure 4: Specific genomic alterations associate with the TERT promoter status. (A) MPM

21 cell models (N=26) were grouped according to TERT promoter status (left) or MPM histology

22 (right) and analyzed for presence of mutations and deletions of the indicated genes based on

23 NGS data (see Material and Methods). Deletions were determined from NGS data by read

24 coverage and in all cases confirmed by array CGH. Differences between the subgroups were

1 tested for significance by Chi-square test. *** = P<0.001; * = P<0.05. (B) Relation between

2 TERT promoter status (wild-type = wt; mutated = mut) and the following parameters:

3 MPM histology (epithelioid = ep; non epithelioid = non-ep; uk = unknown); BAP1 protein

4 expression and subcellular localization by immunohistochemistry (IHC, abbreviations as

5 indicated); presence of mutations/deletions of BAP1, CDKN2A, NF2, TP53, RBFOX1, and

6 GSTT1 in the investigated MPM cell lines as well as confirmation in tumor/healthy tissues, as

7 appropriate (blue asterisk = consistent alterations between tumor tissue and cell line;

8 consistent wild-type status is indicated by blue asterisk below the cell line name; red asterisk

9 = mutation only present in cell line; red question mark = alteration not confirmable due to

10 low coverage; g = germline deletion; na = not analyzed; s = somatic deletion; samples with

11 no remaining tumor specimen after cell culture work up are underlined).

Telomerase reverse transcriptase promoter mutations identify a genomically defined and highly aggressive human pleural mesothelioma subgroup

Christine Pirker, Agnes Bilecz, Michael Grusch, et al.

Clin Cancer Res Published OnlineFirst April 21, 2020.

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