A novel patient-derived cell line of adrenocortical carcinoma shows a pathogenic role of germline MUTYH mutation and high tumour mutational burden.

Background The response of advanced adrenocortical carcinoma (ACC) to current chemotherapies is unsatisfactory and a limited rate of response to immunotherapy was observed in clinical trials. High tumour mutational burden (TMB) and the presence of a specific DNA signature are characteristic features of tumours with mutations in the gene MUTYH encoding the mutY DNA glycosylase. Both have been shown to potentially predict the response to immunotherapy. High TMB in an ACC cell line model has not been reported yet. Design and Methods The JIL-2266 cell line was established from a primary ACC tumour, comprehensively characterised and oxidative damage, caused by a dysfunctional mutY DNA glycosylase, confirmed. Results Here, we characterise the novel patient-derived ACC cell line JIL-2266, which is deficient in MUTYH-dependent DNA repair. JIL-2266 cells have a consistent STR marker profile that confirmed congruousness with primary ACC tumour. Cells proliferate with a doubling time of 41±13 hours. Immunohistochemistry revealed positivity for steroidogenic factor-1. Mass spectrometry did not demonstrate significant steroid hormone synthesis. JIL-2266 have hemizygous mutations in the tumour suppressor gene TP53 (c.859G>T:p.E287X) and MUTYH (c.316C>T:p.R106W). Exome sequencing showed 683 single nucleotide variants and 4 insertions/deletions. We found increased oxidative DNA damage in the cell line and the corresponding primary tumour caused by impaired mutY DNA glycosylase function and accumulation of 8-oxoguanine. Conclusion This model will be valuable as a pre-clinical ACC cell model with high TMB and a tool to study oxidative DNA damage in the adrenal gland.


Abstract Background
The response of advanced adrenocortical carcinoma (ACC) to current chemotherapies is unsatisfactory and a limited rate of response to immunotherapy was observed in clinical trials. High tumour mutational burden (TMB) and the presence of a speci c DNA signature are characteristic features of tumours with mutations in the gene MUTYH encoding the mutY DNA glycosylase. Both have been shown to potentially predict the response to immunotherapy. High TMB in an ACC cell line model has not been reported yet.

Methods
The JIL-2266 cell line was established from a primary ACC tumour, comprehensively characterised and oxidative damage, caused by a dysfunctional mutY DNA glycosylase, con rmed.

Results
Here, we characterise the novel patient-derived ACC cell line JIL-2266, which is de cient in MUTYHdependent DNA repair. JIL-2266 cells have a consistent STR marker pro le that con rmed congruousness with primary ACC tumour. Cells proliferate with a doubling time of 41±13 hours. Immunohistochemistry revealed positivity for steroidogenic factor-1. Mass spectrometry did not demonstrate signi cant steroid hormone synthesis. JIL-2266 have hemizygous mutations in the tumour suppressor gene TP53 (c.859G>T:p.E287X) and MUTYH (c.316C>T:p.R106W). Exome sequencing showed 683 single nucleotide variants and 4 insertions/deletions. We found increased oxidative DNA damage in the cell line and the corresponding primary tumour caused by impaired mutY DNA glycosylase function and accumulation of 8-oxoguanine.

Conclusion
This model will be valuable as a pre-clinical ACC cell model with high TMB and a tool to study oxidative DNA damage in the adrenal gland.

Background
Adrenocortical carcinoma (ACC) is a rare endocrine cancer affecting approximately 0.5 to 2 per million people annually [1]. About 60% of patients present symptoms of adrenal steroid excess, such as Cushing syndrome or virilisation [2]. Surgery is the only curative approach but many patients with ACC experience a relapse, even after complete surgical resection [3,4]. Mitotane is the only approved drug in metastatic disease [5] and used both in a palliative and adjuvant setting [6,7]. Objective response rate of advanced ACC is however low with only 20% [8] and severe adverse effects are common [2,9]. Combination of etoposide, doxorubicin and cisplatin (EDP) is the most effective cytotoxic chemotherapy, but median overall survival is still poor with only 12-15 months [10].
Somatic mutations in the Wnt/ -catenin pathway [11] are present in ~40 % of cases with CTNNB1 mutations present in 25% [12] and ZNRF3 mutations in 21% [13]. Inactivating mutations in the tumour suppressor TP53 occur in at least another 20% of sporadic ACC [11]. Increased expression of IGF2 (Insulin like growth factor 2) that is found in ~90% of ACC [14][15][16] provided the rationale for a phase III clinical trial with the selective small molecule IGF-1R inhibitor linsitinib but the results were disappointing [17]. Only few patients derive clinical bene t from multi-tyrosine kinase inhibitors that have been clinically tested [18].
Genetic predisposition to ACC includes Li-Fraumeni syndrome (LFS) caused by mutations in the tumour suppressor TP53 in 3-7 % of ACC [19,20]. Mutations in the mismatch repair (MMR) genes MSH2, MSH6, MLH1, PMS2 cause Lynch syndrome (LS) and have been found in at least 3% of ACC [1,21]. Impaired MMR protein function leads to high tumour mutational burden (TMB) and -consecutively -the presence of a high number of tumour speci c neoantigens that may trigger an anti-tumoural immune response [22,23].
While the overall response rate of ACC to immunotherapy in clinical trials was heterogeneous [24][25][26], 2/9 of ACC patients responding to the programmed cell death 1 (PD-1) inhibitor pembrolizumab in a clinical phase II trial had LS [27]. In a patient-derived mouse xenograft of a LS-associated ACC, experimental immune checkpoint inhibition led to increased antitumoural immune cell in ltration and consecutive treatment response [28].
Similar to LS-associated DNA repair de ciencies, inactivating mutations in the MUTYH gene involved in oxidative DNA damage repair confer a heritable predisposition to colorectal carcinoma termed MUTYHassociated polyposis. MUTYH-de cient tumours exhibit a high TMB and are responsive to immune checkpoint inhibition [29]. Two genome-wide studies identi ed MUTYH germline mutations in four ACC with high TMB [30].
Yet, cell culture models to investigate factors of response and resistance to T cell-mediated anti-tumoural response in ACC [31] are scarce. Until recently, H295R [32] cells rst described in 1990 were the only available cell line. The rst paediatric ACC patient-derived xenograft (PDX) model (SJ-ACC3) was reported by Pinto et al. [33] and in 2016, Hantel and colleagues successfully developed the rst adult ACC PDX and established a respective cell line, termed MUC-1 [34]. The publication in 2018 of two additional PDXderived ACC cell lines including one from a LS patient [35] increased the number of available human ACC cell lines to four, which now allows comparative studies that better re ect the genetic heterogeneity of ACC [16,[36][37][38].
Here, we present a newly established ACC cell line with high mutational burden, which was generated directly from a patient-derived tumour. Since the development of the H295R cell line 30 years ago, this is the rst ACC cell line that was transferred directly to cell culture.

Patients
All patients included were participants of the ENSAT (European Network for the Study of Adrenal Tumours) registry and biobank. The study was approved by the Ethics Committee of the University of Würzburg (# 88/11) and all patients provided written informed consent for the use of tissue, cells, clinical data and genetic characterisation.

Establishment of the ACC tumour cell line
At the time of surgery, 1.3 mg of the primary tumour were used for cell culture after removal of the surrounding fat tissue. The tumour piece was minced and a single cell suspension was obtained using the gentleMACS Dissociator and Tumour Dissociation Kit (both Miltenyi Biotec), following the manufacturer`s instructions. Culture medium was supplemented with 5 µmol/l Rho-associated protein kinase (ROCK) inhibitor Y-27632 (Sigma-Aldrich), 10 µl/ml penicillin G/streptomycin (P/S, Sigma-Aldrich) and 250 µg/ml amphotericin B (Sigma-Aldrich) in adaption of a published protocol [39]. for 20 min at 4°C and subsequently washed twice with DPBS (1000 rpm, 5 min, RT). After the last washing step, cell pellets were resuspended in 2 ml paraformaldehyde (4 %) and kept in gentle movement at 4°C overnight. Suspensions were pelleted at maximal speed (15.000 rpm) using a microcentrifuge for 5 min. Pellets were incubated in 2 ml 70 % ethanol (EtOH) for 20 min and centrifuged at 1000 rpm for 5 min. This was repeated with 85 %-, 96 % EtOH and 100 % isopropanol. After removal of the supernatant, 1 ml para n was added to cell pellets and the sample placed into a para n tank for 1h at 60°C. Subsequently, embedded pellets were stored until slides were prepared.
For mitotane treatment, 3 x 10 4 JIL-2266 cells were seeded in black 96-well plates with clear bottom. After 24 hours, cells were treated for another 24 h with mitotane (AlsaChim) dissolved in EtOH and viability was determined with CellTiter Glo Assay (Promega) as described before [36].

Chromogenic immunohistochemistry
Formalin-xed, para n-embedded (FFPE) slides of tumour tissue and cell pellets were depara nised twice in xylol for 25 min and subsequently rehydrated. Antigen retrieval was performed in 10 mM citric acid monohydrate buffer (pH 6.0) for 13  Immuno uorescence FFPE slides were depara nised three times in xylol for 5 min and subsequently rehydrated. Slides were incubated with proteinase K for 30 min at RT and afterwards with 100 μg/ml RNAse A for 1 h at 37 °C. DNA was denatured in 2 N HCl for 5 min and neutralized with 1 M Tris-base. Unspeci c binding sites were blocked in 10 % goat serum for 1 h. Incubation with primary antibody (8-oxoG, Trevigen, #15A3), occurred in a dilution of 1:250 in DPBS with 0.1 % BSA (w/v) at 4 °C overnight. The primary antibody was omitted in negative controls. Secondary antibody (goat-anti-mouse Alexa Fluor488, ThermoFisher) was incubated in the dark for 1h at RT at a dilution of 1:200 in PBS with 0.1 % BSA (w/v). Nuclei were stained with DAPI (1:1000) for 3 min in the dark, slides washed with DPBS and mounted with ProLong Gold Antifade (ThermoFisher). Analyses of immune cell in ltrates were performed as described previously [31].

Microscopy
Microphotographs were taken with the Leica Aperio slide scanner (20 x objective) and processed with Aperio Image Analysis software (chromogenic immunohistochemistry) or the Zeiss Axioscope.A1 microscope (40 x objective) equipped with a Zeiss Axiocam 503 mono (immuno uorescence).

Extraction of DNA and RNA, qPCR
Pathological anatomical assessment con rmed high tissue quality and the presence of 90 % vital tumour cells by haematoxylin-eosin staining. Genomic DNA was isolated from fresh frozen ACC tumour, cell line (p 6, 8, 13, 29) and patient-matched leukocytes by using the Maxwell RSC Blood DNA Kit (Promega). DNA concentration was determined by Nanodrop spectrophotometer (Thermo Fisher).
Short tandem repeat (STR) pro ling DNA from JIL-2266 cells and corresponding leukocytes from donor patient was extracted using the Maxwell RSC Blood DNA Kit (Promega). Typing was performed using the STR-loci VWA, THO1, TPOX, CSF1PO, D16S539, D13S317, D7S820, D5S818 and the amelogenin sex-determining marker (AMELlocus) to show patient originality, analyse congruousness and to rule out cross-contamination in the course of cultivation after p 5, 8, 13, and 29. Loci were ampli ed and electrophoretic analysis was carried out with the GeXP-instrument (AB Sciex). Data were analysed using the GeXP fragment analysis software.
Proliferation assay 1 x 10 5 JIL-2266 (p 30, 32, 33) and NCI-H295R per well were seeded into a 12-well plate. After 24 h, 48 h and 72 h, cells were harvested with trypsin and numbers were quanti ed in duplicates by using the Countess® Automated Cell Counter (ThermoFisher). Doubling time was determined using the exponential growth function in GraphPad Prism 8 software [40].
Supernatants were collected and steroids were quanti ed with the MassChrom steroids kit (Chromsystems) on a Qtrap 6500+ (Sciex) mass spectrometer coupled to a 1290 In nity HPLC System (Agilent). Signal analysis was performed with Analyst Software (1.6.3, Sciex) as described elsewhere [41].

Library preparation
SureSelectXT Human All Exon V6 Kit (Agilent) was used for library preparation. Paired end sequencing with a read length of 100 base pairs (bps) was performed on a NovaSeq 6000 (Illumina). For the library preparation of the tumour and matched control sample, the xGen Exome Research Panel v2 (Integrated DNA Technologies) was used and paired end sequencing with a read length of 150 bps was performed on a NextSeq 500 (Illumina).
For germline variant calling, we used GATK3 and GATK4. MuTect2 integrated in the GATK4 package was used for somatic variant calling. All variants were annotated with ANNOVAR, v2019-10-24 [46] and considered if they were below a frequency of 2 % in the databases 1000g2015aug_all, ExAC_nontcga_ALL, gnomAD_exome_ALL and gnomAD_genome_ALL, if the position is covered by at least 20 reads and the alternative allele is covered by at least 8 reads and comprised at least 10 % of the total reads. The ltered MuTect2 results were used for TMB calculations. Mutational signatures were identi ed using MutaGene [47]. For this analysis, synonymous variants were also included.

Case presentation
The donor of the tissue was a female 50 years old patient who presented with severe Cushing´s syndrome including hypertension, muscle weakness, hypokalaemia and lymphopenia. Computer tomography scan indicated a left adrenal mass with a maximum of 9 cm diameter and pulmonary metastases. Endocrine work-up according to current guidelines [4] revealed androgen and cortisol excess (including pathological dexamethasone suppression test). Adrenolytic therapy with mitotane and metyrapone was initiated to ameliorate Cushing's syndrome, followed by left adrenalectomy two weeks later. Routine histological examination con rmed the diagnosis of an advanced high-grade ACC (35 mitoses / 10 high power elds (HPF)), Ki67 proliferation index of 60% up to 90% and a Weiss score of 9. By immunohistochemistry, the tumour was positive for steroidogenic factor-1 (SF-1) and partially for inhibin α. In line with our previous study of immune cells in ACC [31], the donor tumour was in ltrated by CD3 + -, CD4 + -and CD8 + T lymphocytes to a moderate extent (7.8, 3.3 and 5.2 T cells per HPF, respectively; Figure 1 A). Programmed cell death-ligand 1 was expressed in less than 1% of tumour cells and DNA MMR protein expression was normal. After surgery, the patient received chemotherapy with EDP in addition to mitotane. After two cycles, the patient experienced rapid progression of pulmonary metastases, new liver metastases and died shortly thereafter.
The family history was remarkable with a cerebellar tumour not otherwise speci ed in the mother and breast cancer in the sister and grandmother. LFS was excluded by routine germline sequencing of TP53.
Establishment of the JIL-2266 ACC cell line Tumour material was obtained from surgical resection and subsequently enzymatically processed. By using chemically modi ed polystyrene Primaria culture asks with a 1:1 ratio of nitrogen and oxygen and without CO 2 supply, the generated cell suspension was initially cultured in the presence of ROCK inhibitor supporting immortalization of human tumours [39]. Subsequently, cells initially grew in primary culture preferably in colonies. By means of weakly renewal of media up to one half, cells started to proliferate and were passaged rst after 180 days. While the time interval for passaging decreased to 90 days, broblast removal was performed after further 270 days at p 4. By consistent appearance, the new cell line proliferated until con uence for further 50 days. At that point, the cells were termed JIL-2266 and routinely passaged with further declining intervals (from 168 h to 41 h) and characterised.
To con rm match of the cell line and the corresponding human blood sample, STR pro ling was performed. JIL-2266 cells only exhibited alleles expressed in the corresponding human blood DNA, con rming the authenticity and correspondence between cell line and respective human tissue. For three loci -THO1 (chromosomal localization: 11p15.5; intron 1 tyrosine-hydroxylase gene), D16S539 (chromosomal localization: 16q24.1) and TPOX (chromosomal localization: 2p25.3; intron 10 thyroidperoxidase gene) -the JIL-2266 cell line demonstrated loss of heterozygosity (LOH) ( Table 1). Comparison with large cell line STR pro le databases (DSMZ online STR analysis https://www.dsmz.de/services/human-and-animal-cell-lines/online-str-analysis) provided the proof of a unique genomic identity and excluded cross-contamination.

Immunohistochemistry reveals similarity between primary tumour and JIL-2266 cells
For con rmation of the adrenocortical origin of JIL-2266 cells and comparison with the standard ACC cell line H295R, immunohistochemistry of the primary tumour and cell pellets was performed. SF-1 was moderately expressed in the corresponding primary tumour and in JIL-2266 cells, while H295R cells showed a very strong SF-1 expression (Figure 1 B). The primary tumour showed only partial expression of inhibin α with very localized positive areas, while inhibin α expression was completely absent in the corresponding cell line JIL-2266. H295R cells showed strong inhibin α expression (Figure 1 C). Ki-67 and TP53 expression were high in the primary tumour, JIL-2266 and H295R cells (Figure 1 D and E). Analysis of cell pellets revealed a larger size of JIL-2266 compared to H295R cells (Figure 1). JIL-2266 cells grow adherent to the plastic bottom of the culture ask and morphological investigation of p 8, 14 and 30 con rmed morphological stability during multiple passaging steps and over time ( Figure S1).

mRNA expression of adrenal gland markers
Expression of adrenocortical markers transcripts by qPCR showed similar expression of PINK1, BUB1B and CTNNB1 in the matching primary tumour and JIL-2266 cells with insigni cant differences between passages (Figure 2). BUB1B was overexpressed in all ACC samples compared to normal adrenal gland (nAG). Expression of adrenal cortex markers was variable and lower in all ACC samples compared to nAG. MC2R expression was undetectable in JIL-2266 cells and highest expression of steroidogenic markers were observed for SREBP1 and SREBP2 (Figure 2).

Sensitivity to mitotane
The proliferation rate of the H295R was 36±15 hours (Figure 3 A) (Table S1). 48 h treatment with forskolin did not result in detectable steroidogenesis of JIL-2266 cells (Table S1). Yet, when JIL-2266 cells were cultured with adenine, insulin, EGF and cholera toxin, small amounts of the androgens and the precursors progesterone, 17-OH-progesterone, DHEA, androstenedione and testosterone were detectable, which decreased with higher passages (FigureS2 and Table S1). These in vitro data are consistent with the plasma steroid hormone pro le in the donor who had increased androstenedione, testosterone and 17-OH-progesterone. Taken together, this novel human ACC cell line exhibits high TMB caused by oxidative DNA damage that fails to be counteracted by BER due to pathogenic MUTYH mutation.

Discussion
ACC is a very rare and heterogeneous malignancy and the development of new and effective treatment options is signi cantly hampered by the lack of pre-clinical models that mirror the clinically heterogeneous picture of the disease. Here we describe a novel patient-derived ACC cell line, JIL-2266, which is to our knowledge the rst cell line since 30 years that was established by direct transfer into cell culture. Interestingly, JIL-2266 harbour a high TMB due to a pathogenic MUTYH mutation. SF-1 positivity is an essential marker of adrenal cortical origin [50]. Both JIL-2266 cells and its corresponding tumour show moderate SF-1 expression (Figure 1 B). JIL-2266 cells also express additional adrenal markers involved in steroidogenesis albeit at lower levels than H295R cells; however, both less in comparison to normal adrenal glands. This is in line with low expression of steroidogenic enzymes and absent steroid hormone secretion in the JIL-2266 cell line under basal conditions; only seemingly in contrast to the clinical presentation of the patient who had pre-operative pathological elevation of steroid blood concentration. Indeed, the very high tumour burden in the presence of rather low steroidogenic capacity of tumour cells may explain the discrepancy. In general, steroid hormone secretion is variable in the JIL-2266 cells and similarly dependent on cell culture supplements and passage as in H295R cells [41]. Lower steroid secretion was associated with higher passages ( Figure S2). It is conceivable that the accumulation of mutations with increasing passage negatively affects the cells' steroidogenic capacity. ACC markers PINK1, BUB1B and CTNNB1 were lower in the JIL-2266 cell line compared to H295R cells. Yet, expression was similar to that in the primary tumour.
Mutations in DNA repair mechanisms have been found in a small proportion of ACC and mostly affect DNA MMR genes. MUTYH encodes a MutY DNA glycosylase and is part of the BER involved in the recognition and resolution of 8-oxoG-adenine mismatches by excising the mis-paired adenine. Sporadic cases of ACC have been shown to harbour MUTYH mutations. Pilati et al. described two hyper-mutated ACC tumours dominated by C>A transversions and germline MUTYH mutation (ACC33: NM_001128425.1:c.1187G>A, p.Gly396Asp and ACC39: NM_001128425.1:c.721C>T, p.Arg241Trp) [13]. Additionally, by analysing the TCGA cohort consisting of 91 ACCs, once again two tumours with COSMIC signature 18 and rare pathogenic MUTYH mutations (A: NM_001128425.1:c.467G>A; p.Trp156* and B: NM_001128425.1:c.536A>G, p.Tyr179Cys) were observed [11,30].
Yet, the MUTYH (NM_012222.2:exon3:c.316C>T: p.R106W) mutation identi ed in our ACC patient and the derived JIL-2266 cell line has not been described in ACC but was observed in colorectal cancer [51]. This mutation was heterozygous in the germline of the patient and became hemizygous in the tumour. The observed strong accumulation of 8-oxoG in the primary tumour and JIL-2266 cell line, to a comparable extent as observed in H295R cells exposed to oxidative stress (H 2 O 2 treatment), suggests a strong impairment in MutY DNA glycosylase function due to the mutation identi ed. Impaired DNA repair is further indicated by the increase of TMB in the JIL-2266 cell line after excessive passaging and replications compared to the primary tumour as revealed by exome sequencing. Our cell line exhibited the distinctive COSMIC mutational signature 18 associated with oxidative DNA damage and characterised by an enrichment of C>A transversions [30,49] caused by MUTYH mutations. COSMIC signature 18 has been rarely described in ACC and all these tumours were found to harbour MUTYH mutations [30].
PD-1 checkpoint inhibition may be particularly valuable in patients with DNA repair defects; the PD-1 inhibitor pembrolizumab is FDA-approved for MMR de cient or microsatellite instability high (MSI-H) solid tumours regardless of its origin. ACC has been shown to be associated with germline mutations in DNA MMR genes and hence LS-associated tumour [21,52]. While the overall response to immunotherapy in ACC patients is heterogeneous [24,27] and not certainly associated with PD-L1 expression status [27], results from clinical trials [27] and case reports of successful PD-1 inhibition in MMR de cient patients are encouraging [53]. Hence, our novel cell line, derived from a primary tumour with present tumourin ltrating lymphocytes also shows a de cient DNA repair mechanism and high TMB, which combined present a unique model to study antitumoural response to immunotherapy in vitro. Accordingly, a MMR de cient, high TMB humanized CU-ACC2 PDX demonstrated a signi cant increased immune in ltration following PD-1 inhibition and tumoural response to treatment [28].
Consistent with failure of the patient's tumour to respond to mitotane treatment, JIL-2266 cells were unresponsive to mitotane in culture. The patient´s rapid disease progression precluded a therapeutic attempt with immune checkpoint inhibitors that have been demonstrated to be effective in other MUTYHmutated tumours [29].

Conclusion
The newly developed JIL-2266 ACC cell line presents an additional and valuable tool re ecting the genetic heterogeneity of ACC. JIL-2266 will enable a better understanding of mutY DNA glycosylase function in ACC and allows to study further the mechanisms of resistance to mitotane. Moreover, this new ACC cell line exhibits high TMB and COSMIC signature 18 and will be valuable for the assessment of mechanisms underlying response and resistance to immunotherapy in ACC.
MUTYH should be included in the genetic counselling and testing strategy of ACC to identify patients who potentially respond to immunotherapy [29,52,53,56] Tumours) registry and biobank. The study con rmed to the principles of the Declaration of Helsinki, the Good Clinical Practice Guidelines and was approved by the Ethics Committee of the University of Würzburg (# 88/11). All patients provided written informed consent for the use of tissue, cells, clinical data and genetic characterisation.

Consent for publication
This manuscript does not contain personal and/or medical information about identi able living individual. All patients were anonymised and provided written informed consent.

Availability of data and materials
All data generated or analysed during relevant to the current study are included in the article, uploaded as supplementary information or are available from corresponding author on reasonable request.

Competing Interests
The authors declare no competing interest.

Funding
This research work was funded by the German Research Council project 237292849 (to MK and MF) and project 314061271-TRR 205 (project B16, B20 and S1, to M.F. and M.K.).
Author´s contributions IW and MK participated in the study design and supervision. MF and MK were responsible for funding acquisition. LSL, JS, SA, SH, SK and IW performed the experiments and are involved in data analyses.
LSL, JS, MK and IW prepared and edited the nal manuscript. All authors read and approved the nal manuscript for publish.