This study describes the tumor spectrum observed in a cohort of cancer patients harboring the NM_007194.4(CHEK2):c.707T > C p.(Leu236Pro) variant. Among the 58 individuals analyzed, 73 primary malignancies were identified across 12 anatomical sites (Fig. 2). Genetic testing was performed for 10 distinct clinical indications. HBOC was the most common indication, followed by treatment selection in metastatic castration-resistant prostate cancer and personal or family history suggestive of Lynch syndrome. The mean age at initial cancer diagnosis was 43.8 ± 12 years (range: 21–77 years), suggesting a predisposition to early-onset disease, especially breast cancer.

Characterizing the tumor spectrum associated with this variant is particularly relevant for the Mexican population, as CHEK2 p.(Leu236Pro) is the most frequently identified pathogenic CHEK2 variant in Mexican individuals undergoing multi-gene panel testing for hereditary cancer. Based on the internal database of the Genomic Diagnostic Laboratory (GDL) at the National Institute of Genomic Medicine (INMEGEN), this variant accounts for approximately 56% of the pathogenic CHEK2 alleles detected in patients tested for suspected HBOC, suggesting a founder effect in Mexico (unpublished data, GDL, INMEGEN).

Comparative analysis of the variant’s frequency across different ethnic groups supports the hypothesis that CHEK2 p.(Leu236Pro) may have originated in the central region of Mexico within an indigenous population. Data from the Mexico City Prospective Study (MCPS) [21] show that the variant is present exclusively in individuals genetically classified as “Indigenous Mexican” (allele frequency 0.005113) and is absent in Mexican individuals of European or African ancestry. It is also absent from other Latin American subpopulations, including Peruvians from Lima, Colombians from Medellín, Puerto Ricans, and Mexican mestizos from Los Angeles (https://rgc-mcps.regeneron.com/rsid/rs587782471). This observation is consistent with gnomAD data, which reports the variant only in “admixed American populations” (0.17%) and not in other groups (gnomAD v4.1) (https://gnomad.broadinstitute.org/variant/22-28711994-A-G).

To further elucidate the ethnic and geographical origin of the CHEK2 p.(Leu236Pro) variant and its potential founder effect, future studies should incorporate haplotype analysis, assessments of the variant’s geographical distribution, and evaluations of ancestry-informative markers from specific Mexican indigenous groups. Such investigations would provide valuable insights into the demographic history underlying this variant and its implications for cancer risk assessment in the Mexican population.

CHEK2 is classified as a moderate-risk gene for breast cancer [1]. An absolute breast cancer risk of 23–27% is outlined in the NCCN Guidelines for Genetic/Familial High-Risk Assessment: Breast, Ovarian, Pancreatic, and Prostate [22]. Its inclusion in breast cancer multi-gene risk panels highlights its clinical importance [23].

The personal and family cancer histories of individuals carrying the CHEK2 p.(Leu236Pro) variant resembled those typically seen in BRCA1/2-related HBOC. This similarity has been previously reported for CHEK2-positive individuals [4, 6, 23, 24]. In our cohort, most individuals (72.9%) were diagnosed with breast cancer at an early age (mean 41.2 years), and 63.4% reported a positive family history. The majority were referred for genetic testing due to suspected HBOC, including one case of male breast cancer (MBC). Our case-control analysis supports this similarity, with significant associations observed between CHEK2 p.(Leu236Pro) and both breast cancer (OR = 3.2, 95% CI 2.4–4.3) and ovarian cancer (OR = 4.2, 95% CI 1.8–9.5). These associations remained statistically significant even when using a control group composed exclusively of Indigenous Mexican individuals, who have a higher frequency of the variant (Supplementary Material 1, Table S3).

Three breast cancer patients carried an additional pathogenic variant in a cancer gene: CDKN2A (patient ID40), BRCA2 (patient ID21), and ATM (patient ID34). While BRCA2 and ATM are associated with high and moderate breast cancer risk, respectively, the role of CDKN2A in breast cancer risk remains unclear [22]. To further clarify the association between CHEK2 p.(Leu236Pro) and breast cancer risk, the three cases were excluded from the case-control analysis. The results continued to support a significant association (OR = 3.02, 95% CI [2.2-4], p < 0.00001).

The association between pathogenic and likely pathogenic variants of CHEK2 and MBC remains controversial [25, 26]. In a cohort of 715 Caucasian MBC patients, CHEK2 pathogenic variants were present in 4.1% of patients (OR = 3.7, p < 6.24 × 10− 24), representing the second most common predisposition gene after BRCA2 [27]. Hallamies et al. [28] analyzed 68 MBC cases from the Finnish population and showed that CHEK2 c.1100delC was associated with an increased risk of MBC (OR = 4.47, 95% CI 1.51–13.18, p = 0.019). A meta-analysis also supported the association between CHEK2 c.1100delC and an increased risk of MBC (OR = 3.13, 95% CI 1.94–5.07) [29]. Other CHEK2 variants have also been investigated, with some studies identifying a significant association between MBC and pathogenic or likely pathogenic variants [25, 30]. However, other studies do not support an association between CHEK2 variants and MBC risk [31,32,33,34,35,36]. The risk of MBC may vary depending on the specific pathogenic CHEK2 variant.

In our cohort, we identified a female breast cancer patient who was homozygous for the CHEK2 p.(Leu236Pro) variant (patient ID28; Table 1 and Supplementary Table S2). She was diagnosed with IDC at age 42 and had a positive family history of cancer. Two of her sisters were diagnosed with breast cancer before age 50, but they were not genetically tested to confirm the presence of the variant (Table 1 and Supplementary Material Table S2). The clinical consequences of homozygosity for CHEK2 pathogenic variants were initially investigated for NM_007194.4(CHEK2):c.1100del p.(Thr367fs), due to its high carrier frequency in European populations. Findings suggest that homozygosity is associated with a higher breast cancer risk than heterozygosity in female carriers [37]. Additional studies on patients homozygous or compound heterozygous for other CHEK2 pathogenic or likely pathogenic variants indicate increased susceptibility to multiple malignancies and earlier onset in both sexes [19, 38]. Multiple cytogenetic anomalies in peripheral lymphocytes have been reported in such patients; however, karyotyping was not performed in our case [39]. Estimating lifetime risk in CHEK2 homozygous or compound heterozygous individuals remains challenging due to the small sample sizes in the existing studies. Therefore, the NCCN Guidelines recommend incorporating both personal and family history into cancer risk management decisions [40].

Ovarian cancer was a recurrent tumor type among CHEK2 p.(Leu236Pro) carriers, occurring as a primary tumor in two cases and as a second primary tumor in four, with most being serous adenocarcinomas (4/6 cases). A significant association between CHEK2 p.(Leu236Pro) and ovarian cancer was identified in our cohort (OR = 4.2, 95% CI [1.8–9.5]). No additional pathogenic variants related to ovarian cancer risk were identified in these six patients. Analyses of large European and North American cohorts have identified carriers of pathogenic or likely pathogenic CHEK2 variants among ovarian cancer patients [41, 42]. Additionally, a small study in patients of Hispanic ancestry reported ovarian cancer in individuals carrying other pathogenic CHEK2 variants [11]. However, the ACMG and NCCN guidelines (v2.2025) do not currently consider CHEK2 as a gene conferring increased ovarian cancer risk [2, 40]. Prospective studies in diverse populations are required to clarify the contribution of specific pathogenic CHEK2 variants to ovarian cancer susceptibility.

Prostate cancer was the third most frequent tumor type in our study. Among the affected patients, 4 of 5 had metastatic castration-resistant prostate cancer diagnosed between the ages of 65 and 77 and underwent genetic testing to determine eligibility for poly(ADP-ribose) polymerase (PARP) inhibitors. In one case, HBOC was suspected because the patient’s sister had breast cancer at age 32. (Table 2 and Supplementary Material 1, Table S2). Pathogenic germline CHEK2 variants have been associated with increased prostate cancer risk. In one study, CHEK2 pathogenic variants were identified in 2.7% of 1,022 prostate cancer patients, compared to a population prevalence of 1.4% (RR: 1.9, 95% CI: 1.3–2.8, p < 0.001) [43]. A multicenter cross-sectional study of 384 patients with metastatic castration-resistant prostate cancer from 11 cancer centers in seven Latin American countries, including Mexico, identified CHEK2 as the most frequently mutated gene (1%), comparable to BRCA2 (0.8%) and ATM (0.8%) [44].

Prostate cancer risk associations have also been reported for other pathogenic CHEK2 variants. A meta-analysis identified an association with c.1100delC (OR = 3.2; 95% CI: 1.85–5.9) and p.(Ile157Thr) (OR = 1.80; 95% CI: 1.5–2.1) [45]. Additionally, the c.349 A > G (p.Arg117Gly) variant was associated with prostate cancer in Portuguese patients with early-onset disease or a family history (OR = 1.9; 95% CI: 1.1–3.2) [46]. In contrast, no statistically significant association was observed between the CHEK2 p.(Leu236Pro) variant and prostate cancer risk in our case-control analysis. However, the frequency of CHEK2 p.(Leu236Pro) carriers was higher in the patient group than in the control group, suggesting a trend toward increased risk (1.7% of cases vs. 0.7% of controls) (OR = 2.2 95% CI: [0.94–5.4]; p = 0.05, χ2 = 3.5, data not shown in Table 2). Analyzing a larger cohort, including men with early-stage prostate cancer or younger age at diagnosis, may help clarify whether an associated risk exists in specific subgroups.

Although endometrioid carcinoma (EC) is the most common malignancy of the female reproductive tract, it was not a recurrent tumor type among CHEK2 p.(Leu236Pro) carriers in our cohort, with one case occurring as a first tumor and another as a second primary malignancy. No definitive association has been established between CHEK2 pathogenic variants and EC risk; similarly, our results showed no statistical association. A multi-gene panel analysis in EC patients reported CHEK2 as the most frequently mutated gene after excluding cases with pathogenic variants in the mismatch repair genes MLH1, MSH2, MSH6, and PMS2 [47]. Conversely, an analysis of EC risk associated with the CHEK2 p.(Ile157Thr) variant in a cohort of 268 European patients and 449 female controls found no association. However, the variant was more frequently observed in EC patients over 75 years of age and those with deep myometrial invasion [48]. Depending on the specific CHEK2 pathogenic variant, heterogeneity in EC risk may exist. In our cohort, the patient with EC as a primary tumor was diagnosed with adenocarcinoma at age 30 and referred for genetic testing due to suspected Lynch syndrome. In addition to CHEK2 p.(Leu236Pro), a loss-of-function pathogenic variant in BRCA1 was identified, which likely explains the family history of cancers in this patient and may have contributed to EC development [49]. Given the clinical overlap between Lynch syndrome and other hereditary cancer syndromes, multi-gene panel testing provides a more comprehensive approach than syndrome-specific testing. This strategy enables the detection of additional clinically actionable mutations, as illustrated by this patient [47].

Previous NCCN guidelines reported a 5–10% increased risk of colorectal cancer in individuals with pathogenic or likely pathogenic CHEK2 variants. Additionally, a colon cancer phenotype has been described in CHEK2 c.1100delC carriers, leading some authors to refer to it as ‘hereditary breast and colorectal cancer [50]. However, the 2024 NCCN Genetic/Familial High-Risk Assessment: Colorectal, Endometrial, and Gastric guidelines now state that individuals with CHEK2 pathogenic or likely pathogenic variants are not at increased risk for colorectal cancer [40, 51]. No significant association with colon cancer was found in our study. The only case of colon adenocarcinoma occurred in a 33-year-old patient who also carried a pathogenic ATM variant. This variant may have contributed to the early-onset disease, as ATM is associated with a low-to-moderate increased risk of colorectal and other cancers [52, 53]. No pathogenic variants or variants of uncertain significance were identified in Lynch syndrome genes or other colorectal cancer predisposition genes in this patient.

Additionally, gastric and thyroid cancers were observed in Mexican patients carrying the CHEK2 p.(Leu236Pro) variant. Several studies have suggested a role for pathogenic CHEK2 variants in gastric cancer predisposition [54,55,56]. In a Polish population, an increased risk (OR = 1.6; p = 0.004) was reported [57], and a separate study found a stronger association for the loss-of-function CHEK2 variant NM_007194.4:c.444 + 1G > A (OR = 3.5) [1]. A literature review using natural language processing also identified an association between CHEK2 variants and gastric cancer [55]. Two studies of Chinese patients with stomach cancer identified CHEK2 as one of the most frequently mutated genes [56, 58]. Our findings also support an association between CHEK2 p.(Leu236Pro) and gastric cancer (OR = 9.9; 95% CI: 3.1–32.2). These results contrast with a Japanese study that found no pathogenic CHEK2 variants in gastric cancer patients. However, the limited sample size and use of single-strand conformation polymorphism analysis may have affected its results [54].

The association between thyroid cancer and germline pathogenic variants in CHEK2 remains uncertain. A case-control study reported an increased risk for papillary carcinoma (OR = 4.54; p = 0.0116) and a higher risk among carriers of CHEK2 loss-of-function alleles (OR = 5.7; p = 0.006) [8, 59]. However, thyroid cancer was infrequent in our cohort, with only one case of medullary carcinoma as a primary tumor and one case of papillary carcinoma as a second primary tumor. In the case-control analysis, the association between CHEK2 p.(Leu236Pro) and thyroid cancer was not significant when compared with the “Indigenous Mexican” control group (Supplementary Table S3). In the case of medullary thyroid carcinoma, pathogenic variants or variants of uncertain significance in the coding region or exon–intron boundaries of RET were excluded. These findings align with a recent review concluding that the risk of thyroid cancer associated with CHEK2 is low and that routine thyroid cancer screening is not currently recommended [60].

Non-melanoma skin cancer, non-Hodgkin lymphoma (NHL), sacrococcygeal bone cancer, and lung cancer were each observed in a single patient. A moderately increased risk of NHL and poorer progression-free survival have been suggested in CHEK2 carriers [7]. However, the role of CHEK2 in the development of NHL and Hodgkin lymphoma remains unclear. Although some studies have explored the risk of melanoma and non-melanoma skin cancers in CHEK2 carriers [61], current evidence is insufficient to draw definitive conclusions. Further research is warranted to clarify the risk associated with these tumor types.

The patient with a sacrococcygeal bone tumor developed three primary malignancies, with breast cancer diagnosed as the second and third tumors. In addition to CHEK2 p.(Leu236Pro), a pathogenic PALB2 variant was detected, fulfilling the criteria for multi-locus inherited neoplasia allele syndrome (MINAS). Approximately 28% of reported MINAS cases develop multiple primary tumors, and some exhibit atypical tumor phenotypes. The tumor spectrum in MINAS generally reflects the independent effects of the germline-mutated cancer genes. However, the occurrence of unusual phenotypes or multiple primary tumors, as in our patient, may indicate complex interactions between coexisting mutations [62].

Lung cancer was observed in only one patient, and no significant association between CHEK2 p.(Leu236Pro) and lung cancer risk was identified. However, recent evidence suggests that pathogenic CHEK2 variants may contribute to lung cancer predisposition. A large retrospective study of 7,788 lung cancer patients reported a significantly higher prevalence of pathogenic variants in BRCA2, ATM, CHEK2, and BRCA1 compared with controls [63]. Additionally, cross-sectional cohort study of 201 Mexican patients with lung adenocarcinoma, selected based on a family history of lung cancer, early-onset disease, a history of never or light smoking, or actionable genomic alterations, identified pathogenic CHEK2 variants in 6.9% of positive cases (3/43), including one CHEK2 p.(Leu236Pro) carrier [64]. Germline testing is not currently recommended for all lung cancer patients [19], except for untreated carriers of the EGFR p.(T790M) variant [65]. However, prospective studies involving germline testing of lung cancer patients from different ethnic backgrounds may help clarify the contribution of the CHEK2 gene and specific pathogenic variants to lung cancer risk.