Short Report: The Variants in CHEK2 in Metastatic Uveal Melanoma

Abstract

Background: Uveal melanoma (UM) is a rare subtype of melanoma with distinct clinical and molecular features compared to other melanoma subtypes. UM tumors are frequently detected with mutations in GNA11, GNAQ, EIF1AX, BAP1, and SF3B1 instead of the typical mutations associated with cutaneous melanoma. Although hereditary UM is rare, germline BAP1 loss predisposes patients to UM and various other cancers. The CHEK2 (Checkpoint kinase 2) gene that encodes the protein CHK2, a serine-threonine kinase, is a cell cycle checkpoint regulator that acts as a tumor suppressor. CHK2 is involved in DNA repair, cell cycle arrest, or apoptosis in response to DNA damage. CHEK2 mutations have been linked to various cancers. While there is no strong evidence that CHEK2 mutations increase the risk of melanoma, two cases of germline CHEK2 mutations in UM patients have been reported. However, the incidence of CHEK2 variants in metastatic UM (MUM) has not been investigated. Thus, we conducted a retrospective analysis of patients with MUM and CHEK2 variants to understand this link better. Methods: We collected MUM cases from 2016 to 2024 from institutional databases. Tissues underwent analyses of molecular and genomic features, including tumor mutational burden, and were performed by a Clinically Certified Laboratory. Next-generation sequencing and variant calling were conducted to identify CHEK2 variants. Results: In this study, we reported ten patients with CHEK2 variants among 740 metastatic UM patients (1.4%) and four primary UM patients with CHEK2 germline mutations. Conclusions: Although rare, UM patients with an abnormal ATM–CHEK2 axis might receive clinical benefits from medications that target DNA repair mechanisms.

1. Introduction

Uveal melanoma (UM), the most common primary ocular malignancy in adults, originates from melanocytes derived from neural crest cells and later inhabits the choroid, ciliary body, and iris. The cause of UM remains unknown. UM is particularly prevalent in Caucasians, with increased incidence observed in older age groups. The median age at diagnosis of primary UM ranges from 58 to 61 years [1,2,3]. Notably, UM typically has a lower mutational burden compared to cutaneous melanoma [4]. The most frequent genetic mutations are in guanine nucleotide-binding protein alpha 11 (GNA11) and guanine nucleotide-binding protein q polypeptide (GNAQ), which are somatic in tumor cells. Both GNA11 and GNAQ genes encode proteins involved in the Gα signaling pathway, and their expression is mutually exclusive in most tumors.

Cancer prognosis can be determined based on the mutation status of additional genes like BRCA1-associated protein-1 (BAP1), Splicing Factor 3B Subunit 1 (SF3B1), and eukaryotic translation initiation factor 1A x-linked (EIF1AX). For instance, patients with a BAP1 mutation have a high risk of metastasis, while patients with an EIF1AX mutation have a low risk [5,6]. Germline mutations or variants can be passed from generation to generation (inheritance) and can cause a specific phenotype or disease. A small group of UM patients has been found to harbor genes of familial predisposition. BAP1 has been identified as one of these predisposition genes, associated with a high susceptibility to various malignancies such as UM, malignant mesothelioma, cutaneous melanoma, renal cell carcinoma, meningioma, and other cancers [7,8,9]. Other germline mutations in UM have been found in the BRCA2, TP53, and MBD4 genes, but these mutations are rare, and their roles need further investigation [10,11,12].

The CHEK2 gene is located on chromosome 22q12.1 and encodes the CHK2 protein, which is involved in responding to DNA double-strand breaks [13]. Human CHK2 is a serine-threonine kinase and consists of 543 amino acids with three distinct functional domains: an N-terminal SQ/TQ cluster domain (SCD), a forkhead-associated domain (FHA), and a C-terminal Ser/Thr kinase domain [14]. CHK2 is activated by signals from ataxia-telangiectasia mutated kinase (ATM) (Figure 1), which is phosphorylated in response to double-stranded DNA breaks, DNA alkylation, or replicative stress. The ATM–CHEK2-p53 axis has been documented as a backbone for DNA damage response (DDR) and hypothesized as a barrier against cancer initiation [15]. CHK2 contributes to the phosphorylation of downstream target CDC25 to help regulate cell cycle checkpoints and apoptosis [16]. Thus, CHEK2 mutations severely impair DNA damage repair and cell cycle control. These mutations may impact tumor mutational burden (TMB) and microsatellite instability (MSI), both of which are critical indicators of genomic instability and potential predictors of response to immunotherapies [17]. Pathogenic CHEK2 variants are associated with an increased risk of several malignancies, including breast, colorectal, prostate, kidney, bladder, and thyroid cancers, depending on whether the mutation is a frameshift mutation or a missense substitution [18]. The two most frequent CHEK2 variants are missense p.I157T (c.470T > C) and truncating c.1100delC (p.T367Mfs*) [16].

To our knowledge, two cases of germline CHEK2 and PALB2 mutations in patients in UM were reported previously [19]. However, the actual incidence of CHEK2 mutation and variants in UM has not been investigated. Identifying a link between CHEK2 mutations and MUM may have important implications for both early diagnosis and targeted treatment, potentially improving patient outcomes. In this short report, we retrospectively analyzed and reported 10 metastatic uveal melanoma (MUM) patients with CHEK2 variants among 740 MUM cases (1.4%) in our MUM database and Caris database.

2. Materials and Methods

2.1. Participants and Ethical Standards

This study was a retrospective analysis of MUM patients with CHEK2 variants. The Institutional Review Board (IRB) approved this investigation (Jefferson iRISID-2024-3144). The data were obtained from a clinical laboratory system at Thomas Jefferson University and the database of MUM patients from Caris Life Sciences (Phoenix, AZ, USA).

2.2. Patient Specimens and DNA Sequencing

We collected data on MUM cases between 2016 and 2024 from the databases of Thomas Jefferson University and sequenced database Caris Life Sciences (Phoenix, AZ, USA). MUM tissue specimens were retrieved from paraffin-embedded archival core biopsy or surgically removed specimens for mutation analysis. Analyses of molecular and genomic features, including tumor mutational burden, were performed by a CLIA/CAP-certified lab (Caris Life Sciences). Next-generation sequencing (NGS; NextSeq or NovaSeq 6000, Illumina, Inc, San Diego, CA, USA) was performed on genomic DNA using a 592-gene panel and whole-exome sequencing (WES), with over 700 genes analyzed at high coverage and read depth. Genetic variant calling was conducted by board-certified molecular geneticists, as previously described [20]. TMB and MSI were calculated and called as previously described [21,22]. We also collected data on UM patients without systemic recurrence who underwent germline mutation testing.

3. Results

3.1. UM Patients with Metastasis

First, we identified three cases with variants in CHEK2 from our institutional database of 162 MUM patients (1.9%) with available NGS and WES of metastatic UM specimens. The following is a summary of three cases from the database at Thomas Jefferson University. Table 1 and Supplementary Table S1 show the summary of key DNA mutations.

3.1.1. Case 1

A 61-year-old male presented with metastasis in the liver. He was diagnosed with choroidal melanoma at the age of 59 and treated with radioactive plaque. A biopsy of the liver tumor was performed three months after the initial diagnosis of primary UM, confirming he had MUM. The tumor had mutations in GNA11 (p.Q209P) and BAP1 (p.S583fs). In addition, a likely pathogenic missense variant in CHEK2 (p.I157T) and a BRCA2 (c.475 + 1G > T) mutation were found. The total mutational load in the metastatic tissue was 11 mut/Mb and MSI was deemed stable. His familial history showed that his father had leukemia. The patient was treated with immunoembolization with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2. Upon disease progression, he was treated with percutaneous hepatic perfusion with HEPZATO KIT™, SIR-Spheres^® Y-90 resin microspheres, and chemoembolization with Carmustine (BCNU) to the liver [23,24,25]. The patient passed away 1 year and 10 months after the diagnosis of metastasis.

3.1.2. Case 2

A 67-year-old male presented with MUM in the liver. He was found to have a choroidal nevus in the right eye in his 40s. At the age of 53, he was diagnosed with choroidal melanoma and subsequently treated with radioactive plaque. A biopsy of the liver tumor performed 14 years after his initial diagnosis of primary UM confirmed he had MUM. The tumor had mutations in GNA11 (p.Q209L) and BAP1 (c.68-13_82del28). In addition, an MITF (c.952G > A/p.E318K) mutation, a CHEK2 (c.904G > A/p.E302K) variant of uncertain significance (VUS), a BRCA1 (p.D695Y) mutation, and a deletion of PALB2 were found. The total mutational load in the tumor tissue was 4 mut/Mb and MSI was stable. The patient’s familial history showed that his mother had stomach cancer, his brother had Hodgkin’s lymphoma, his maternal grandmother had pancreatic cancer, and his maternal grandfather had colon cancer. The patient was treated with chemoembolization with BCNU and was briefly on a clinical trial with FHD-286 [NCT04879071]. The patient passed away 1 year and 4 months after the diagnosis of metastasis.

3.1.3. Case 3

A 57-year-old male presented with MUM in the liver. He was found to have choroidal melanoma in the left eye at the age of 55 and subsequently treated with radioactive plaque. A biopsy of the liver tumor performed 1 year and 4 months after his initial diagnosis of primary UM confirmed the diagnosis of MUM. The tumor had mutations in GNAQ (p.Q209P) and BAP1 (p.P510fs). In addition, we also found pathogenic mutations in CHEK2 (p.S428F) and RAD51C (p.Y216fs). The total mutational load in the MUM tissue was 1 mut/Mb and MSI was stable. The patient’s familial history showed that his father died of glioblastoma. His mother had skin melanoma and died of lung cancer. His sister also had skin melanoma. The patient was enrolled in a clinical trial with a PKC inhibitor [NCT05987332] and is alive.

3.2. CHEK2 Variants in the Caris Database

Subsequently, we identified CHEK2 variants in seven additional MUM cases from the Caris Life Sciences tumor database between 2016 and 2023. We confirmed that these variants were derived from melanoma cells in MUM tissue specimens. CHEK2 variants were found in seven specimens from 578 cases (1.2%). The mutations of Gαq family members, BAP1, SF3B1, and EIF1AX, were present in the tumor specimens of these patients except for one case (Case 7 in Table 1 and Supplementary Table S1). This patient had a PLCB4 (p.G517E) mutation. ATM somatic or germline mutations were not identified in these patients.

Combining our database with the Caris database, we identified 10 patients with CHEK2 variants among 740 MUM patients (1.4%).

Table 1. The summary of key DNA variants or deletions.

ID	1	2	3	4	5	6	7	8	9	10	11	12	13	14
Group
CHEK2
BRCA2
BRCA1
RAD51C
PALB2
GNA11
GNAQ
PLCB4
BAP1
SF3B1
EIF1AX
ATM

footer: MUM (tumor specimen); Primary UM (PBMCs*); CHEK2; DNA damage repair gene; Gaq signaling pathway; Other.

Table 1. The summary of key DNA variants or deletions.

ID	1	2	3	4	5	6	7	8	9	10	11	12	13	14
Group
CHEK2
BRCA2
BRCA1
RAD51C
PALB2
GNA11
GNAQ
PLCB4
BAP1
SF3B1
EIF1AX
ATM

footer: MUM (tumor specimen); Primary UM (PBMCs*); CHEK2; DNA damage repair gene; Gaq signaling pathway; Other.

Case 1 to 3: Date from Thomas Jefferson University; Case 4 to 10 from Caris Life Science. *PBMCs: peripheral blood mononuclear cells.

3.3. Patients Without Metastatic UM (Primary Uveal Melanoma Patients Without Systemic Recurrence)

In addition to patients with UM metastasis, our database contained four primary uveal melanoma patients with CHEK2 germline mutations.

3.3.1. Case 11

A 66-year-old female presented with uveal melanoma in the right eye. She noticed reduced peripheral vision. She was subsequently diagnosed with choroidal melanoma. The tumor measured 16 mm in maximum diameter and 8.2 mm in thickness (stage IIB, T3aN0M0) at the age of 58, and she was treated with plaque radiation. Whole genome copy number analysis of DNA isolated from the primary uveal melanoma showed disomies of chromosomes 3 and 8, as well as chromosome 6p amplification. Her family history was significant for her paternal uncle and grandfather, who had uveal melanoma. In addition, her paternal aunt had breast cancer, and her uncle had prostate cancer. She had genetic counseling and germline testing completed, which reported pathogenic CHEK2 mutations (c.1100delC:p.Thr367Mfs and c.7C > T:p.Arg3Trp). BAP1, BRCA1, and BRCA2 germline mutations were negative. The patient is increasing the frequency of cancer screening. The patient has not had a local or distant recurrence of the disease for eight years after diagnosis of primary uveal melanoma.

3.3.2. Case 12

A 57-year-old female presented with uveal melanoma in the left eye. She developed left eye photopsia. She was referred to a retina specialist and subsequently diagnosed with choroidal melanoma. The tumor measured 8 × 7 mm in base and 2.7 mm in thickness (stage I, T1aN0M0) at the age of 54, and the patient was treated with plaque radiation. The tumor was analyzed with whole genome copy number analysis of DNA isolated from the primary uveal melanoma, which showed mosaic monosomy 3 and disomies of chromosomes 6 and 8. Her personal and family history was remarkable for her brother, who was diagnosed with uveal melanoma and developed metastasis 10 years after his primary eye tumor diagnosis. He passed away roughly 15 months after developing systemic metastasis. Her mother had a meningioma. Her paternal grandmother has breast cancer, and her grandfather had lung cancer. Her maternal grandfather had an unknown cancer. She has one healthy sister. The patient also has a personal history of cervical cancer at the age of 43 and had a hysterectomy. She had genetic counseling and subsequently found a germline pathogenic CHEK2 mutation (c.1100delC) and a BAP1 (c.542_543delTT) mutation. BRCA1 and BRCA2 germline mutations were negative. She has a stable 1 cm mass in the right hepatic lobe favored to represent a flash-filling hemangioma or vascular malformation. The patient has not had a local or distant recurrence of the disease for four years after diagnosis of primary uveal melanoma.

3.3.3. Case 13

A 46-year-old female presented with uveal melanoma in the right eye. She had a routine eye exam and identified an abnormality in the right eye. She was subsequently diagnosed with choroidal melanoma. The tumor measured 8 × 7 mm in base and 3.1 mm in thickness (stage I, T1aN0M0) at the age of 40, and the patient was treated with plaque radiation. The primary uveal tumor showed partial monosomy 3, as well as disomies of chromosomes 1, 6, and 8. Her personal and family history was remarkable for her mother, who was diagnosed with thyroid cancer. Her maternal grandmother had leukemia. Her son had sarcoma with a CHEK2 mutation. She had genetic counseling and found a germline pathogenic CHEK2 mutation (c.1215 C > A, p. N405K). There were no other cancer-related germline mutations, including BAP1, BRCA1, or BRCA2. The patient has not had a local or distant recurrence of the disease for seven years after diagnosis of primary uveal melanoma.

3.3.4. Case 14

A 67-year-old female presented with uveal melanoma in the left eye. She had a routine eye exam and identified an amelanotic nevus in her left eye. She was subsequently diagnosed with choroidal melanoma. The tumor measured 10 × 8 mm in base and 2.7 mm in thickness (stage I, T1aN0M0) at the age of 67, and the patient was treated with plaque radiation. The tumor analysis showed disomies of chromosomes 3, 6, and 8. The patient has a personal history of two breast cancers. The first one was a stage IIA right breast carcinoma diagnosed at the age of 40. At the age of 54, she was found to have a stage 0 left breast carcinoma (LCIS versus DCIS). Her family history was significant for her mother and maternal aunt, as well as her brother’s daughter, who were all diagnosed with breast cancer. Her maternal grandfather had lymphoma. She had genetic counseling and found a germline CHEK2 mutation (c.349A > G, p. R117G), which is considered a pathogenic missense mutation associated with an increased risk of developing certain cancers. Additionally, the patient has a germline ATM mutation (c.519G > T, p.R173S). Other cancer germline mutations, such as BAP1, BRCA1, or BRCA2, were negative. The patient has not had a local or distant recurrence of the disease for half a year after diagnosis of primary uveal melanoma.

Since we have not tested all primary uveal melanoma patients with germline CHEK2 variations, we do not know the incidence of CHEK2 variation in the general population of primary uveal melanoma patients.

4. Discussion

In this study, we report 10 patients among 740 MUM patients (1.4%) whose metastatic specimens exhibited variants in the CHEK2 gene and four primary UM patients without systemic recurrence with CHEK2 germline mutations. Although confirmatory germline testing with normal or non-malignant cells was not performed on the metastatic specimens, the CHEK2 variants could be germline origin based on the high variable allele frequency (VAF). Three of these metastatic patients from our institution had at least one member in the family with clearly documented cancer. The sample number in this study is too small to make any broad conclusions; however, the median age of onset of primary UM with CHEK2 variations in our database was 55 years old (n = 7, range 40–67), which is only slightly younger than the reported median age of onset of UM of 58 to 62 years [1,2,3]. To our knowledge, only two UM cases have previously been reported in the literature with a germline mutation in CHEK2 [c.1100delC:p.Thr367Metfs and c.T470C:p.Ile157Thr] [19]. These patients were identified in a study of 154 UM patients with a high risk of hereditary cancer, using germline DNA analysis with WES. It is well known that a BAP1 germline variant predisposes patients to UM and other cancers, including cutaneous melanoma, malignant mesothelioma, meningioma, and renal cell carcinoma [7,8,9]. CHEK2 variants are also known to increase the risk of breast cancer, leukemia, and other cancers [26,27,28]. Variants in the CHEK2 gene were first reported as truncating CHEK2 mutations (c.1100delC) that confer a greater than twofold increase in the risk of breast cancer. Clinical studies evaluating the risk of cutaneous melanoma in CHEK2 mutation carriers have been reported in the literature. The authors concluded that individuals with CHEK2*1100delC heterozygosity have a twofold risk of melanoma compared with non-carriers (OR 2.01 (1.03–3.91) in the Danish, 1.42 (0.46–4.31) in Germans, and 1.79 (1.02–3.17) in the Danish and Germans combined) [29]. In contrast, three common low-risk missense variants, I157T, S428F, and T476M, are not commonly associated with an increased risk of breast cancer [30,31]. These germline CHEK2 variants occur in 0.6–1.1% of the general population [16,32]. Besides the well-known and most studied CHEK2 mutations described above, other nonsense variants (e.g., Q20X, E85X) and missense mutations (H371Y and D438Y) have been associated with breast cancer [33,34]. Investigating primary UM cases to assess the incidence and risk of UM in individuals with CHEK2 mutations may provide valuable insights.

The CHEK2 variants with p.I157T in Case 1, E302K in Case 2, and p.S428F in Case 3 have been frequently observed as germline variants. I157T (470 T > C) in the FHA domain was reported in the normal Finnish population [35]. The American College of Medical Genetics and Genomics (ACMG) recently published CHEK2 management guidelines highlighting that the low penetrance of I157T does not meet the 2-fold relative risk threshold for clinical significance [36]. The very rare BRCA2 (c.475 + 1G > T) mutation was also found in Case 1. The role of CHEK2 mutations in the presence of BRCA2 mutations should be further investigated.

The CHEK2 variant in Case 2 is located within a kinase domain (E302K). Myriad classifies it as likely pathogenic [37], but others classify it as a VUS [38,39]. There is still a need for further investigation of this variant to determine its level of pathogenicity. The risk of cancer associated with CHEK2 missense variants is relatively low, with some notable exceptions. Data from the studies on the CHEK2 (p.E302K) are insufficient to prove that it is one of these exceptions. It is of note that this patient also had a MITF variant. MITF (E318K) is also a low penetrance variant frequently observed in the germline of the general population. It is associated with a moderately increased risk of cutaneous melanoma [40]. In addition, Case 2 exhibited a deletion of the PALB2 gene and a BRCA1 (D695Y) mutation with a family history of cancer affecting four members, including one diagnosed with pancreatic cancer. The CHEK2 S428F mutation in the kinase domain is a missense variant in Case 3. It is associated with lower rates of non-breast cancers [30,31]. Additionally, Case 3 has a RAD51C mutation that disrupts DNA repair.

It is important to note that the CHEK2 variant data of MUM were analyzed from tumor tissue sections, and pathogenic somatic mutations in GNA11 or GNAQ, along with BAP1, were detected alongside the CHEK2 variants in the UM metastatic tissues. In general, the detectable time for metastasis in UM had been reported to peak differently in BAP1 and SF3B1 mutations. The earlier peaks appear to be associated with the BAP1 mutation (usually between 1 and 3.5 years), while the later peak is most likely associated with the SF3B1 mutation [41]. While Case 1, Case 2, and Case 3 all had somatic BAP1 mutations in the tumor, the time it took to develop distant metastasis was very different. It took only 4 months after the primary diagnosis in Case 1, 1 year and 4 months in Case 3, and 14 years and 8 months in Case 2. A current limitation is the insufficient number of patients available to assess this interesting association with the CHEK2 variant.

Although the role of CHEK2 abnormalities in UM patients is not clear and needs to be further investigated, the presence of alternations in the ATM–CHK2 axis might justify the usage of previously untested medications in UM such as ataxia telangiectasia and Rad-3-related (ATR) kinase inhibitors and ATM kinase inhibitors, as CHEK2 is part of the ATM–CHK2-p53 axis (Figure 1) [42,43]. Furthermore, the role of CHEK2 abnormality in BAP1-mutated and BRCA1/2-mutated tumors should also be investigated to justify the usage of PARP inhibitors for UM tumors with these mutations [43,44,45]. Our findings may contribute to a better understanding of a potentially significant link between CHEK2 mutations and UM patients to ultimately improve patients’ quality of health and provide implications for both early diagnosis and targeted treatments. To further elucidate the clinical implications of CHEK2 variants, investigating a large cohort of primary UM cases would facilitate a comprehensive assessment of the incidence and clinical relevance of UM in affected individuals.

5. Conclusions

Investigations of a dysregulated CHK2 in UM could increase our understanding of its biological role and pave the way for personalized therapy for this disease. In this study, we do not have enough samples to conclude how these tumor-specific CHKE2 variants may influence UM metastasis. Although rare, UM with abnormalities in the CHEK2 mutation might benefit from inhibitors of DNA damage repair, warranting further investigation.