Circulating tumor DNA in patients with Uveal Melanoma (UM) may be associated with the tumor recurrence: Co- accordance of pathogenic variants GNAQ, GNAS and IDH1 in recurrent UM

doi:10.21203/rs.3.rs-4362659/v1

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Research Article

Circulating tumor DNA in patients with Uveal Melanoma (UM) may be associated with the tumor recurrence: Co- accordance of pathogenic variants GNAQ, GNAS and IDH1 in recurrent UM

https://doi.org/10.21203/rs.3.rs-4362659/v1

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Background

Uveal melanoma (UM) is the most common primary intraocular malignancy. Prediction of recurrence risk in UM patients may be done by tumor genotyping for early diagnosis, increasing clinical and pathological accuracy. The purpose of this pilot study is to investigate the importance of pathogenic variants of ctDNA in predicting early diagnosis of recurrent in UM and increasing the globe survival rate of UM by early treatment.

Methods

Variant allelic frequency (VAF) of circulating tumor DNA (ctDNA) was investigated in three patients with choroidal uveal melanoma using the ion ampliseq cancer hotspot panel v2 and Ion Proton™ sequencing. Serial sampling of ctDNA was done from 12 blood samples in 3 cases (Two recurrent patients + one non-recurrent patient) in 4 time periods.

Results

Variant Call Format (VCF) was analyzed through the Franklin Genoox platform. The variant allele frequency (VAF) of ctDNA was examined at each case in 4 phases. After applying filters, we reached 10 important genes out of 50 genes in the panel. Finally, by comparing ctDNA variants, we found the co-occurrence of GNAQ, GNAS and IDH1 mutations at the time of recurrence. Minimum VAF of co-occurrence these genes were reported 0.51%, 0.25% and 0.1%, respectively. Also, in recurrent cases tumor was located at the posterior fundus, but non-recurrent lesion were located in peripheral part. In patient non-recurrent disease, the pathogenic variants related to UM invasion was negative and the VAF of other important genes was decreased from baseline to the last phase.

Conclusion

Considering the poor prognosis associated with the development of recurrent UM, investigating the level of pathogenic variants in GNAQ, GNAS and IDH1 variants in ctDNA may be useful in predicting UM recurrence and proper management of with adjuvant treatment. It is suggested that further studies are needed to develop the best panel and determine the best cut-off level of VAF for early diagnosis of recurrent UM to confirm the implications of GNAS and IDH1 in UM tumorigenesis.

Uveal melanoma (UM) is one of the most common intraocular malignancies in adults, which includes 3–5% of all melanomas. Predominant disease site originates from choroidal melanocytes [1, 2]. The incidence rate in North America and Europe is between 5.74 and 7.30 cases per 1 million [3].

More than 92% of UMs contain oncogenic mutations in the alpha subunit of GNAQ and GNA11 genes at position Q209 and R183 [4–6], which induce cell proliferation, tumor growth and progression by targeting the Akt/ mTOR, Wnt/β-catenin, Yes-associated protein (YAP), and MAPK signaling pathway [7]. BAP1 (BRCA1 Associated Protein 1) is a tumor suppressor gene located on 3p21 and is mutated in approximately 80% of UM metastasizing patients [8]. In addition to point mutations, UM is associated with chromosomal abnormalities and cytogenetic changes. Monosomy 3, loss of 1p, gain of 1q, gain of 6p, loss of 6q, loss of 8p, gain of 8q are common chromosomal abnormalities in UM. These genetic changes can lead to an increase in genomic instability and increased metastasis [1, 9–11]. Currently, UM can be divided into 4 classes Based on the Cancer Genome Atlas (TCGA) classification: class A [disomy 3 and 8q], class B (disomy 3/partial extra 8q), class C (monosomy 3/8q gain) and class D (monosomy 3/multiple 8q gain) [6, 12]. However, The American Joint Committee on Cancer (AJCC) staging considers clinical factors of the tumor, such as tumor size, extrascleral extension and ciliary body involvement [13]. Constellation of information from these two classifications of anatomical features and genetic information improve the prognostication of patients with UM [14].

The local recurrences rate was appeared at 6% within 5- years of initial treatment [15]. Tumor size (largest basal diameter (LBD) and thickness (TH)) are two important prognostic factors that increase the likelihood of recurrence [16–21]. When the initial doses of radiation were delivered via brachytherapy (BRT) at the time of diagnosis are insufficient to reach the base of the tumor, leading to frequent local and/or distant recurrences and increased need for systemic therapy or enucleation [22]. Several studies suggest that following BRT for choroidal melanomas there is often more than 90% local tumor control [23–25]. However, this rate after BRT has a wide range of variation between ocular oncology centers, which has been reported from 0–22% [25–28].

Cell-free DNA (cfDNA) introduced as a non-invasive biomarker in tumor personalized medicine [29]. Then ctDNA was detected in the cfDNA components of a cancer patient's blood that caused by apoptosis and necrosis of tumor cells [30, 31]. ctDNA analysis enables diagnosis and identification of non-invasive tumor detection, prediction of treatment response, monitoring of disease recurrence and identification of mechanisms of resistance for development targeted therapies [32].

Several studies on cutaneous melanoma have shown that ctDNA, in addition to being correlated with tumor size and accurately indicating tumor mutations, also detect recurrence [33, 34] and treatment failure prior to radiological imaging [35–37]. In addition, to identify stage III cutaneous melanoma patients at risk of recurrence, ctDNA detection can be helpful [38, 39].

Today, there is increasing evidence that ctDNA has the ability to detect disease recurrence earlier or at the same time as conventional methods [40–43]. The use of ctDNA remains finite for recurrence and monitoring of residual disease in clinically disease-free patients [40, 42, 43]. Therefore, whether ctDNA is detectable at early stages and/or can provide early evidence of disease advancement is unclear. We aimed to investigate the pathogenic variants of ctDNA in predicting early diagnosis of recurrence UM and improving the patient's survival.

Patients and method

In this prospective Pilot Study between June 2020 and July 2022, ctDNA were measured in all 3 patients that only two recurrent and one without clinical evidence of recurrence in patients with UM. Overall, we collected 12 blood samples from a cohort of 3 consecutive choroidal uveal melanoma at 4 phases (Baseline, 6th month, 12th month and 24th month after treatment).

Variant allele fraction (VAF) was checked using Ion Proton™ Sequencer in any phases. The ctDNA extraction samples of the patients were collected at each phase of the disease and then the sequencing was done in the Genome Center of Nilou Laboratory using Ion Torrent platform. Our study with the Ethics code IR.IUMS.REC.1398.730 was approved by the "Iran University of Medical Sciences ", and written informed consent was received from participating cases in this study.

Indirect ophthalmoscopy, fundus photography, B-scan ultrasound evaluation, or ultrasonic biomicroscopy (Ellex, Adelaide, Australia) was analyzed for clinical status, along with abdominal magnetic resonance imaging (MRI) and chest computed tomography (CT) scans The clinical features and demographics included age, gender, and clinical status, while tumor features included the largest basal diameter, height, location, laterality of disease, and primary treatment type (enucleation vs. plaque brachytherapy).

Sample processing and extraction of cfDNA

10 ml of peripheral blood was collected from all the patients in Norgen cfDNA blood collection tube (Cat. 63950) at each phase. The time it took for the blood collection and cfDNA isolation was 2–3 days. According to studies, the half-life of cfDNA in circulation is between 16 minutes and 2.5 hours [44, 45].

After centrifugation (400 ×g in 10 min, 12000 ×g in 10 min, 20000 ×g in 10 min) at least 4 ml of plasma was collected for the next step.

The supernatants were collected and were kept at -80°C until DNA extraction. cfDNA was extracted using the HiPure Circulating DNA Midi Kit C(Magen Biotech, China). Then, cfDNA concentration was read using Qubit 2.0 Fluorometer (ThermoFisher, Paisly, UK) with dsDNA High sensitivity assay kit. After ensuring the proper concentration, we started preparation of Ampliseq libraries. Sequencing library preparation is provided by Ion ampliseq cancer hotspot panel v2 that contains a set of primers designed to probe hotspots in genes commonly mutated in human cancer, which targeted 50 oncogenes and tumor suppressor genes to amplify 207 amplicons covering nearly 2,800 COSMIC mutations (https://tools.lifetechnologies.com/content/sfs/brochures/Ion-AmpliSeq-Cancer-Hotspot-Panel-Flyer.pdf, ThermoFisher). Out of 50 genes in this panel, 29 genes related to UM (Supplementary Information, Table-S1).

Manufacturer's protocol was used for the preparation of Ion AmpliSeq™ DNA libraries ((Life Technologies, Carlsbad, CA, USA). Multiplex PCR was done with the Ion Ampliseq library kit 2.0 using 3ng DNA and the panel single primer pool. The steps of library preparation and sequencing are briefly described in the following Supplementary Information, Table-S2.

Using the FFPE extraction kit (QIAamp DNA FFPE Tissue kit, QIAGEN), we extracted genomic DNA from a paraffin-embedded retinal tissue sections of patient 1. Then, to look for copy number variations we conducted the MLPA-UM kit (P027-C2, MRC-Holland, Amsterdam, and The Netherlands), according to the manufacturer's protocol.

Analysis method

The data from the Ion Proton™ Sequencer (Thermo Fisher Scientific – US) were first run using the Ion Torrent platform-specific pipeline software, followed by reprocessed to read the sequences, cut the adapter sequences, filtration, and retransfer weak signals. The sequences were aligned to the Homo sapiens hg19 reference genome and stored as BAM files.

Initial variant calling from Ion AmpliSeq sequencing data was performed using Torrent Suite version 5.12 software with the “variant caller” plugin program using key parameters: minimum allele frequency = 0.1 for variant calling of single nucleotide polymorphisms (SNPs) and 5% for small insertions and deletion (indels), quality control > 99.99%, mean read depth > 1500x with coverage > 100 reads and P -value < 0.01.

Sequencing data provided a Variant Call Format (VCF) with a list of variants for the final clinical report. Open-source FRANKLIN database (https://franklin.genoox.com) takes VCF file and patient's phenotypes. All variants were analyzed through the Franklin Genoox platform to obtain proposed classifications using the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) guidelines for interpretation and evidence associated with each variant.

According to ACMG guideline, the variants are classified into 5 main categories including Pathogenic, Likely Pathogenic, Uncertain Significance, Likely Benign and Benign. We also classified the sequence variants in somatic conditions into four categories based on their clinical effect according to AMP guidelines: Tier I, variants with strong clinical significance; tier II, variants with potential clinical significance; tier III, variants with unknown clinical significance and tier IV, variants that are benign or probably benign.

Furthermore, from a wide range of filter parameters, including variant annotation (Chromosome, effect), variant calling (read coverage, genotype quality), internal frequency (minor allele frequency (MAF) and filters use external population datasets (gnomAD، 1000 Genomes).

Variant type prediction via dbscSNV, Splice AI, VARITY, MetaLR and Revel; and clinical interpretation is done through ClinVar, ClinGen, GeneReviews and OMIM databases.

In this study, we chose Pathogenic, Likely Pathogenic, Uncertain Significance for germinal classification and Tier I, Tier II for somatic classification. Next, Genetic changes potentially effective in malignancy (PAF, Potentially actionable findings) were identified and prioritized based on the level of pathogenicity. Finally, variants that placed in poly-nucleotide site and missing data (no calls) were excluded during the analysis.

Clinical Finding

Patient 1:

A 57-year-old man was referred with posterior choroidal malignant melanoma of the left eye in July 2015. In indirect ophthalmoscopy of fundus, a prominent pigmented mass was noted in the macula with dimensions of 14 x 13 mm by 8 mm in height which was subjected to Ru-106 plaque radiotherapy. The patient also underwent systemic examination for possible metastasis. Abdominal sonography and triphasic CT scan of the liver revealed two hemangiomas with dimensions of 50x70 mm in the left lobe and another with a size of 7x16 mm. These lesions were confirmed by MRI. Because of inadequate response and suspicious recurrent disease, secondary Ru-106 plaque was placed in July 2020, and the first sampling of the patient was done two days before placing the 2nd plaque, and it was followed every 6 months until July 2022 (Supplementary Information, Figure-S1).

Due to presence of a new satellite lesion and pigmented vitreous seeding (clumps) development in follow up a vitreous biopsy was done; active malignant pigmented cell was reported in pathology report. So, after discussion with patient, he scheduled for enucleation. After enucleation, pathology categorized the tumor type into mixed spindle cell A & B. Blood tests such as lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALK) were reported as normal. The tissue sample was subjected to MLPA for cytogenetic changes.

Patient 2:

A 49-year-old man was referred for posterior choroidal malignant melanoma of the right eye, with a prominent pigmented mass in fundoscopy. The first sampling was done in September of 2020, a few days before BRT and serial sampling was done every 6 months until September of 2022. A systemic examination was also performed, which did not show any metastasis during these two years. One year after plaque radiotherapy, UBM test revealed a slight increase in tumor thickness. To further consolidation of the tumor, adjuvant Transpupillary thermotherapy (TTT) was done in phase 3(12th month). In the last phase, the thickness of the tumor was slightly reduced compared to phase 3. Also, Blood tests (LDH, AST, ALT and ALK) were reported as normal.

Patient 3:

A 61-year-old woman was referred with diagnosis of iris nevi in the right eye and large vascularized mass of the left eye. Clinical exam and UBM test result revealed a large Iridociliochoroidal malignant melanoma at nasal part (Supplementary Information, Figure-S4-S5). The patient refused fundus imaging in the early stages of treatment. Similar to other patients, preliminary workup of metastasis was reported as normal. One week before BRT, the first sampling was done, and during the 24-month follow-up, UBM and clinical exam showed Progressive reduction in tumor thickness and tumor was under control.

Genetics Finding

The serial analysis of ctDNA levels of patients with UM after applying filters (somatic classification, germinal classification, region, effect and variant allele frequency above 0.1%) [46–48], revealed 10 important variants out of 50 genes in the panel.

According to the KEGG pathway database and GeneCards, the important genes obtained were divided into two groups: oncogenic (70% (7/10)) and tumor suppressor genes (TSG) (30% (3.10)), and all of them are located in MAPK, PI3K/AKT7/mTOR signaling pathway (Table-1). Subsequently, these important genes were analyzed in each patient at each phase.

In patient 1, in time of recurrence at baseline variants related to UM recurrence were GNAS: c.602G > A, IDH1; c.394C > T, GNAQ: c.626A > G and FGFR2: c.1144T > C (Figure-1-A).

In the 6th month, VAF for all variants were undetectable. In the 12th month VAF in GNAS increased and then in last phase it's decreased (VAF = 0.05, Allele Depth = 2000). In the last phase VAF in other variants (IDH1, GNAQ and FGFR2) re-increased.

The reason for decreasing the GNAS in the 3 phase may be due to the elimination of the subclones containing this variant by the dominant subclones due to positive selection [49].

VAF in Other variants related to UM including of STK11: c.996G > A, EGFR: c.2234A > G, GNA11: c.625C > A, EZH2: c.1937A > G, ATM: c.9139C > T and PTEN: c.328C > T increased in phase 3 (12th month) and 4 (24th month) (Figure-1-B). Next, the tissue sample was subjected to MLPA for cytogenetic changes and was evident the removal of the short arm of chromosome 8 (8p) (Supplementary Information, Figure-S6).

In patient 2, in time of recurrence at 12th month, variants related to UM recurrence were GNAS: c.601C > A, IDH1; c.395G > A, GNAQ: c.626A > C, STK11: c.109C > T, EGFR: c.2281G > A, GNA11: c.625C > A and EZH2: c.1937A > G. After TTT (2 weeks later) except of STK11 and EGFR variants, other variants decreased in last phases (Figure-2-A).

In the 6th month, VAF for all variants were undetectable. In the 12th month frequencies of GNAS increased, and then in last phase it's decreased (VAF = 0.05, Depth = 2000). In the last phase VAF in other variants (IDH1, GNAQ and FGFR2) re-increased.

VAF in other variants related to UM including of ATM: c.9139C > T, and FGFR2: c.1124A > G increased in phase 3 and 4. But, PTEN: c.328C > T increased in phase 4 only (Figure-2-B). By applying TTT in phase 3 after tumor recurrence, we observed a decrease in ctDNA level in the last phase (GNAS, IDH1, GNAQ/11 and EZH2).

In patient 3 (as a non-recurrent patient), the pathogenic variants related to UM invasion (GNAS, GNA11, EZH2, IDH1, and ATM) were negative and the VAF in other important genes (STK11: c.109C > T, EGFR: c.2356G > A, FGFR2: c.1124A > G and PTEN: c.992A > G) decreased from baseline to the last phase (Figure-3). Finally, we found only co-occurrence 3 genes including GNAQ, GNAS and IDH1 can be considered as important genes involved in UM recurrence (Table-2).

Next, using the BioGRID4.4 database, the interaction between proteins was drawn online. GNAS protein interacted with the GNAQ/11 and BAP1 proteins (Supplementary Information, Figure-S7) andIDH1 interacted with BAP1 protein (Supplementary Information, Figure-S8).

Our study detected ctDNA alterations before and after treatment of TTT, BRT and enucleation for two recurrent and a non-recurrent UM in 4 phases. VAF was checked using Ion Proton™ in the cell-free DNA and the proportion of mutant DNA/ total number (mutated + wild-type sequences) that represents a percentage alteration in the plasma of the patients.

Interestingly, after analyzing the 50-gene panel, there were 10 important genes involve in UM recurrence including GNAQ, GNA11, GNAS, EGFR.FGFR2, IDH1and EZH2 as oncogenic genes and STK11, PTEN and ATM as suppressor genes, that took part in the UM pathway. Several studies showed that these genes are involved in UM tumorigenesis [46, 50–54]. Only 2 studies to date have shown that UM tumors harbor IDH1 mutations with variants V178I [52] and R132C [54].

We identified that similar to other studies after BRT and enucleation, ctDNA has a positive effect on variants load [44, 45]. In addition, our study revealed that after TTT, load of some variants was also decreased.

By comparing ctDNA variants in 2 recurrent and one non-recurrent uveal melanoma cases, it was found that the co-occurrence of GNAQ (Q209P/R), GNAS (R201H/C) and IDH1 (R132H/C) may lead to recurrence UM that is involved in the BAP1 pathway, which in turn is an important factor in UM metastasis and invasion. Jiang, et al. 2022 have proved that somatic co-mutation is one of the mechanisms of Tumorigenesis [55]. But, one study showed that co-occurrence of BAP1 and SF3B1mutations has the inhibitory effect of UM invasion [56].

In the patient with no recurrent lesion, the pathogenic variants GNAS, GNA11, EZH2, IDH1, and ATM related to UM invasion were negative. In addition an assessment of important genes in this patient with no recurrent lesion showed a descending of VAF trend from baseline to the 24th month.

Our results showed, there is a probability of UM recurrence with the minimum co-occurrence VAF in GNAQ, GNAS, and IDH1 is 0.51, 0.25%, and 0.1%, respectively.

Claudia H. D. et al. in 2021 reported recurrence/ metastatic patients prior to the clinical diagnosis of disease progression had a positive ctDNA biomarker (GNAQ or GNA11) (ranging between 2 and 10 months (mean 5.7 months). Minimum VAF for recurrence/ metastatic patients was reported 0.13%. However, they did not evaluate the VAF trend from the baseline to the 24th month in different phases [47].

Jasmine et al. in 2022 evaluate the presence of ctDNA in plasma of UM patients and found two patients have GNAS mutation, but a patient with metastatic had initial ctDNA VAF = 0.29% and 0.72% around the time of metastases [46]. In contrast, our study showed ctDNA VAF in GNAS was 0.25% at the time of recurrence. So, it may indicate that the desired cut-off for metastasis development has not yet been achieved.

Two known risk factors for tumor recurrence are the tumor size (largest diameter and tumor thickness) and the tumor location (commonly the posterior position) [16, 57]. In our study, 2 recurrent patients had an increase in tumor size at the time of recurrence and the location of the tumor was at the posterior fundus, but in a patient with no recurrent UM located in the anterior position.

In addition, monosomy 8p was observed in a case that received BRT twice and was enucleated after recurrence. Loss of the 8p12-22 region is associated with a rapid metastasis in primary UMs with aggressive morphology due to deletion of multiple genes such as LZTS1 [58–60].

The main limitations of this study are the relatively short follow-up time, the small number of investigated patients because of budget limitation and lack of normal tissue in patients as a control. Nevertheless, our experience confirms that the presence of ctDNA levels with an increasing trend may be a harbinger of recurrent disease. Early detection of patients with recurrent lesion and subsequent management may increase the anatomical success rate following BRT.

Co-accordance GNAQ, GNAS, and IDH1 variants might be an important biomarker for UM recurrence after treatment. To make a more accurate prediction ctDNA panels should be expanded beyond the several genes commonly considered to be related to UM.

The increase in the level of these variants might be related to the development and progression of recurrent disease. To prepare the best panel and best VAF cut-off for early diagnosis of UM recurrence, further studies in a larger number size cohort longer follow up are required to confirm GNAS andIDH1 implications in UM tumorigenesis.

Funding

This work supported by grant form research deputy of eye research center, Tehran, Iran.

Conflict of interest

The authors declare that they have no conflict of interest.

Code availability

IR.IUMS.REC.1398.730

Ethics approval

The study protocol was approved by the institutional ethics committee of Iran University of Medical Sciences (Iran) (IR.IUMS.REC.1398.730).

Consent to participate

Informed consent was obtained from the parents or guardians of all patients who participated in the study.

Consent for publication

All authors and patients

Availability of data and materials

All datasets generated for this study are included in the article.

Acknowledgments

This work supported by grant form research deputy of eye research center, Tehran, Iran.

Author’s contributions:

F.A: wrote the initial draft of the manuscript, samples collection and genetic analysis, genetic tests interpretation; S.T: suggested the main conceptual ideas and proofed outline and critically reviewed and revised the article for important intellectual content and contributed in the data; R.M & G.KH: revised the manuscript text. M.N: Introduced and treated the patients and revised the final draft of the manuscript; All authors read and approved the final manuscript.

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Table-1: Important genes

Oncogenic	Chromosome location	Class	Signaling pathway	TSG	Chromosome location	Class	Signaling pathway
GNAS p.R201H/C	20q13.32	G Protein, Alpha	CREB Pathway	STK11 p. Trp332* p. Gln37*	19p13.3	Serine/ Threonine Kinase 11	PI3K-Akt signaling pathway
EGFR k745R Asp761Asn Val786Met	7p11.2	GPCR Pathway	GPCR Pathway	ATM p. Arg3047*	11q22.3	ATM Serine/ Threonine Kinase	Wnt / Hedgehog / Notch
GNA11 Q209K	19p13.3	G Protein, Alpha 11 (Gq Class)	GPCR downstream signaling	PTEN p.Q110*	10q23.31	Phosphatase And Tensin Homolog	PI3K-Akt-mTOR Pathway
EZH2 p.Tyr646C	7q36.1	Enhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit	PIP3 activates AKT signaling
IDH1 p.R132C	2q34	Isocitrate Dehydrogenase (NADP (+)) 1	PI3K/AKT/mTOR pathway
GNAQ p.Q209R/P	9q21	G Protein Subunit Alpha Q	GPCR downstream signaling
FGFR2 p.C382R p.M497V	10q26.13	Fibroblast Growth Factor Receptor 2	GPCR Pathway

Table-2: important genes involved in UM recurrence

Genes	VAF
	Patient 1 (UM recurrence: Base line)	Patient 2 (UM recurrence: 12 month)
GNAQ (Q209P/R)	0.51%	0.70%
GNAS (R201H/C)	0.25%	0.25%
IDH1(R132H/C)	0.25%	0.1%

No competing interests reported.

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Circulating tumor DNA in patients with Uveal Melanoma (UM) may be associated with the tumor recurrence: Co- accordance of pathogenic variants GNAQ, GNAS and IDH1 in recurrent UM

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Patient 3:

Genetics Finding

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