Prevalence of pathogenic variants in cancer‐predisposing genes in second cancer after childhood solid cancers

Abstract Background Second malignant neoplasms (SMNs) are one of the most severe late complications after pediatric cancer treatment. However, the effect of genetic variation on SMNs remains unclear. In this study, we revealed germline genetic factors that contribute to the development of SMNs after treatment of pediatric solid tumors. Methods We performed whole‐exome sequencing in 14 pediatric patients with SMNs, including three brain tumors. Results Our analysis revealed that five of 14 (35.7%) patients had pathogenic germline variants in cancer‐predisposing genes (CPGs), which was significantly higher than in the control cohort (p < 0.01). The identified genes with variants were TP53 (n = 2), DICER1 (n = 1), PMS2 (n = 1), and PTCH1 (n = 1). In terms of the type of subsequent cancer, leukemia and multiple episodes of SMN had an exceptionally high rate of CPG pathogenic variants. None of the patients with germline variants had a family history of SMN development. Mutational signature analysis showed that platinum drugs contributed to the development of SMN in three cases, which suggests the role of platinum agents in SMN development. Conclusions We highlight that overlapping effects of genetic background and primary cancer treatment contribute to the development of second cancers after treatment of pediatric solid tumors. A comprehensive analysis of germline and tumor samples may be useful to predict the risk of secondary cancers.


| INTRODUCTION
Second malignant neoplasms (SMNs) are the most severe late complications in childhood cancer survivors and are the leading cause of death in long-term survivors. 1 The cumulative incidence of SMNs does not reach plateau even at 10 years after the treatment of primary cancer, more than 10% of long-term survivors develop SMNs. [1][2][3] Several known risk factors for SMN development have been reported. In addition to age at the first cancer, gender, radiation therapy, and DNA-damaging agents (most notably alkylating agents) are established risk factors for SMNs. [4][5][6][7] In addition to these factors, germline pathogenic variants of cancer-related genes are associated with the development of SMNs. Recent comprehensive analyses have revealed that pediatric cancer patients have a high frequency of germline pathogenic variants in cancerpredisposing genes (CPGs). 8,9 Since a history of cancer is often observed in the family members of SMNs, 10 genetic variations in CPGs are believed to be associated with the development of SMNs after childhood cancer. For example, TP53 variants are frequently found in the patient with pediatric SMNs after treatment for solid tumors and acute lymphoblastic leukemia (ALL). 11,12 Additionally, several studies have reported the results of germline variant analyses in the development of SMNs. A comprehensive study of childhood cancer survivors reported that 6.4% of the patients had germline pathogenic variants of CPGs. 13 In a study of pediatric therapy-related myeloid neoplasms, pathogenic or likely pathogenic variants were identified in 13 of 84 patients (15%). 14 Thus, the influence of germline sequence alterations in the development of SMNs is gradually being elucidated, but the data are still limited. Here, we report an analysis of germline alterations in CPGs and somatic mutation profiles in pediatric patients with SMNs after treatment for solid tumors.

| Patient enrollment
Patients who met the following eligibility criteria were enrolled in this retrospective study: (i) developed SMNs after treatment for pediatric solid tumors, (ii) specimens were

Methods:
We performed whole-exome sequencing in 14 pediatric patients with SMNs, including three brain tumors.
Results: Our analysis revealed that five of 14 (35.7%) patients had pathogenic germline variants in cancer-predisposing genes (CPGs), which was significantly higher than in the control cohort (p < 0.01). The identified genes with variants were TP53 (n = 2), DICER1 (n = 1), PMS2 (n = 1), and PTCH1 (n = 1). In terms of the type of subsequent cancer, leukemia and multiple episodes of SMN had an exceptionally high rate of CPG pathogenic variants. None of the patients with germline variants had a family history of SMN development. Mutational signature analysis showed that platinum drugs contributed to the development of SMN in three cases, which suggests the role of platinum agents in SMN development.

Conclusions:
We highlight that overlapping effects of genetic background and primary cancer treatment contribute to the development of second cancers after treatment of pediatric solid tumors. A comprehensive analysis of germline and tumor samples may be useful to predict the risk of secondary cancers.

K E Y W O R D S
cancer predisposition, childhood solid cancer, second malignant neoplasms available, and (iii) provided required informed consent. SMN is defined as a new, independent cancer that occurs in a person who has had cancer in the past. Germline samples (n = 14) with available somatic samples (n = 5) that met these criteria were obtained.

| Germline analysis of pathogenic variants of CPGs
This retrospective study was designed to clarify the genetic risk factors for SMNs. To investigate potential pathogenic variants of CPGs in SMN cases, we performed wholeexome sequencing (WES) in 14 germline samples collected from the tumor-free specimens (Supplementary method and Table S1). Complete methods of sample preparation and WES are shown in detail in the Supplementary files. The selection of the CPG genes and the evaluation of the pathogenicity were performed as previously reported. 15 In brief, variants of 162 known CPGs (Table S2) listed in previous reports 9,16 were extracted from each sample. Then, the pathogenicity of each variant was manually evaluated using online databases and the guideline of the American College of Medical Genetics and Genomics 17 (detailed in the Supplementary methods). Detailed genomic data are available on request for corresponding author.

| Comparison of the prevalence of germline variants between the experimental group and control samples
To compare the prevalence of germline variants between the experimental group and the control cohort, WES data of 104 adults with no cancer history or a family history of hematologic disorders, consecutively enrolled in the National Center Biobank Network (NCBN) project were analyzed using the same process as that used for the experimental cohort. 15

| Genomic analysis of SMN samples
To identify somatic mutations in SMN specimens, available tumor samples at SMN diagnosis from five patients were analyzed using WES. Using paired tumor-normal WES data, somatic mutations were extracted. Driver mutations were identified by reference to the Catalogue of Somatic Mutations in Cancer (COSMIC, http://cancer.sanger.ac.uk/cance rgeno me/proje cts/cosmic), NCBI ClinVar (http://www.ncbi.nlm.nih.gov/clinv ar/), and previous reports.

| Copy number analysis
We also analyzed the WES data using CNVkit (version 0.9.6) to estimate the copy number status. 18 In this analysis, the number of reads mapped to each target region of the tumor genome was calculated and compared with the value obtained from multiple normal reference samples. Copy number segmentation was performed using the circular binary segmentation method with default parameter settings.

| Tumor mutational burden analysis
Tumor mutational burden (TMB) was calculated as the total number of somatic, coding, base substitution, and indel mutations per mega base, using paired tumornormal WES data.

| Mutational signature analysis
To investigate the etiology of SMN development, mutational signature analysis was performed using a previously described method. 15 Known mutational signatures were obtained from the COSMIC database (Mutational signatures V3, synap se.org ID: syn12009743) and from Li et al. 19

| Statistical analysis
The comparison of the prevalence of pathogenic variants between SMN patients and NCBN controls was performed using Fisher's exact test. A p value of <0.05 was considered statistically significant.

| Patient characteristics
The characteristics of the 14 patients with SMNs are presented in Figure 1 and in Tables 1 and 2. Brain tumors (n = 3) and nonbrain solid tumors (n = 11) were the first cancers in these patients. As for subsequent cancers, five patients had leukemia (n = 4) or lymphoma (n = 1), and nine patients had solid tumors, including a brain tumor (n = 1) and nonbrain solid tumors (n = 8). Four of the 14 patients had multiple episodes of SMNs including both solid and hematologic malignancies (SMN03-05, and 07). The median age at diagnosis of primary cancer and SMNs was 3 years (range, 9 months-13 years) and 10 years (range, 4-19 years), respectively. The median time from diagnosis of the primary malignancy to diagnosis of SMN was 6 years (range, 1-11 years). Radiation therapy was administered in five cases. Three patients developed a second solid tumor within the radiation fields, while the remaining SMNs were hematological ( Figure 1; Table 2).

| Germline pathogenic variants in CPGs
WES identified 25 nonsilent germline variants in the 110 cancer-associated genes with a dominant inheritance pattern. Five variants were deemed to be pathogenic and were detected in five patients (Figure 1; Table S3). Notably, neither of the five patients with pathogenic variants had a family history of cancer at the time of SMN development. Pathogenic variants were detected in TP53 (n = 2), DICER1 (n = 1), PMS2 (n = 1), and PTCH1 (n = 1). Four patients experienced a third SMN, and of these, three had pathogenic variants (SMN 04-06). In terms of treatment for their prior malignancy, three of five patients with a CPG variant had not received radiation therapy (Figure 1).
The prevalence of pathogenic variants in the SMN cohort was estimated to be 35.7% (5 of 14), which was significantly higher than that in the 104 control cases in the NCBN cohort (1.0%; p < 0.01) (Figure 2A; Table S4). In terms of cancer type, subsequent leukemia cases and cases with multiple episodes of SMN exhibited a particularly high rate of CPG pathogenic variants (71.4% and 75%) ( Figure 2B,C).
Of the 52 genes with a recessive inheritance pattern, none were detected as homozygous or compound heterozygous mutations in either the SMN or the control cohort.

| Genomic features of SMN tumor samples
We performed WES for the five cases for which SMN tumor specimens were available, including cases of AML (n = 2), ALL (n = 2), and renal cell carcinoma (n = 1) ( Figure 3A). Two of the available specimens were a third cancer (AML in SMN03 and ALL in SMN04). Known somatic driver mutations in PTPN11 (NM_002834: c.T211C: p.F71L) and KRAS (NM_004985: c.G35A: p.G12D) were detected in the ALL (SMN04) and AML (SMN08) samples.

| Copy number alterations
The results of the copy number variant analysis are shown in Figure S1. In SMN03, in which a germline TP53 pathogenic variant was identified, a deletion was detected in  17p13, including the remaining allele in the TP53 region ( Figure 3B and Figure S1).

| TMB analysis
A high TMB (>10 mutations/Mb) was observed in two patients (SMN04 and SMN10) (Table S5), including one patient with a germline pathogenic variant of a mismatch repair (MMR) gene (PMS2 in SMN04).

| Mutational signature analysis
To evaluate the contribution of anticancer, agentinduced DNA damage to SMN development, a mutational signature analysis was performed. A platinum drug-related signature was observed in three cases ( Figure 3C).

| DISCUSSION
Our analysis revealed that germline genetic alterations contribute to SMN development following treatment of childhood solid cancers. First, we identified pathogenic variants in 35.7% of our SMN cohort. The prevalence was higher than that reported in a previous comprehensive study of primary cancer in pediatric patients (8.5%), 8 and it was especially high in subsequent leukemia cases and cases with multiple episodes of SMN. The prevalence was also higher than that observed in a previous study of cancer survivors with SMN (6.4%). 13 This discrepancy might result from differences in the SMN subtypes between cohorts due to different eligibility criteria. In the cancer survivor study cohort, 13 most patients had survived longer than 10 years after diagnosis to at least 18 years of age, and few patients experienced subsequent leukemia. By contrast, our cohort was not limited to survivors, but included deceased patients. In fact, in our study, the median interval from primary cancer to SMN was 5.5 years, and more than 50% of the SMNs in our cohort were leukemia. Moreover, all five patients with pathogenic variants in CPGs subsequently developed leukemia, and three of them were deceased. These findings suggest a high prevalence of CPGs among patients with subsequent leukemia. Another study showed a high prevalence of pathogenic variants in CPGs in pediatric therapy-related myeloid neoplasms (15%). 14 The highest frequency of CPG variants in our cohort might be due to the selection bias that results from the retrospective collection of patients with second cancers and the inclusion of four cases with multiple SMNs. Tumor genomic analysis revealed high TMB (>10 mutations/Mb) in an SMN specimen from SMN04 with a PMS2 variant. Generally, compared with adult cancer, pediatric cancer is known for its low TMB. 20 Moreover, the tumors of patients with a germline mutation in MMR genes, including PMS2, typically demonstrate high TMB. 21 Interestingly, another case also had high TMB, although no pathogenic variant in CPGs was detected in the patient (SMN10). In this case, a MMR abnormality that cannot be detected by WES might be present in the germline. Evaluation of TMB in SMN samples may be contribute to the therapeutic strategy. In treating patients with high TMB, the use of immune checkpoint inhibitors has been suggested. 20,22 We found that three of five patients with a CPG variant had not received radiation therapy. Although radiation therapy is a risk factor for SMN development, avoiding such  c Since the third cancer was leukemia, determination of the irradiation range was not applicable.
therapy is insufficient to reduce the risk of SMN in patients carrying CPG variants. Our analysis showed that a platinuminduced mutational signature was common in SMN specimens from the patients treated with platinum during primary treatment, which is concordant with a previous study that reported that platinum-based chemotherapy confers a risk of secondary leukemia development. 23 The use of not only known high-risk agents, such as topoisomerase II inhibitors, but also platinum agents might need to be minimized in treatment strategies for patients with a CPG variant.  cancer-predisposing genes. We highlight that overlapping effects of genetic background and primary cancer treatment contribute to the development of second cancers after treatment of pediatric solid tumors. These results support universal germline genetic screening for children with cancer to assess the risk of SMN development.