Functional pri-miR-34b/c rs4938723 and KRAS 3'UTR rs61764370 SNPs: Novel phenotype modifiers in Li-Fraumeni Syndrome?

PURPOSE: Li-Fraumeni Syndrome (LFS) is a rare cancer predisposing condition caused by germline pathogenic TP53 variants, in which core tumors comprise sarcomas, breast, brain and adrenocortical neoplasms. Clinical manifestations are highly variable in carriers of the Brazilian germline founder variant TP53 p.R337H, possibly due to the influence of modifier genes such as miRNA genes involved in the regulation of the p53 pathway. Herein, we investigated the potential phenotypic effects of two miRNA-related functional SNPs, pri-miR-34b/c rs4938723 and 3'UTR KRAS rs61764370, in a cohort of 273 LFS patients from Southern and Southeastern Brazil. METHODS: The genotyping of selected SNPs was performed by TaqMan® allelic discrimination and subsequently custom TaqMan® genotyping results were confirmed by Sanger sequencing in all SNP-positive LFS patients. RESULTS: Although the KRAS SNP showed no effect as a phenotype modulator, the rs4938723 CC genotype was significantly associated with development of LFS non-core tumors (first tumor diagnosis) in p.R337H carriers (p = 0.039). Non-core tumors were also more frequently diagnosed in carriers of germline TP53 DNA binding domain variants harboring the rs4938723 C variant allele. Previous studies described pri-miR-34b/c rs4938723 C as a risk allele for sporadic occurrence of thyroid and prostate cancers (non-core tumors of the LFS spectrum). CONCLUSION: With this study, we presented additional evidence about the importance of analyzing miRNA genes that could indirectly regulate p53 expression, and, therefore, may modulate the LFS phenotype, such as those of the miR-34 family.

Analysis of the Li-Fraumeni Spectrum Based on an International Germline TP53 Variant Data Set: An International Agency for Research on Cancer TP53 Database Analysis.

IMPORTANCE: Li-Fraumeni syndrome is a cancer predisposition syndrome that is associated with a high, lifelong risk of a broad spectrum of cancers that is caused by pathogenic TP53 germline variants. A definition that reflects the broad phenotypic spectrum that has evolved since the gene discovery is lacking, and mechanisms leading to phenotypic differences remain largely unknown. OBJECTIVE: To define the phenotypic spectrum of Li-Fraumeni syndrome and conduct phenotype-genotype associations across the phenotypic spectrum. DESIGN, SETTING, AND PARTICIPANTS: We analyzed and classified the germline variant data set of the International Agency for Research on Cancer TP53 database that contains data on a cohort of 3034 persons from 1282 families reported in the scientific literature since 1990. We defined the term Li-Fraumeni spectrum to encompass (1) phenotypic Li-Fraumeni syndrome, defined by the absence of a pathogenic/likely pathogenic TP53 variant in persons/families meeting clinical Li-Fraumeni syndrome criteria; (2) Li-Fraumeni syndrome, defined by the presence of a pathogenic/likely pathogenic TP53 variant in persons/families meeting Li-Fraumeni syndrome testing criteria; (3) attenuated Li-Fraumeni syndrome, defined by the presence of a pathogenic/likely pathogenic TP53 variant in a person/family with cancer who does not meet Li-Fraumeni syndrome testing criteria; and (4) incidental Li-Fraumeni syndrome, defined by the presence of a pathogenic/likely pathogenic TP53 variant in a person/family without a history of cancer. Data analysis occurred from November 2020 to March 2021. MAIN OUTCOMES AND MEASURES: Differences in variant distribution and cancer characteristics in patients with a germline TP53 variant who met vs did not meet Li-Fraumeni syndrome testing criteria. RESULTS: Tumor spectra showed significant differences, with more early adrenal (n = 166, 6.5% vs n = 0), brain (n = 360, 14.17% vs n = 57, 7.46%), connective tissue (n = 303, 11.92% vs n = 56, 7.33%), and bone tumors (n = 279, 10.98% vs n = 3, 0.39%) in patients who met Li-Fraumeni syndrome genetic testing criteria (n = 2139). Carriers who did not meet Li-Fraumeni syndrome genetic testing criteria (n = 678) had more breast (n = 292, 38.22% vs n = 700, 27.55%) and other cancers, 45% of them occurring after age 45 years. Hotspot variants were present in both groups. Several variants were exclusively found in patients with Li-Fraumeni syndrome, while others where exclusively found in patients with attenuated Li-Fraumeni syndrome. In patients who met Li-Fraumeni syndrome genetic testing criteria, most TP53 variants were classified as pathogenic/likely pathogenic (1757 of 2139, 82.2%), whereas 40.4% (404 of 678) of TP53 variants identified in patients who did not meet the Li-Fraumeni syndrome genetic testing criteria were classified as variants of uncertain significance, conflicting results, likely benign, benign, or unknown. CONCLUSIONS AND RELEVANCE: The findings of this cohort study suggest that this new classification, Li-Fraumeni spectrum, is a step toward understanding the factors that lead to phenotypic differences and may serve as a model for other cancer predisposition syndromes.

Variable population prevalence estimates of germline TP53 variants: A gnomAD-based analysis.

Reports of variable cancer penetrance in Li-Fraumeni syndrome (LFS) have raised questions regarding the prevalence of pathogenic germline TP53 variants. We previously reported higher-than-expected population prevalence estimates in sequencing databases composed of individuals unselected for cancer history. This study aimed to expand and further evaluate the prevalence of pathogenic and likely pathogenic germline TP53 variants in the gnomAD dataset (version r2.0.2, n = 138,632). Variants were selected and classified based on our previously published algorithm and compared with alternative estimates based on three different classification databases: ClinVar, HGMD, and the UMD_TP53 database. Conservative prevalence estimates of pathogenic and likely pathogenic TP53 variants were within the range of one carrier in 3,555-5,476 individuals. Less stringent classification increased the approximate prevalence to one carrier in every 400-865 individuals, mainly due to the inclusion of the controvertible p.N235S, p.V31I, and p.R290H variants. This study shows a higher-than-expected population prevalence of pathogenic and likely pathogenic germline TP53 variants even with the most conservative estimates. However, these estimates may not necessarily reflect the prevalence of the classical LFS phenotype, which is based upon family history of cancer. Comprehensive approaches are needed to better understand the interplay of germline TP53 variant classification, prevalence estimates, cancer penetrance, and LFS-associated phenotype.

Prevalence of pathogenic/likely pathogenic variants in the 24 cancer genes of the ACMG Secondary Findings v2.0 list in a large cancer cohort and ethnicity-matched controls.

BACKGROUND: Prior research has established that the prevalence of pathogenic/likely pathogenic (P/LP) variants across all of the American College of Medical Genetics (ACMG) Secondary Findings (SF) genes is approximately 0.8-5%. We investigated the prevalence of P/LP variants in the 24 ACMG SF v2.0 cancer genes in a family-based cancer research cohort (n = 1173) and in cancer-free ethnicity-matched controls (n = 982). METHODS: We used InterVar to classify variants and subsequently conducted a manual review to further examine variants of unknown significance (VUS). RESULTS: In the 24 genes on the ACMG SF v2.0 list associated with a cancer phenotype, we observed 8 P/LP unique variants (8 individuals; 0.8%) in controls and 11 P/LP unique variants (14 individuals; 1.2%) in cases, a non-significant difference. We reviewed 115 VUS. The median estimated per-variant review time required was 30 min; the first variant within a gene took significantly (p = 0.0009) longer to review (median = 60 min) compared with subsequent variants (median = 30 min). The concordance rate was 83.3% for the variants examined by two reviewers. CONCLUSION: The 115 VUS required database and literature review, a time- and labor-intensive process hampered by the difficulty in interpreting conflicting P/LP determinations. By rigorously investigating the 24 ACMG SF v2.0 cancer genes, our work establishes a benchmark P/LP variant prevalence rate in a familial cancer cohort and controls.

Mouse Homolog of the Human TP53 R337H Mutation Reveals Its Role in Tumorigenesis.

Inheritance of germline mutations in the tumor suppressor gene TP53 causes Li-Fraumeni syndrome (LFS), a cancer predisposition disorder. The arginine to histidine substitution at amino acid position 337 of p53 (R337H) is a founder mutation highly prevalent in southern and southeastern Brazil and is considered an LFS mutation. Although this mutation is of significant clinical interest, its role in tumorigenesis using animal models has not been described. Here, we generate a knockin mouse model containing the homologous R337H mutation (mouse R334H). De novo tumorigenesis was not significantly increased in either heterozygous (p53334R/H ) or homozygous (p53334H/H ) p53 R334H knockin mice compared with wild-type mice. However, susceptibility to diethylnitrosamine (DEN)-induced liver carcinogenesis was increased in a mutant allele dose-dependent manner. In parallel, p53334H/H mice exposed to DEN exhibited increased DNA damage but decreased cell-cycle regulation in the liver. Oligomerization of p53, which is required for transactivation of target genes, was reduced in R334H liver, consistent with its decreased nuclear activity compared with wild-type. By modeling a TP53 mutation in mice that has relatively weak cancer penetrance, this study provides in vivo evidence that the human R337H mutation can compromise p53 activity and promote tumorigenesis.Significance: A germline mutation in the oligomerization domain of p53 decreases its transactivation potential and renders mice susceptible to carcinogen-induced liver tumorigenesis. Cancer Res; 78(18); 5375-83. ©2018 AACR.

Complex Landscape of Germline Variants in Brazilian Patients With Hereditary and Early Onset Breast Cancer.

Pathogenic variants in known breast cancer (BC) predisposing genes explain only about 30% of Hereditary Breast Cancer (HBC) cases, whereas the underlying genetic factors for most families remain unknown. Here, we used whole-exome sequencing (WES) to identify genetic variants associated to HBC in 17 patients of Brazil with familial BC and negative for causal variants in major BC risk genes (BRCA1/2, TP53, and CHEK2 c.1100delC). First, we searched for rare variants in 27 known HBC genes and identified two patients harboring truncating pathogenic variants in ATM and BARD1. For the remaining 15 negative patients, we found a substantial vast number of rare genetic variants. Thus, for selecting the most promising variants we used functional-based variant prioritization, followed by NGS validation, analysis in a control group, cosegregation analysis in one family and comparison with previous WES studies, shrinking our list to 23 novel BC candidate genes, which were evaluated in an independent cohort of 42 high-risk BC patients. Rare and possibly damaging variants were identified in 12 candidate genes in this cohort, including variants in DNA repair genes (ERCC1 and SXL4) and other cancer-related genes (NOTCH2, ERBB2, MST1R, and RAF1). Overall, this is the first WES study applied for identifying novel genes associated to HBC in Brazilian patients, in which we provide a set of putative BC predisposing genes. We also underpin the value of using WES for assessing the complex landscape of HBC susceptibility, especially in less characterized populations.

Revisiting tumor patterns and penetrance in germline TP53 mutation carriers: temporal phases of Li-Fraumeni syndrome.

PURPOSE OF REVIEW: Germline pathogenic TP53 mutation may predispose to multiple cancers but penetrance and cancer patterns remain incompletely documented. We have analyzed international agency for research on cancer TP53 database to reevaluate age and variant-dependent tumor patterns. RECENT FINDINGS: Genome-wide studies suggest that germline variants are more frequent than estimated prevalence of Li-Fraumeni syndrome (LFS), suggesting that many carriers of potentially pathogenic mutations may not develop the syndrome. Carriers of a germline TP53 mutation who are detected in a clinical context have a penetrance of 80% at age 70. Penetrance varies according to age, sex and mutation type. Temporal tumor patterns show distinct phases, with childhood phase (0-15 years, 22% of all cancers) characterized by adrenal cortical carcinoma, choroid plexus carcinoma, rhabdomyosarcoma and medulloblastoma; early adulthood phase (16-50 years, 51%) including breast cancer, osteosarcoma, soft tissue sarcomas, leukemia, astrocytoma and glioblastoma, colorectal and lung cancer; late adulthood phase (51-80 years, 27%) including pancreatic and prostate cancer. SUMMARY: Germline pathogenic variants in TP53 gene have different consequences according to cell, tissue, context and age. The occurrence of frequent variants in patients with no criteria suggestive of LFS calls for attention in predicting individual risk and highlights the need of additional predictors for assigning carriers to appropriate surveillance programs.

Rare germline alterations in cancer-related genes associated with the risk of multiple primary tumor development.

Multiple primary tumors (MPT) have been described in carriers of inherited cancer predisposition genes. However, the genetic etiology of a large proportion of MPT cases remains unclear. We reviewed 267 patients with hereditary cancer predisposition syndromes (HCPS) that underwent genetic counseling and selected 22 patients with MPT to perform genomic analysis (CytoScan HD Array, Affymetrix) aiming to identify new alterations related to a high risk of developing MPT. Twenty patients had a positive family history of cancer and 11 met phenotypic criteria for HCPS. Genetic testing for each of the genes associated with these syndromes revealed negative results for pathogenic mutations. Seventeen rare germline copy number variations (CNVs) covering 40 genes were identified in 11 patients, including an EPCAM/MSH2 deletion in one Lynch syndrome patient. An enrichment analysis revealed a significant number of genes (where the CNVs are mapped) associated with carcinogenesis and/or related to functions implicated with tumor development, such as proliferation and cell survival. An interaction network analysis highlighted the importance of TP53 pathway in cancer emergence. A high number of germline copy-neutral loss of heterozygosity (cnLOH) was identified in nine cases, particularly in two patients. Eighteen genes were covered by both rare CNVs and cnLOH, including 14 related to tumorigenesis and seven genes (ABCC1, KDM4C, KIAA0430, MYH11, NDE1, PIWIL2, and ULK2) specifically associated with cellular growth and proliferation. Overall, we identified 14 cases with rare CNVs and/or cnLOH that may contribute to the risk of MPT development. KEY MESSAGE: CNVs may explain the risk of hereditary cancer syndromes in MPT patients. CNVs affecting genes related to cancer are candidates to be involved in MPT risk. EPCAM/MSH2 deletions should be investigated in patients suspected to have LS. Gene enrichment related to the TP53 network is associated with MPT development. cnLOH and CNVs contribute to the risk of MPT development.

Early-onset breast cancer patients in the South and Southeast of Brazil should be tested for the TP53 p.R337H mutation.

Germline TP53 mutations are associated with Li-Fraumeni syndrome (LFS), a disease that predisposes carriers to a wide variety of early onset tumors. In southern and southeastern Brazil, a high frequency of a germline TP53 mutation, p.R337H, was diagnosed in 0,3% of the population due to a founder effect. Carriers are at risk for developing cancer but the penetrance is lower than in typical DNA binding domain mutations. To date, only a few families were detected and diagnosis of carriers remains a challenge. Therefore, the inclusion of additional criteria to detect p.R337H carriers is necessary for the Brazilian population. We assessed the A.C. Camargo Cancer Center Oncogenetics Department database in search of common characteristics associated with p.R337H families that did not fulfill LFS/LFL clinical criteria. Among 42 p.R337H families, three did not meet any LFS/LFL criteria. All cases were young female patients with breast cancer diagnosed before age 45 and with no family history of LFS linked-cancers. Our results suggest that screening for the germline TP53 p.R337H mutation should be indicated, along with BRCA1 and BRCA2 genetic testing, for this group of patients, especially in the South and Southeast of Brazil.

ROBO1 deletion as a novel germline alteration in breast and colorectal cancer patients.

Despite one third of breast (BC) and colorectal cancer (CRC) cases having a hereditary component, only a small proportion can be explained by germline mutations. The aim of this study was to identify potential genomic alterations related to cancer predisposition. Copy number variations (CNVs) were interrogated in 113 unrelated cases fulfilling the criteria for hereditary BC/CRC and presenting non-pathogenic mutations in BRCA1, BRCA2, MLH1, MSH2, TP53, and CHEK2 genes. An identical germline deep intronic deletion of ROBO1 was identified in three index patients using two microarray platforms (Agilent 4x180K and Affymetrix CytoScan HD). The ROBO1 deletion was confirmed by quantitative PCR (qPCR). Six relatives were also evaluated by CytoScan HD Array. Genomic analysis confirmed a co-segregation of the ROBO1 deletion with the occurrence of cancer in two families. Direct sequencing revealed no pathogenic ROBO1 point mutations. Transcriptomic analysis (HTA 2.0, Affymetrix) in two breast carcinomas from a single patient revealed ROBO1 down-expression with no splicing events near the intronic deletion. Deeper in silico analysis showed several enhancer regions and a histone methylation mark in the deleted region. The ROBO1 deletion in a putative transcriptional regulatory region, its down-expression in tumor samples, and the results of the co-segregation analysis revealing the presence of the alteration in affected individuals suggest a pathogenic effect of the ROBO1 in cancer predisposition.

Contribution of rare germline copy number variations and common susceptibility loci in Lynch syndrome patients negative for mutations in the mismatch repair genes.

In colorectal carcinoma (CRC), 35% of cases are known to have a hereditary component, while a lower proportion (∼ 5%) can be explained by known genetic factors. In this study, copy number variations (CNVs) were evaluated in 45 unrelated patients with clinical hypothesis of Lynch syndrome (Amsterdam or Bethesda criteria); negative for MLH1, MSH2, MSH6, PMS2, CHEK2*1100delC and TP53 pathogenic mutations; aiming to reveal new predisposing genes. Analyses with two different microarray platforms (Agilent 180K and Affymetrix CytoScan HD) revealed 35 rare CNVs covering 67 known genes in 22 patients. Gains (GALNT6 and GALNT11) and losses (SEMA3C) involving the same gene families related to CRC susceptibility were found among the rare CNVs. Segregation analysis performed on four relatives from one family suggested the involvement of GALNT11 and KMT2C in those at risk of developing CRC. Notably, in silico molecular analysis revealed that 61% (41/67) of the genes covered by rare CNVs were associated with cancer, mainly colorectal (17 genes). Ten common SNPs, previously associated with CRC, were genotyped in 39 index patients and 100 sporadic CRC cases. Although no significant, an increased number of risk alleles was detected in the index cases compared with the sporadic CRC patients. None of the SNPs were covered by CNVs, suggesting an independent effect of each alteration in cancer susceptibility. In conclusion, rare germline CNVs and common SNPs may contribute to an increased risk for hereditary CRC in patients with mismatch repair proficiency.

DNA Methylation Levels of Melanoma Risk Genes Are Associated with Clinical Characteristics of Melanoma Patients.

In melanoma development, oncogenic process is mediated by genetic and epigenetic mutations, and few studies have so far explored the role of DNA methylation either as predisposition factor or biomarker. We tested patient samples for germline CDKN2A methylation status and found no evidence of inactivation by promoter hypermethylation. We have also investigated the association of clinical characteristics of samples with the DNA methylation pattern of twelve genes relevant for melanomagenesis. Five genes (BAP1, MGMT, MITF, PALB2, and POT1) presented statistical association between blood DNA methylation levels and either CDKN2A-mutation status, number of lesions, or Breslow thickness. In tumors, five genes (KIT, MGMT, MITF, TERT, and TNF) exhibited methylation levels significantly different between tumor groups including acral compared to nonacral melanomas and matched primary lesions and metastases. Our data pinpoint that the methylation level of eight melanoma-associated genes could potentially represent markers for this disease both in peripheral blood and in tumor samples.

Genomic profile of a Li-Fraumeni-like syndrome patient with a 45,X/46,XX karyotype, presenting neither mutations in TP53 nor clinical stigmata of Turner syndrome.

Li-Fraumeni syndrome (LFS) is a hereditary disorder that predisposes patients to several types of cancer and is associated with TP53 germline mutations. Turner syndrome (TS) is one of the most common aneuploidies in women. Patients with TS have a higher risk of developing cancer, although multiple malignant tumors are extremely rare. Herein, we describe a patient with a 45,X/46,XX karyotype with no classic phenotype of TS. She presented with a clinical diagnosis of Li-Fraumeni-like syndrome (LFL), showing papillary thyroid carcinoma and fibrosarcoma of the left flank, and had no TP53 germline mutations. Genome-wide analysis of copy number variations (CNVs) was assessed in DNA from peripheral blood cells and saliva. A total of 109 rare CNVs in the blood cells, including mosaic loss of the X chromosome (76% of cells), were identified. In saliva, three rare CNVs were detected, all of them were also detected in the blood cells: loss of 8q24.11 (EXT1), gain of 16q24.3 (PRDM7 and GAS8), and the mosaic loss of the X chromosome (50% of cells). Results of conventional G-banding confirmed the 45,X/46,XX karyotype. Surprisingly, the patient presented with an apparently normal phenotype. The PRDM and GAS8 genes are potential candidates to be associated with the risk of developing cancer in this LFL/TS patient.

LINE-1 hypermethylation in peripheral blood of cutaneous melanoma patients is associated with metastasis.

Aberrant DNA methylation pattern is a well-known epigenetic marker of cancer cells. Recently, aberrant methylation was also reported in the peripheral blood of cancer patients and it could potentially serve as a biomarker for cancer risk. We investigated the methylation pattern of LINE-1 and other repetitive DNA elements in peripheral blood of cutaneous melanoma patients in order to search for an association with clinical characteristics. The patient cohort was composed by 69 unrelated melanoma patients, 28 of whom were hereditary cases (with or without CDKN2A mutations) and 41 were isolated (sporadic) melanoma cases. Methylation of LINE-1 was evaluated by pyrosequencing, whereas additional repetitive DNA sequences were assessed using Illumina 450K methylation microarray. Melanoma patients exhibited a higher, albeit heterogeneous, LINE-1 methylation level compared with controls. Hereditary melanoma patients carrying CDKN2A mutations showed a hypermethylated pattern of both LINE-1 and repetitive DNA elements compared with other patients. In particular, the methylation level at one specific CpG of LINE-1 was found to be correlated with the occurrence of metastasis. Our data suggest that LINE-1 hypermethylation in peripheral blood of melanoma patients is a potential epigenetic biomarker for metastasis occurrence.

Hereditary breast and ovarian cancer: assessment of point mutations and copy number variations in Brazilian patients.

BACKGROUND: Germ line mutations in BRCA1 and BRCA2 (BRCA1/2) and other susceptibility genes have been identified as genetic causes of hereditary breast and ovarian cancer (HBOC). To identify the disease-causing mutations in a cohort of 120 Brazilian women fulfilling criteria for HBOC, we carried out a comprehensive screening of BRCA1/2, TP53 R337H, CHEK2 1100delC, followed by an analysis of copy number variations in 14 additional breast cancer susceptibility genes (PTEN, ATM, NBN, RAD50, RAD51, BRIP1, PALB2, MLH1, MSH2, MSH6, TP53, CDKN2A, CDH1 and CTNNB1). METHODS: Capillary sequencing and multiplex ligation-dependent probe amplification (MLPA) were used for detecting point mutations and copy number variations (CNVs), respectively, for the BRCA1 and BRCA2 genes; capillary sequencing was used for point mutation for both variants TP53 R337H and CHEK2 1100delC, and finally array comparative genomic hybridization (array-CGH) was used for identifying CNVs in the 14 additional genes. RESULTS: The positive detection rate in our series was 26%. BRCA1 pathogenic mutations were found in 20 cases, including two cases with CNVs, whereas BRCA2 mutations were found in 7 cases. We also found three patients with the TP53 R337H mutation and one patient with the CHEK2 1100delC mutation. Seven (25%) pathogenic mutations in BRCA1/2 were firstly described, including a splice-site BRCA1 mutation for which pathogenicity was confirmed by the presence of an aberrant transcript showing the loss of the last 62 bp of exon 7. Microdeletions of exon 4 in ATM and exon 2 in PTEN were identified in BRCA2-mutated and BRCA1/2-negative patients, respectively. CONCLUSIONS: In summary, our results showed a high frequency of BRCA1/2 mutations and a higher prevalence of BRCA1 (64.5%) gene. Moreover, the detection of the TP53 R337H variant in our series and the fact that this variant has a founder effect in our population prompted us to suggest that all female breast cancer patients with clinical criteria for HBOC and negative for BRCA1/2 genes should be tested for the TP53 R337H variant. Furthermore, the presence of genomic structural rearrangement resulting in CNVs in other genes that predispose breast cancer in conjunction with BRCA2 point mutations demonstrated a highly complex genetic etiology in Brazilian breast cancer families.