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The emerging landscape of germline variants in urothelial carcinoma: Implications for genetic testing
Cancer Treatment and Research Communications 23 (2020) 100165
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Cancer Treatment and Research Communications journal homepage: www.elsevier.com/locate/ctarc
The emerging landscape of germline variants in urothelial carcinoma: Implications for genetic testing
T
Panagiotis J. Vlachostergios (M.D., Ph.D.)a, Bishoy M. Faltas (M.D.)a,b,c, Maria I. Carlo (M.D.)d, Amin H. Nassar (M.D.)e, Sarah Abou Alaiwi (M.D.)f, Guru Sonpavde (M.D.)f,⁎ a
Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, NY, United States Department of Cell and Developmental Biology, Weill Cornell Medicine, New York, NY, United States c Caryl and Israel Englander Institute for Precision Medicine, Weill Cornell Medicine, New York, NY, United States d Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, United States e Department of Medicine, Brigham and Women's Hospital, Boston, MA, United States f Lank Center for Genitourinary Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave, DANA 1230, Boston, MA 02215, United States b
ARTICLE INFO
ABSTRACT
Keywords: Bladder cancer Genetic testing Germline variants Urothelial carcinoma Lynch syndrome
Urothelial carcinoma (UC) of the bladder and upper tract (ureter, renal pelvis) is one of the most frequently occurring malignancies. While the majority of UC are chemically induced by smoking, accumulating evidence from genetic studies have demonstrated a small, but consistent impact of heritable gene variants and family history of UC on the development of the disease. Beyond the established association between upper tract UC and germline mismatch DNA repair defects as a defining feature of Lynch syndrome, newer investigations focusing on moderate- and high-risk cancer-related gene variants in DNA damage repair and other signaling pathways are expanding our knowledge on the heritable genetic basis of UC, opening new avenues in the breadth of genetic testing and in clinical counseling of these patients. Overcoming existing challenges in the interpretation of uncertain findings and family cascade testing may help expand our testing approach and guidelines. Following the paradigm of other tumor types, such as breast and ovarian cancers, germline genetic testing, particularly when combined with somatic testing, has the potential to directly benefit affected UC patients and their families in the future through therapeutic targeting (i.e. with poly(ADP-ribose)) polymerase inhibitors, immune checkpoint inhibitors) and genetically informed screening/surveillance, respectively.
1. Introduction
populations supported an increased risk of developing UC in subjects with a positive family history of bladder cancer. The Spanish casecontrol study reported an odds ratio of 2.34 among patients with at least one first-degree relative with bladder UC [3]. The Nordic study examined prospectively 80,309 monozygotic and 123,382 same-sex dizygotic twin individuals within the population-based registers of Denmark, Finland, Norway, and Sweden. After a median follow up of 32 years, the familial risk of developing bladder cancer was higher in monozygotic (9.9%) compared to dizygotic (5.5%) twins, with an estimated heritability of 30% for bladder cancer [4].
Urothelial carcinoma (UC) is the fourth most common malignancy among men in the United States with 158,220 estimated new cases in both sexes in 2019, one fifth of which (33,420) are predicted to be lethal [1]. The main environmental risk factors are tobacco and aromatic amines [2]. Nevertheless, UC develops in a minority of these cases, suggesting a potential role of genetic predisposition to urothelial carcinogenesis. Previous observations from large studies in Spanish and Nordic
Disclosures: Guru Sonpavde: research grants from Boehringer-Ingelheim, Bayer, Onyx-Amgen, grants and advisory board fees from Pfizer, advisory board for Genentech, advisory board for Novartis, research grants and advisory board for Merck, research grants and advisory board for Sanofi, advisory board and steering committee of trial for Seattle Genetics/Astellas, speaking fees from Clinical Care Options, grants, trial steering committee, advisory board and travel fees from Astrazeneca, writing fees from Uptodate, advisory board for Exelixis, advisory board and travel costs from Bristol-Myers-Squibb (BMS), research grants and advisory board Janssen, advisory board for Amgen, advisory board for Eisai, advisory board for NCCN, research grants from Celgene, speaking fees from Physicians Education Resource, Onclive, Research to Practice, Steering committee of trials sponsored by Bavarian Nordic, Debiopharm, QED. Bishoy Faltas, Advisory board for Immunomedics. Research grants: Eli-Lilly. Honoraria: Urotoday. ⁎ Corresponding author. E-mail address: [email protected] (G. Sonpavde). https://doi.org/10.1016/j.ctarc.2020.100165
2468-2942/ © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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Genetic variations in UC can be germline or somatic [5]. A germline variant is defined as a genetic alteration that occurs within the germ cells (egg or sperm), such that the alteration can be passed to subsequent generations [5]. Genetic variants may be activating, resulting in a gain of function of an oncogene (such as a missense variant in its functional or kinase domain), or inactivating (such as nonsense, splicesite, and frameshift insertion/deletion variants, thereby causing a loss of function of a tumor-suppressor gene). The types of variants observed include single-nucleotide variants (SNVs) that cause missense, silent, or nonsense amino acid substitutions, or splice site alterations which affect normal splicing of the mRNA transcript. Alternatively, one or more nucleotides may be involved in duplications, deletions, insertions, or even more complex combinations of these changes, including for example a nucleotide(s) deletion coupled with a nucleotide(s) insertion (indels) at a specific gene location [5]. Curating gene variants in knowledge bases is critical for deepening our understanding of their role in cancer predisposition and providing genetic counseling to patients [6]. ClinVar is a publicly-available database that associates germline variants with their phenotypic characteristics and couples that with clinical and experimental evidence. Moreover, various in silico prediction algorithms have been developed to better predict whether a nucleotide change alters a protein's structure and/or function [7]. However, variants of uncertain clinical significance (VUS) pose a particular challenge in clinical medicine since the functional impact cannot be inferred from sequence information alone. For this reason, patients with no pathogenic variants or with presence of a VUS are counseled based on the family history only [8]. In this review we discuss recent advances in germline variant testing that are shaping the current landscape of heritable cancer-susceptibility genes in patients with UC, and their potential implications for testing and family screening recommendations.
Table 1 Estimated risks for development of Lynch syndrome by canonical MMR gene variant [13]. Gene
Risk of Lynch syndrome (%)
Age at diagnosis
MLH1 MSH2 MSH6 PMS2 Non-carrier (general population)
0.2–5 2–18 0.7–7 Not well established <1
52–60 52–61 52–69 Not well established
risk of a number of malignancies. LS is the most common hereditary cause of colorectal (CRC) (1–3%) and endometrial (2–5%) cancers. Upper tract UC (UTUC) is considered a core cancer in LS [11-14]. The reported lifetime risk of UTUC varies according to the MMR gene involved. MSH2 confers the highest lifetime risk, which ranges between 2% and 18% [13] (Table 1). Traditionally, the diagnosis of LS is a two-step process which includes: a) screening family history using the Amsterdam II or/and revised Bethesda criteria, and b) immunohistochemical (IHC) staining for absence of MMR protein expression, and/or a polymerase chain reaction (PCR) for presence of microsatellite instability. LS can subsequently be confirmed by germline genetic testing for deleterious MMR variants [11–14]. The Amsterdam II criteria follow a 3–2–1 rule: ≥1 relative must be a first-degree relative of another two, ≥2 successive generations must be affected, and ≥1 LS-associated diagnosis must be at age <50. The updated Bethesda criteria are less strict to minimize the proportion of patients that can be missed by family history alone. They are as follows: CRC in a patient <50 years old, synchronous or metachronous CRC or other LS-associated cancer, CRC with microsatellite instability-high (MSI-H) histology at age <60, and CRC in a patient with one a relative with family history of LS-associated cancer diagnosed at age <50, or ≥2 relatives at any age [11–14]. On top of the clinical criteria, risk estimation statistical models have been developed to assist with patient selection, including the PREMM5, MMRpredict, and MMRpro models [11–14]. In view of the high prevalence of LS in patients with UTUC, evaluation is recommended for high-risk patients based on family history. Universal screening has also been proposed, based on detection of up to 14% of potential LS-associated UTUC cases in a universally screened cohort of 115 patients [15]. However, it is important to note that contrary to the prevalent notion, sporadic UTUCs (which constitute the vast majority of UTUCs) are not frequently MSI-H and may have a lower TMB compared to bladder UC [16,17]. Patients with LS may also be at higher risk of bladder cancer, with a risk of up to 6.2% in MSH2 carriers reported in a Canadian registry cohort [18]. However, these studies are limited due to the presence of synchronous tumors and drop metastases, and the true association of LS and bladder cancer risk has been difficult to quantify. There also appears to be a risk for prostate, testicular and adrenocortical carcinoma which requires further investigation [19,20]. There is currently no clear evidence to support routine surveillance for UC in LS. Surveillance in select patients may be considered such as patients with a family history of UC or individuals with MSH2 pathogenic variants (especially males) as these groups appear to be at higher risk, up to 18% (Table 1) [11–14]. Surveillance options may include annual urinalysis (UA) starting at age 30–35 years [11–14]. Frequent UA with a threshold of 3 red blood cells per high-power field (RBC/HPF) or greater to pursue further testing has been suggested [11]. However, the optimal screening strategy is unclear. The detection of suspicious urinary or imaging findings should prompt computed tomography urogram (CTU) and a cystoscopy. Additional retrograde studies should be performed if the ureters are not fully imaged, with ureteroscopic evaluation, selective washings and biopsy when indicated [11]. In the therapeutic context, programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) immune checkpoint inhibitors are
2. Known cancer-susceptibility genes in UC Genome wide association studies (GWAS) have been the mainstay of studying associations between putative variants and risk of cancer [9]. After genotyping of DNA samples from patients and controls, association test statistics are generated to identify genetic risk loci. Overall, within the number of cancer cases that are present in a particular population at a given time (prevalence), the proportion of individuals with a specific cancer-associated genotype who also develop cancer within specific time period (penetrance) is small [10]. In other words, most genotypes for common, complex diseases are incompletely penetrant, and correlations between the genotype and the phenotype vary [9,10]. Numerous GWAS conducted in nearly all common malignancies have identified three major groups of genes that shape the genetic architecture of cancer risk in all cancers: a) rare, high-penetrance genetic variants that confer a higher relative risk (RR), such as pathogenic variants in BRCA1 and BRCA2 associated with hereditary breast and ovarian cancer, and MLH1 and MSH2 associated with Lynch syndrome (LS); b) uncommon, moderate-penetrance genetic variants that confer a moderate RR, such as those in ATM and CHEK2, and c) common, lowpenetrance genetic variants resulting in low RR, such as single nucleotide polymorphisms (SNPs) [9]. 2.1. Germline variants in mismatch repair (MMR) genes - Lynch syndrome Germline variants in the MMR genes MLH1, MSH2, MSH6, PMS2, and deletions in EPCAM gene, resulting in hypermethylation-induced silencing of MSH2, are all diagnostic of LS, also known as hereditary non-polyposis colon cancer (HNPCC) [11–14]. Defective MMR proteins lead to the accumulation of DNA replication errors within microsatellite regions, which are short repeating sequences of DNA that frequently serve as molecular markers in various genetic tests. MMR gene variants are inherited in an autosomal dominant pattern and cause increased 2
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extensively used in clinical practice for treatment of patients with advanced UC [21]. Interestingly, the presence of microsatellite instability (MSI) resulting from defective MMR, confers a high likelihood of response to PD-1 inhibition, with the most prevalent underlying mechanism being a high tumor mutational burden resulting in increased neoantigenesis and immune recognition [22,23]. The FDA approved the PD-1 inhibitor pembrolizumab for the treatment of patients with progressive advanced, MSI-H, or MMR-deficient tumors independent of origin [24,25].
patients with UC. Among the entire cohort, 15% of patient harbored germline pathogenic or likely pathogenic variants [32], the majority (92%) of which involved DDR genes. Additionally, variants in LS-associated MMR genes (4%), BRCA1/2 (2.5%), CHEK2 (2.3%), and MUTYH (1.9%) were detected [32]. Overall, 90 pathogenic or likely pathogenic variants affecting 18 DDR genes were detected in 12.2% of patients [32]. This high frequency of germline DDR variants in patients with UC may open new avenues for therapeutic targeting via exploitation of poly (ADP-ribose) polymerase (PARP) inhibitors which are already in clinical use for other cancer types with DDR alterations such as ovarian or breast cancer [33,34]. A case-control study conducted in an Eastern European population also supports an association between the presence of variants in the moderate-risk CHEK2 gene and increased risk of bladder cancer, as founder CHEK2 variants were more significantly detected in UC cases (10.6%) compared with controls (5.9%) [35]. Analysis of 100 UC bladder cases using Tumor-Only Boosting Identification (TOBI), a unifying framework that uses a machine learning algorithm to identify oncogenic germline variants in patients with tumor-normal paired DNA, revealed germline variants in 5% of patients, affecting BRCA2, FANCM, and FANCD2 genes [36]. This enrichment of germline inactivating variants in the Fanconi anemia (FA) pathway was unique for bladder UC compared to six other cancer types [36]. This supports a potential genetic predisposition of FA gene mutation carriers to UC. Taking family history into account, a study tested 73 UC patients, two thirds of whom had first-degree and one third had second-degree (or higher) relatives with UC of the bladder or upper tract [37]. Overall, half of the patients (52%) in the cohort were found to have germline alterations, with pathogenic variants seen in 18% of subjects [37]. Among patients with positive family history in a first-degree relative 17% were carriers of pathogenic variants while 20% of patients with second-degree (or higher) relatives tested positive for pathogenic variants. These affected several DDR genes, including BRCA1 (1.4%), BRCA2 (1.4%), BRIP1 (1.4%), CHEK2 (1.4%), MSH2 (4.1%), MUTYH (2.7%), as well as other known cancer-predisposition genes such as MITF (2.7%), PTCH (1.4%), and SDHC (1.4%) [37]. The largest study to date of germline genetic testing in UC included a total of 1038 patients with high suspicion of heritable pathogenic variants, based on history of at least one other non-urothelial cancer, young age, or family history of cancer or cancer predisposition syndrome [38]. Most patients (97%) underwent multigene testing of at least 5 genes among a total panel of 130 genes [38]. One fourth (24%) of these patients were found to be carriers of germline pathogenic and likely pathogenic variants, the majority of which (78% of carriers or 20% of total cohort) involved various highand moderate-penetrance DDR genes: MSH2 (3.5%), FANCC (3.3%), BRCA1 (2.3%), BRCA2 (2.1%), MUTYH (2%), ATM (1.6%), CHEK2 (1.4%), MLH1 (1%) [38]. Other cancer-predisposition moderate- and low-risk genes were also affected, including MITF (1.2%) and FH (1.3%), respectively [38]. Importantly, a significant enrichment of pathogenic variants in MSH2 (OR: 15.4), MLH1 (OR: 15.9), BRCA2 (OR: 5.7), and ATM (OR: 3.8) was found in UC patients compared to non-cancer subjects from the ExAC database [38]. Although these findings warrant validation in dedicated case-control studies jointly analyzing both groups and stratifying for ancestry, BRCA2 and ATM are highlighted as potential UC predisposition genes, in addition to the known MMR genes. An overview of the frequencies of various inherited DDR gene variants in patients with UC across different targeted sequencing studies is presented in Table 2. The average prevalence of moderate- and high-penetrance pathogenic or likely pathogenic DDR variants that are shared between the two largest studies to date are depicted in Fig. 1.
2.2. Germline variants in other DNA repair genes in UC Beyond deleterious MMR gene variants, heritable variants in other DNA repair genes are also frequent in UC and may play a role in its pathogenesis. In a cohort of 53 patients who were unselected for family history, consisting of 43 bladder and 10 upper tract UC, 12 different DNA repair gene germline variants were detected by whole-exome sequencing. These included genes involved in non-homologous endjoining such as RECQL4 (54.5%) and POLQ (6%), nucleotide excision repair, including ERCC6 (6%), XPA (3%), CCNH (3%) and POLK (3%), homologous recombination, including RNF168 (3%), RAD17 (3%) and POLE (3%), Fanconi anemia pathway such as POLN (3%), non-canonical mismatch repair represented by EXO1 (3%) [26,27]. Overall, the presence of these DDR variants in the cohort occurred at a significantly higher frequency (33/53, 62.3%) compared to the Exome Aggregation Consortium (ExAC) database, which consists of a large (60,706) repository of unrelated individuals’ DNA sequences as part of various disease-specific and population genetic studies [26–28]. In another cohort, 113 patients with UC involving bladder , or ureter and renal pelvis , unselected for suspicion of inherited cancer syndrome, underwent next generation sequencing of tumor-normal DNA pairs using a targeted germline gene panel that consisted of 76 genes associated with hereditary cancer predisposition [29]. Germline variants were found in 22% of patients. The majority of germline variants affected DNA damage response and repair (DDR) genes (58%), several of which were of high penetrance, such as BRCA1 (2.6%), BRCA2 (1.8%), MMR (7%), TP53 (0.8%), FH (0.8%) or moderate penetrance, such as CHEK2 (3.5%), ATM (0.8%), BRIP1 (0.8%), or NBN (0.8%) [29]. An expansion of the same study included 176 patients (cohort 1) and 327 de-identified patients without clinical annotation (cohort 2). Highly- or moderately-penetrant, pathogenic or likely pathogenic DDR gene variants, including MSH2, BRCA1, CHEK2, BRCA2, MSH6, MLH1, MUTYH, ERCC2, ERCC3 were found in 17% of cohort 1 patients and 9.8% of patients from cohort 2 [30].These results further highlight that DDR germline variants are not as rare in UC as previously thought, and that their presence should trigger a genetic consultation and counseling for risk assessment of hereditary cancer syndromes associated with germline variants in the same genes [30]. An expanded version (n=586) of this largely unselected group of UC patients was reported, the majority of whom did not have a family history of UC (n=544) or personal history of second primary (n=474), underwent paired tumor and germline testing [31]. Sixty-six of 80 patients (83%) were carriers of germline pathogenic or likely pathogenic variants in DDR genes, including: BRCA2 (1.5%), MSH2 (1.4%), BRCA1 (1.4%), CHEK2 (1.0%), ERCC3 (0.7%), NBN (0.5%) and RAD50 (0.5%) [31]. When these DDR genes' allele frequencies were compared to the those from non-cancer subjects in the ExAC database, variants in BRCA2 (odds ratio [OR]: 3.68) and MSH2 (OR: 4.58) were enriched in UC [31]. These findings suggest that non-MMR DDR gene variants (9% of the total cohort) may also play a previously unreported role in genetic predisposition to UC, particularly BRCA2. These pathogenic variant carriers were more likely to present at an early age of ≤45 [31]. Additionally, there was a quarter of patients (26%) harboring high-penetrance germline variants that would not have been detected using current guidelines based on family history, suggesting a potential value for expanded germline testing in UC [31]. Another large scale study used a commercially available targeted sequencing platform, Invitae, to and analyzed a set of 42 genes in 645
3. Common germline variants and polygenic risk scores in UC Common low-penetrance variants (risk allele frequency >5% and OR <1.5) [9], such as SNPs have been extensively studied for potential 3
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Table 2 Prevalence of pathogenic/likely pathogenic heritable variants in high- and moderate-penetrance DNA damage response and repair (DDR) genes in UC patients. Gene
Pathway
Mutation frequency (%)
Number of patients (N)
Ref
BRCA1 BRCA2 BAP1 NBN FANCD2, FANCM, BRCA2 BRIP1 FANCC MSH2, MSH6, MLH1, PMS2 MUTYH ERCC2 ERCC3 ATM CHEK2 TP53 Total DDR genes
HR
1.4–2.6 1.4–2.1 0.5–1.4 0.4–0.8 2.1–5 0.8–1.4 3.3 2–7 1.1–2.7 0.5–1.2 0.7–1.2 0.5–1.6 1.0–3.5 0.2–0.8 11.3–22.0
113–867 113–867 73–398 113–784 100–885 73–764 60 73–969 176–754 176–586 176–586 113–827 73–862 113–929 73–1038
[29,30,31,37,38] [29,30,31,37,38] [31,37,38] [29,31,38] [36,38] [29,31,37,38] [38] [29,30,31,37,38] [29,30,31,37,38] [30,31] [30,31] [29,31,38] [29,30,31,35,37,38] [29,31,38] [29,30,31,37,38]
FA MMR BER NER Checkpoint activation Cell Cycle Total DDR pathways
HR: homologous recombination; FA: Fanconi anemia; MMR: mismatch repair; BER: base excision repair; NER: nucleotide excision repair; DDR: DNA damage response and repair.
contribution to heritability of several cancers, including UC. Two large meta-analyses of several available datasets from UC GWAS have dissected the most commonly involved genes and pathways [39,40]. Interestingly, specific SNPs in the DDR genes ERCC2, NBN, and XPC are all weakly (OR: 1.10) but significantly associated with bladder cancer risk (p-value < 0.05) [41]. Additionally, several SNPs located in genes belonging to pathways such as chemical carcinogenesis (e.g. NAT2, UGT1A), and cell cycle (e.g. CCND1, CCNE1) were identified [42]. Occasionally, functional studies have further supported a causal
association for specific genotypes, including APOBEC-rs1014971, GSTM1-null, NAT2-slow, UGT1A-rs1189203, and CCNE1-rs8102137 [41–46]. The APOBEC3 gene family (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3) encodes proteins with cytidine deaminase activity (A3A, A3B, A3C, A3D, A3F, A3G, and A3H) which results in a particular mutagenesis pattern, found in several cancer types [44]. The APOBEC-rs1014971 SNP was not only associated with bladder cancer risk, but also with increased APOBEC3B gene expression and enrichment of APOBEC-signature mutations in bladder UC tumors
Fig. 1. Average frequency of shared pathogenic/likely pathogenic germline DDR variants across two large UC cohorts. 4
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their true effect on development of UC. It might also be useful to broaden our family history-based testing to be more inclusive, since a substantial proportion of patients with germline variants is not identified by currently available criteria. Paired germline – tumor testing represents a more comprehensive approach to assess potential interactions between biallelic or combined germline and somatic alterations that may accelerate carcinogenesis or/and tumor progression in a patient (i.e. a germline DDR mutation and a somatic mutation in an oncogene or tumor-suppressor gene). In a prospectively accrued cohort of 907 patients who underwent tumor targeted sequencing during four consecutive years (2013-2017), the frequency of germline testing and genetic counseling visits showed a significantly and consistently increasing trend [56]. This supports an integrated clinical cancer genetic approach to improve detection rates and ability to inform therapeutic decisions for patients, as well as counseling on cancer screening and prevention strategies for affected families [56]. Finally, it is key to take into account patients’ preferences and potential financial and emotional impact of testing for themselves and their families [57]. Further studies are needed to refine the selection of appropriate patients for germline evaluation, and elucidate the impact of clinical (e.g. age and family history of UC and other malignancies) and tumor characteristics. The increasing availability of clinical whole-exome and wholegenome sequencing is expected to increase opportunities for discovery of rare deleterious variants and challenges for the clinical management of patients harboring such variants. Providing a better framework for clinical decision-making based on an improved understanding of genetic predisposition of UC will enable a more rationalized use of interventions for individuals with inherited gene variants associated with an increased risk of UC. Our proposed framework for germline genetic testing in UC is provided in Table 3.
Table 3 Proposed framework for germline genetic testing in urothelial carcinoma (UC). Which patients with urothelial carcinoma (UC) should be offered germline genetic testing? 1) All patients who meet the Amsterdam/Bethesda criteria or Hereditary breast and ovarian cancer/BRCA guidelines or have a close relative who would have met guidelines. 2) All patients with MSI-H or MMR-deficient tumors. 3) Patients with early onset diagnosis ≤45 years, which is outside the 5% confidence interval of median age of onset for UC.
from The Cancer Genome Atlas (TCGA) [43]. The plethora of SNPs potentially involved in bladder carcinogenesis, as well as the contribution of environmental factors like smoking, have led to the development of polygenic risk scores (PRS). A PRS is a number that reflects the variability and weight of several genetic loci and non-genetic contributors in the prediction of risk for developing a particular condition or trait [47]. Testing 3,942 cases and 5,680 controls of European background for multiplicative and additive interactions between smoking and 12 susceptibility loci revealed that NAT2 and UGT1A6 variants have significant additive gene-environment interactions. Additionally, bladder cancer risk ranged from 2.9% for current smokers in the lowest quartile of the PRS to 9.9% for current smokers in the upper quartile. This demonstrates that prevention strategies targeting smoking cessation may be more successful in reducing the number of bladder cancer cases for subjects exhibiting higher genetic risk compared to those with lower risk [48]. A more recent study used genotype and phenotype data from TCGA and Electronic Medical Records and Genomics (eMERGE) to estimate 11 cancer‐specific PRS, including bladder UC. The mean cancer‐specific PRS was significantly higher among UC patients in TCGA (1.04) than controls in eMERGE (0.98) [49].
Declaration of Competing Interest
4. Perspectives and challenges of germline genetic testing in UC
No other conflicts of interest to declare by rest authors.
At present, screening for UC in asymptomatic non-high-risk adults is not recommended by several major organizations including The US Preventive Services Task Force (USPSTF), the National Cancer Institute (NCI), and the American Cancer Society (ACS), in the absence of sufficient evidence to support it [50–52]. Efforts have rather focused on better identifying high-risk groups that might benefit from screening. Such models have typically included age, sex, smoking history, and family history of bladder cancer and indicated a risk score > 6 as a threshold for future design of screening trials [53]. Hence, outside the spectrum of LS, genetic testing of UC patients potentially plays an important role in identifying high-risk patients based on the presence of pathogenic or likely pathogenic variants. In addition to the obvious implications for family cascade testing in the case of UC pathogenic mutation carriers, there may be additional benefit from therapeutic targeting of these defects. For instance, beyond its FDA-approved use in patients with advanced BRCA-mutated breast or ovarian cancer, the PARP inhibitor olaparib has shown activity in UC patients harboring germline DNA repair gene DRR variants [54]. Also, there are ongoing clinical trials of PARP inhibitors such as olaparib in bladder UC patients with germline or somatic with germline or somatic DDR defects (NCT33755307). As technological advances continue to enhance our understanding of the genetic determinants of UC and as cost of genetic testing is further reduced, the question of performing universal sequencing of heritable cancer-predisposing genes in patients with UC (particularly at advanced stage or/and younger age at diagnosis) versus family historybased germline testing is open. Accumulating evidence favors the first, as this approach may identify patients that would have otherwise been missed or excluded from current guideline-based testing [55]. However, there are additional steps required, such as large-scale functional assessment of several VUS and low-penetrance gene variants to elucidate
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60–65. [16] B.D. Robinson, P.J. Vlachostergios, B. Bhinder, et al., Upper tract urothelial carcinoma has a luminal-papillary T-cell depleted contexture and activated FGFR3 signaling, Nat. Commun. 10 (1) (2019) 2977. [17] T.F. Donahu, A. Bagrodia, F. Audenet, et al., Genomic characterization of uppertract urothelial carcinoma in patients with lynch syndrome, JCO Precis. Oncol. 2018 (2018), https://doi.org/10.1200/PO.17.00143. [18] S.C. Skeldon, K. Semotiuk, M. Aronson, et al., Patients with Lynch syndrome mismatch repair gene mutations are at higher risk for not only upper tract urothelial cancer but also bladder cancer, Eur. Urol. 63 (2) (2013) 379–385. [19] D. Huang, S.F. Matin, N. Lawrentschuk, M. Roupret, Systematic review: an update on the spectrum of urological malignancies in lynch syndrome, Bladder Cancer 4 (3) (2018) 261–268. [20] A. Lim, P. Rao, S.F. Matin, Lynch syndrome and urologic malignancies: a contemporary review, Curr. Opin. Urol. 29 (4) (2019) 357–363. [21] A. Siefker-Radtke, B. Curti, Immunotherapy in metastatic urothelial carcinoma: focus on immune checkpoint inhibition, Nat. Rev. Urol. 15 (2) (2018) 112–124. [22] A. Latham, P. Srinivasan, Y. Kemel, et al., Microsatellite instability is associated with the presence of lynch syndrome pan-cancer, J. Clin. Oncol. 37 (4) (2019) 286–295. [23] D.T. Le, J.N. Durham, N. Kellie, et al., Mismatch-repair deficiency predicts response of solid tumors to PD-1 blockade, Science 357 (6349) (2017) 409–413. [24] L. Marcus, S.J. Lemery, P. Keegan, R. Pazdur, FDA approval summary: pembrolizumab for the treatment of microsatellite instability-high solid tumors, Clin. Cancer Res. 25 (13) (2019) 3753–3758. [25] https://www.fda.gov/drugs/resources-information-approved-drugs/fda-grantsaccelerated-approval-pembrolizumab-first-tissuesite-agnostic-indication. [26] P. Vlachostergios, B. Faltas, T. Zhang, et al., Germline DNA repair single nucleotide polymorphisms in urothelial cancer patients, J. Urol. 197 (4S) (2017) e644–e645, https://doi.org/10.1016/j.juro.2017.02.1499. [27] B.M. Faltas, P.J. Vlachostergios, L. Lam, et al., Germline single nucleotide polymorphisms in DNA repair genes in urothelial cancer patients, Cancer Res. 77 (13 Suppl) (2017), https://doi.org/10.1158/1538-7445.AM2017-1115 abstr 1115. [28] http://exac.broadinstitute.org/. [29] M.I. Carlo, L. Zhang, D. Mandelker, et al., Cancer predisposing germline mutations in patients (pts) with urothelial cancer (UC) of the renal pelvis (R-P), ureter (U) and bladder (B), J. Clin. Oncol. 35 (15Suppl) (2017), https://doi.org/10.1200/JCO. 2017.35.15_suppl.4510 4510-4510. [30] M.I. Carlo, J. Vijai, D. Mandelker, et al., DNA damage repair (DDR) germline mutations in patients (Pts) with urothelial carcinoma (UC), J. Clin. Oncol. 36 (15Suppl) (2018), https://doi.org/10.1200/JCO.2018.36.15_suppl.1516 1516-1516. [31] M.I. Carlo, V. Ravichandran, P. Srinavasan, et al., Cancer Susceptibility Mutations in Patients With Urothelial Malignancies. J. Clin. Oncol. (2019), https://doi.org/10. 1200/JCO.19.01395 In press. [32] S. Abou Alaiwi, A. Nassar, K.W. Mouw, et al., Germline variants in urothelial carcinoma: analysis of pathogenic and likely pathogenic variants in 645 subjects, J. Clin. Oncol. 37 (15Suppl) (2019), https://doi.org/10.1200/JCO.2019.37.15_suppl. 1528 1528-1528. [33] https://www.fda.gov/drugs/resources-information-approved-drugs/fda-approvesolaparib-germline-brca-mutated-metastatic-breast-cancer. [34] https://www.fda.gov/drugs/fda-approved-olaparib-lynparza-astrazenecapharmaceuticals-lp-maintenance-treatment-adult-patients. [35] E. Złowocka, C. Cybulski, B. Górski, et al., Germline mutations in the CHEK2 kinase gene are associated with an increased risk of bladder cancer, Int. J. Cancer. 122 (3) (2008) 583–586. [36] C.J. Madubata, A. Roshan-Ghias, T. Chu, et al., Identification of potentially oncogenic alterations from tumor-only samples reveals Fanconi anemia pathway mutations in bladder carcinomas, NPJ Genom. Med. 2 (2017) 29, https://doi.org/10. 1038/s41525-017-0032-5.
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Cancer Treatment and Research Communications journal homepage: www.elsevier.com/locate/ctarc
The emerging landscape of germline variants in urothelial carcinoma: Implications for genetic testing
T
Panagiotis J. Vlachostergios (M.D., Ph.D.)a, Bishoy M. Faltas (M.D.)a,b,c, Maria I. Carlo (M.D.)d, Amin H. Nassar (M.D.)e, Sarah Abou Alaiwi (M.D.)f, Guru Sonpavde (M.D.)f,⁎ a
Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, NY, United States Department of Cell and Developmental Biology, Weill Cornell Medicine, New York, NY, United States c Caryl and Israel Englander Institute for Precision Medicine, Weill Cornell Medicine, New York, NY, United States d Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, United States e Department of Medicine, Brigham and Women's Hospital, Boston, MA, United States f Lank Center for Genitourinary Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave, DANA 1230, Boston, MA 02215, United States b
ARTICLE INFO
ABSTRACT
Keywords: Bladder cancer Genetic testing Germline variants Urothelial carcinoma Lynch syndrome
Urothelial carcinoma (UC) of the bladder and upper tract (ureter, renal pelvis) is one of the most frequently occurring malignancies. While the majority of UC are chemically induced by smoking, accumulating evidence from genetic studies have demonstrated a small, but consistent impact of heritable gene variants and family history of UC on the development of the disease. Beyond the established association between upper tract UC and germline mismatch DNA repair defects as a defining feature of Lynch syndrome, newer investigations focusing on moderate- and high-risk cancer-related gene variants in DNA damage repair and other signaling pathways are expanding our knowledge on the heritable genetic basis of UC, opening new avenues in the breadth of genetic testing and in clinical counseling of these patients. Overcoming existing challenges in the interpretation of uncertain findings and family cascade testing may help expand our testing approach and guidelines. Following the paradigm of other tumor types, such as breast and ovarian cancers, germline genetic testing, particularly when combined with somatic testing, has the potential to directly benefit affected UC patients and their families in the future through therapeutic targeting (i.e. with poly(ADP-ribose)) polymerase inhibitors, immune checkpoint inhibitors) and genetically informed screening/surveillance, respectively.
1. Introduction
populations supported an increased risk of developing UC in subjects with a positive family history of bladder cancer. The Spanish casecontrol study reported an odds ratio of 2.34 among patients with at least one first-degree relative with bladder UC [3]. The Nordic study examined prospectively 80,309 monozygotic and 123,382 same-sex dizygotic twin individuals within the population-based registers of Denmark, Finland, Norway, and Sweden. After a median follow up of 32 years, the familial risk of developing bladder cancer was higher in monozygotic (9.9%) compared to dizygotic (5.5%) twins, with an estimated heritability of 30% for bladder cancer [4].
Urothelial carcinoma (UC) is the fourth most common malignancy among men in the United States with 158,220 estimated new cases in both sexes in 2019, one fifth of which (33,420) are predicted to be lethal [1]. The main environmental risk factors are tobacco and aromatic amines [2]. Nevertheless, UC develops in a minority of these cases, suggesting a potential role of genetic predisposition to urothelial carcinogenesis. Previous observations from large studies in Spanish and Nordic
Disclosures: Guru Sonpavde: research grants from Boehringer-Ingelheim, Bayer, Onyx-Amgen, grants and advisory board fees from Pfizer, advisory board for Genentech, advisory board for Novartis, research grants and advisory board for Merck, research grants and advisory board for Sanofi, advisory board and steering committee of trial for Seattle Genetics/Astellas, speaking fees from Clinical Care Options, grants, trial steering committee, advisory board and travel fees from Astrazeneca, writing fees from Uptodate, advisory board for Exelixis, advisory board and travel costs from Bristol-Myers-Squibb (BMS), research grants and advisory board Janssen, advisory board for Amgen, advisory board for Eisai, advisory board for NCCN, research grants from Celgene, speaking fees from Physicians Education Resource, Onclive, Research to Practice, Steering committee of trials sponsored by Bavarian Nordic, Debiopharm, QED. Bishoy Faltas, Advisory board for Immunomedics. Research grants: Eli-Lilly. Honoraria: Urotoday. ⁎ Corresponding author. E-mail address: [email protected] (G. Sonpavde). https://doi.org/10.1016/j.ctarc.2020.100165
2468-2942/ © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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Genetic variations in UC can be germline or somatic [5]. A germline variant is defined as a genetic alteration that occurs within the germ cells (egg or sperm), such that the alteration can be passed to subsequent generations [5]. Genetic variants may be activating, resulting in a gain of function of an oncogene (such as a missense variant in its functional or kinase domain), or inactivating (such as nonsense, splicesite, and frameshift insertion/deletion variants, thereby causing a loss of function of a tumor-suppressor gene). The types of variants observed include single-nucleotide variants (SNVs) that cause missense, silent, or nonsense amino acid substitutions, or splice site alterations which affect normal splicing of the mRNA transcript. Alternatively, one or more nucleotides may be involved in duplications, deletions, insertions, or even more complex combinations of these changes, including for example a nucleotide(s) deletion coupled with a nucleotide(s) insertion (indels) at a specific gene location [5]. Curating gene variants in knowledge bases is critical for deepening our understanding of their role in cancer predisposition and providing genetic counseling to patients [6]. ClinVar is a publicly-available database that associates germline variants with their phenotypic characteristics and couples that with clinical and experimental evidence. Moreover, various in silico prediction algorithms have been developed to better predict whether a nucleotide change alters a protein's structure and/or function [7]. However, variants of uncertain clinical significance (VUS) pose a particular challenge in clinical medicine since the functional impact cannot be inferred from sequence information alone. For this reason, patients with no pathogenic variants or with presence of a VUS are counseled based on the family history only [8]. In this review we discuss recent advances in germline variant testing that are shaping the current landscape of heritable cancer-susceptibility genes in patients with UC, and their potential implications for testing and family screening recommendations.
Table 1 Estimated risks for development of Lynch syndrome by canonical MMR gene variant [13]. Gene
Risk of Lynch syndrome (%)
Age at diagnosis
MLH1 MSH2 MSH6 PMS2 Non-carrier (general population)
0.2–5 2–18 0.7–7 Not well established <1
52–60 52–61 52–69 Not well established
risk of a number of malignancies. LS is the most common hereditary cause of colorectal (CRC) (1–3%) and endometrial (2–5%) cancers. Upper tract UC (UTUC) is considered a core cancer in LS [11-14]. The reported lifetime risk of UTUC varies according to the MMR gene involved. MSH2 confers the highest lifetime risk, which ranges between 2% and 18% [13] (Table 1). Traditionally, the diagnosis of LS is a two-step process which includes: a) screening family history using the Amsterdam II or/and revised Bethesda criteria, and b) immunohistochemical (IHC) staining for absence of MMR protein expression, and/or a polymerase chain reaction (PCR) for presence of microsatellite instability. LS can subsequently be confirmed by germline genetic testing for deleterious MMR variants [11–14]. The Amsterdam II criteria follow a 3–2–1 rule: ≥1 relative must be a first-degree relative of another two, ≥2 successive generations must be affected, and ≥1 LS-associated diagnosis must be at age <50. The updated Bethesda criteria are less strict to minimize the proportion of patients that can be missed by family history alone. They are as follows: CRC in a patient <50 years old, synchronous or metachronous CRC or other LS-associated cancer, CRC with microsatellite instability-high (MSI-H) histology at age <60, and CRC in a patient with one a relative with family history of LS-associated cancer diagnosed at age <50, or ≥2 relatives at any age [11–14]. On top of the clinical criteria, risk estimation statistical models have been developed to assist with patient selection, including the PREMM5, MMRpredict, and MMRpro models [11–14]. In view of the high prevalence of LS in patients with UTUC, evaluation is recommended for high-risk patients based on family history. Universal screening has also been proposed, based on detection of up to 14% of potential LS-associated UTUC cases in a universally screened cohort of 115 patients [15]. However, it is important to note that contrary to the prevalent notion, sporadic UTUCs (which constitute the vast majority of UTUCs) are not frequently MSI-H and may have a lower TMB compared to bladder UC [16,17]. Patients with LS may also be at higher risk of bladder cancer, with a risk of up to 6.2% in MSH2 carriers reported in a Canadian registry cohort [18]. However, these studies are limited due to the presence of synchronous tumors and drop metastases, and the true association of LS and bladder cancer risk has been difficult to quantify. There also appears to be a risk for prostate, testicular and adrenocortical carcinoma which requires further investigation [19,20]. There is currently no clear evidence to support routine surveillance for UC in LS. Surveillance in select patients may be considered such as patients with a family history of UC or individuals with MSH2 pathogenic variants (especially males) as these groups appear to be at higher risk, up to 18% (Table 1) [11–14]. Surveillance options may include annual urinalysis (UA) starting at age 30–35 years [11–14]. Frequent UA with a threshold of 3 red blood cells per high-power field (RBC/HPF) or greater to pursue further testing has been suggested [11]. However, the optimal screening strategy is unclear. The detection of suspicious urinary or imaging findings should prompt computed tomography urogram (CTU) and a cystoscopy. Additional retrograde studies should be performed if the ureters are not fully imaged, with ureteroscopic evaluation, selective washings and biopsy when indicated [11]. In the therapeutic context, programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) immune checkpoint inhibitors are
2. Known cancer-susceptibility genes in UC Genome wide association studies (GWAS) have been the mainstay of studying associations between putative variants and risk of cancer [9]. After genotyping of DNA samples from patients and controls, association test statistics are generated to identify genetic risk loci. Overall, within the number of cancer cases that are present in a particular population at a given time (prevalence), the proportion of individuals with a specific cancer-associated genotype who also develop cancer within specific time period (penetrance) is small [10]. In other words, most genotypes for common, complex diseases are incompletely penetrant, and correlations between the genotype and the phenotype vary [9,10]. Numerous GWAS conducted in nearly all common malignancies have identified three major groups of genes that shape the genetic architecture of cancer risk in all cancers: a) rare, high-penetrance genetic variants that confer a higher relative risk (RR), such as pathogenic variants in BRCA1 and BRCA2 associated with hereditary breast and ovarian cancer, and MLH1 and MSH2 associated with Lynch syndrome (LS); b) uncommon, moderate-penetrance genetic variants that confer a moderate RR, such as those in ATM and CHEK2, and c) common, lowpenetrance genetic variants resulting in low RR, such as single nucleotide polymorphisms (SNPs) [9]. 2.1. Germline variants in mismatch repair (MMR) genes - Lynch syndrome Germline variants in the MMR genes MLH1, MSH2, MSH6, PMS2, and deletions in EPCAM gene, resulting in hypermethylation-induced silencing of MSH2, are all diagnostic of LS, also known as hereditary non-polyposis colon cancer (HNPCC) [11–14]. Defective MMR proteins lead to the accumulation of DNA replication errors within microsatellite regions, which are short repeating sequences of DNA that frequently serve as molecular markers in various genetic tests. MMR gene variants are inherited in an autosomal dominant pattern and cause increased 2
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extensively used in clinical practice for treatment of patients with advanced UC [21]. Interestingly, the presence of microsatellite instability (MSI) resulting from defective MMR, confers a high likelihood of response to PD-1 inhibition, with the most prevalent underlying mechanism being a high tumor mutational burden resulting in increased neoantigenesis and immune recognition [22,23]. The FDA approved the PD-1 inhibitor pembrolizumab for the treatment of patients with progressive advanced, MSI-H, or MMR-deficient tumors independent of origin [24,25].
patients with UC. Among the entire cohort, 15% of patient harbored germline pathogenic or likely pathogenic variants [32], the majority (92%) of which involved DDR genes. Additionally, variants in LS-associated MMR genes (4%), BRCA1/2 (2.5%), CHEK2 (2.3%), and MUTYH (1.9%) were detected [32]. Overall, 90 pathogenic or likely pathogenic variants affecting 18 DDR genes were detected in 12.2% of patients [32]. This high frequency of germline DDR variants in patients with UC may open new avenues for therapeutic targeting via exploitation of poly (ADP-ribose) polymerase (PARP) inhibitors which are already in clinical use for other cancer types with DDR alterations such as ovarian or breast cancer [33,34]. A case-control study conducted in an Eastern European population also supports an association between the presence of variants in the moderate-risk CHEK2 gene and increased risk of bladder cancer, as founder CHEK2 variants were more significantly detected in UC cases (10.6%) compared with controls (5.9%) [35]. Analysis of 100 UC bladder cases using Tumor-Only Boosting Identification (TOBI), a unifying framework that uses a machine learning algorithm to identify oncogenic germline variants in patients with tumor-normal paired DNA, revealed germline variants in 5% of patients, affecting BRCA2, FANCM, and FANCD2 genes [36]. This enrichment of germline inactivating variants in the Fanconi anemia (FA) pathway was unique for bladder UC compared to six other cancer types [36]. This supports a potential genetic predisposition of FA gene mutation carriers to UC. Taking family history into account, a study tested 73 UC patients, two thirds of whom had first-degree and one third had second-degree (or higher) relatives with UC of the bladder or upper tract [37]. Overall, half of the patients (52%) in the cohort were found to have germline alterations, with pathogenic variants seen in 18% of subjects [37]. Among patients with positive family history in a first-degree relative 17% were carriers of pathogenic variants while 20% of patients with second-degree (or higher) relatives tested positive for pathogenic variants. These affected several DDR genes, including BRCA1 (1.4%), BRCA2 (1.4%), BRIP1 (1.4%), CHEK2 (1.4%), MSH2 (4.1%), MUTYH (2.7%), as well as other known cancer-predisposition genes such as MITF (2.7%), PTCH (1.4%), and SDHC (1.4%) [37]. The largest study to date of germline genetic testing in UC included a total of 1038 patients with high suspicion of heritable pathogenic variants, based on history of at least one other non-urothelial cancer, young age, or family history of cancer or cancer predisposition syndrome [38]. Most patients (97%) underwent multigene testing of at least 5 genes among a total panel of 130 genes [38]. One fourth (24%) of these patients were found to be carriers of germline pathogenic and likely pathogenic variants, the majority of which (78% of carriers or 20% of total cohort) involved various highand moderate-penetrance DDR genes: MSH2 (3.5%), FANCC (3.3%), BRCA1 (2.3%), BRCA2 (2.1%), MUTYH (2%), ATM (1.6%), CHEK2 (1.4%), MLH1 (1%) [38]. Other cancer-predisposition moderate- and low-risk genes were also affected, including MITF (1.2%) and FH (1.3%), respectively [38]. Importantly, a significant enrichment of pathogenic variants in MSH2 (OR: 15.4), MLH1 (OR: 15.9), BRCA2 (OR: 5.7), and ATM (OR: 3.8) was found in UC patients compared to non-cancer subjects from the ExAC database [38]. Although these findings warrant validation in dedicated case-control studies jointly analyzing both groups and stratifying for ancestry, BRCA2 and ATM are highlighted as potential UC predisposition genes, in addition to the known MMR genes. An overview of the frequencies of various inherited DDR gene variants in patients with UC across different targeted sequencing studies is presented in Table 2. The average prevalence of moderate- and high-penetrance pathogenic or likely pathogenic DDR variants that are shared between the two largest studies to date are depicted in Fig. 1.
2.2. Germline variants in other DNA repair genes in UC Beyond deleterious MMR gene variants, heritable variants in other DNA repair genes are also frequent in UC and may play a role in its pathogenesis. In a cohort of 53 patients who were unselected for family history, consisting of 43 bladder and 10 upper tract UC, 12 different DNA repair gene germline variants were detected by whole-exome sequencing. These included genes involved in non-homologous endjoining such as RECQL4 (54.5%) and POLQ (6%), nucleotide excision repair, including ERCC6 (6%), XPA (3%), CCNH (3%) and POLK (3%), homologous recombination, including RNF168 (3%), RAD17 (3%) and POLE (3%), Fanconi anemia pathway such as POLN (3%), non-canonical mismatch repair represented by EXO1 (3%) [26,27]. Overall, the presence of these DDR variants in the cohort occurred at a significantly higher frequency (33/53, 62.3%) compared to the Exome Aggregation Consortium (ExAC) database, which consists of a large (60,706) repository of unrelated individuals’ DNA sequences as part of various disease-specific and population genetic studies [26–28]. In another cohort, 113 patients with UC involving bladder , or ureter and renal pelvis , unselected for suspicion of inherited cancer syndrome, underwent next generation sequencing of tumor-normal DNA pairs using a targeted germline gene panel that consisted of 76 genes associated with hereditary cancer predisposition [29]. Germline variants were found in 22% of patients. The majority of germline variants affected DNA damage response and repair (DDR) genes (58%), several of which were of high penetrance, such as BRCA1 (2.6%), BRCA2 (1.8%), MMR (7%), TP53 (0.8%), FH (0.8%) or moderate penetrance, such as CHEK2 (3.5%), ATM (0.8%), BRIP1 (0.8%), or NBN (0.8%) [29]. An expansion of the same study included 176 patients (cohort 1) and 327 de-identified patients without clinical annotation (cohort 2). Highly- or moderately-penetrant, pathogenic or likely pathogenic DDR gene variants, including MSH2, BRCA1, CHEK2, BRCA2, MSH6, MLH1, MUTYH, ERCC2, ERCC3 were found in 17% of cohort 1 patients and 9.8% of patients from cohort 2 [30].These results further highlight that DDR germline variants are not as rare in UC as previously thought, and that their presence should trigger a genetic consultation and counseling for risk assessment of hereditary cancer syndromes associated with germline variants in the same genes [30]. An expanded version (n=586) of this largely unselected group of UC patients was reported, the majority of whom did not have a family history of UC (n=544) or personal history of second primary (n=474), underwent paired tumor and germline testing [31]. Sixty-six of 80 patients (83%) were carriers of germline pathogenic or likely pathogenic variants in DDR genes, including: BRCA2 (1.5%), MSH2 (1.4%), BRCA1 (1.4%), CHEK2 (1.0%), ERCC3 (0.7%), NBN (0.5%) and RAD50 (0.5%) [31]. When these DDR genes' allele frequencies were compared to the those from non-cancer subjects in the ExAC database, variants in BRCA2 (odds ratio [OR]: 3.68) and MSH2 (OR: 4.58) were enriched in UC [31]. These findings suggest that non-MMR DDR gene variants (9% of the total cohort) may also play a previously unreported role in genetic predisposition to UC, particularly BRCA2. These pathogenic variant carriers were more likely to present at an early age of ≤45 [31]. Additionally, there was a quarter of patients (26%) harboring high-penetrance germline variants that would not have been detected using current guidelines based on family history, suggesting a potential value for expanded germline testing in UC [31]. Another large scale study used a commercially available targeted sequencing platform, Invitae, to and analyzed a set of 42 genes in 645
3. Common germline variants and polygenic risk scores in UC Common low-penetrance variants (risk allele frequency >5% and OR <1.5) [9], such as SNPs have been extensively studied for potential 3
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Table 2 Prevalence of pathogenic/likely pathogenic heritable variants in high- and moderate-penetrance DNA damage response and repair (DDR) genes in UC patients. Gene
Pathway
Mutation frequency (%)
Number of patients (N)
Ref
BRCA1 BRCA2 BAP1 NBN FANCD2, FANCM, BRCA2 BRIP1 FANCC MSH2, MSH6, MLH1, PMS2 MUTYH ERCC2 ERCC3 ATM CHEK2 TP53 Total DDR genes
HR
1.4–2.6 1.4–2.1 0.5–1.4 0.4–0.8 2.1–5 0.8–1.4 3.3 2–7 1.1–2.7 0.5–1.2 0.7–1.2 0.5–1.6 1.0–3.5 0.2–0.8 11.3–22.0
113–867 113–867 73–398 113–784 100–885 73–764 60 73–969 176–754 176–586 176–586 113–827 73–862 113–929 73–1038
[29,30,31,37,38] [29,30,31,37,38] [31,37,38] [29,31,38] [36,38] [29,31,37,38] [38] [29,30,31,37,38] [29,30,31,37,38] [30,31] [30,31] [29,31,38] [29,30,31,35,37,38] [29,31,38] [29,30,31,37,38]
FA MMR BER NER Checkpoint activation Cell Cycle Total DDR pathways
HR: homologous recombination; FA: Fanconi anemia; MMR: mismatch repair; BER: base excision repair; NER: nucleotide excision repair; DDR: DNA damage response and repair.
contribution to heritability of several cancers, including UC. Two large meta-analyses of several available datasets from UC GWAS have dissected the most commonly involved genes and pathways [39,40]. Interestingly, specific SNPs in the DDR genes ERCC2, NBN, and XPC are all weakly (OR: 1.10) but significantly associated with bladder cancer risk (p-value < 0.05) [41]. Additionally, several SNPs located in genes belonging to pathways such as chemical carcinogenesis (e.g. NAT2, UGT1A), and cell cycle (e.g. CCND1, CCNE1) were identified [42]. Occasionally, functional studies have further supported a causal
association for specific genotypes, including APOBEC-rs1014971, GSTM1-null, NAT2-slow, UGT1A-rs1189203, and CCNE1-rs8102137 [41–46]. The APOBEC3 gene family (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3) encodes proteins with cytidine deaminase activity (A3A, A3B, A3C, A3D, A3F, A3G, and A3H) which results in a particular mutagenesis pattern, found in several cancer types [44]. The APOBEC-rs1014971 SNP was not only associated with bladder cancer risk, but also with increased APOBEC3B gene expression and enrichment of APOBEC-signature mutations in bladder UC tumors
Fig. 1. Average frequency of shared pathogenic/likely pathogenic germline DDR variants across two large UC cohorts. 4
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their true effect on development of UC. It might also be useful to broaden our family history-based testing to be more inclusive, since a substantial proportion of patients with germline variants is not identified by currently available criteria. Paired germline – tumor testing represents a more comprehensive approach to assess potential interactions between biallelic or combined germline and somatic alterations that may accelerate carcinogenesis or/and tumor progression in a patient (i.e. a germline DDR mutation and a somatic mutation in an oncogene or tumor-suppressor gene). In a prospectively accrued cohort of 907 patients who underwent tumor targeted sequencing during four consecutive years (2013-2017), the frequency of germline testing and genetic counseling visits showed a significantly and consistently increasing trend [56]. This supports an integrated clinical cancer genetic approach to improve detection rates and ability to inform therapeutic decisions for patients, as well as counseling on cancer screening and prevention strategies for affected families [56]. Finally, it is key to take into account patients’ preferences and potential financial and emotional impact of testing for themselves and their families [57]. Further studies are needed to refine the selection of appropriate patients for germline evaluation, and elucidate the impact of clinical (e.g. age and family history of UC and other malignancies) and tumor characteristics. The increasing availability of clinical whole-exome and wholegenome sequencing is expected to increase opportunities for discovery of rare deleterious variants and challenges for the clinical management of patients harboring such variants. Providing a better framework for clinical decision-making based on an improved understanding of genetic predisposition of UC will enable a more rationalized use of interventions for individuals with inherited gene variants associated with an increased risk of UC. Our proposed framework for germline genetic testing in UC is provided in Table 3.
Table 3 Proposed framework for germline genetic testing in urothelial carcinoma (UC). Which patients with urothelial carcinoma (UC) should be offered germline genetic testing? 1) All patients who meet the Amsterdam/Bethesda criteria or Hereditary breast and ovarian cancer/BRCA guidelines or have a close relative who would have met guidelines. 2) All patients with MSI-H or MMR-deficient tumors. 3) Patients with early onset diagnosis ≤45 years, which is outside the 5% confidence interval of median age of onset for UC.
from The Cancer Genome Atlas (TCGA) [43]. The plethora of SNPs potentially involved in bladder carcinogenesis, as well as the contribution of environmental factors like smoking, have led to the development of polygenic risk scores (PRS). A PRS is a number that reflects the variability and weight of several genetic loci and non-genetic contributors in the prediction of risk for developing a particular condition or trait [47]. Testing 3,942 cases and 5,680 controls of European background for multiplicative and additive interactions between smoking and 12 susceptibility loci revealed that NAT2 and UGT1A6 variants have significant additive gene-environment interactions. Additionally, bladder cancer risk ranged from 2.9% for current smokers in the lowest quartile of the PRS to 9.9% for current smokers in the upper quartile. This demonstrates that prevention strategies targeting smoking cessation may be more successful in reducing the number of bladder cancer cases for subjects exhibiting higher genetic risk compared to those with lower risk [48]. A more recent study used genotype and phenotype data from TCGA and Electronic Medical Records and Genomics (eMERGE) to estimate 11 cancer‐specific PRS, including bladder UC. The mean cancer‐specific PRS was significantly higher among UC patients in TCGA (1.04) than controls in eMERGE (0.98) [49].
Declaration of Competing Interest
4. Perspectives and challenges of germline genetic testing in UC
No other conflicts of interest to declare by rest authors.
At present, screening for UC in asymptomatic non-high-risk adults is not recommended by several major organizations including The US Preventive Services Task Force (USPSTF), the National Cancer Institute (NCI), and the American Cancer Society (ACS), in the absence of sufficient evidence to support it [50–52]. Efforts have rather focused on better identifying high-risk groups that might benefit from screening. Such models have typically included age, sex, smoking history, and family history of bladder cancer and indicated a risk score > 6 as a threshold for future design of screening trials [53]. Hence, outside the spectrum of LS, genetic testing of UC patients potentially plays an important role in identifying high-risk patients based on the presence of pathogenic or likely pathogenic variants. In addition to the obvious implications for family cascade testing in the case of UC pathogenic mutation carriers, there may be additional benefit from therapeutic targeting of these defects. For instance, beyond its FDA-approved use in patients with advanced BRCA-mutated breast or ovarian cancer, the PARP inhibitor olaparib has shown activity in UC patients harboring germline DNA repair gene DRR variants [54]. Also, there are ongoing clinical trials of PARP inhibitors such as olaparib in bladder UC patients with germline or somatic with germline or somatic DDR defects (NCT33755307). As technological advances continue to enhance our understanding of the genetic determinants of UC and as cost of genetic testing is further reduced, the question of performing universal sequencing of heritable cancer-predisposing genes in patients with UC (particularly at advanced stage or/and younger age at diagnosis) versus family historybased germline testing is open. Accumulating evidence favors the first, as this approach may identify patients that would have otherwise been missed or excluded from current guideline-based testing [55]. However, there are additional steps required, such as large-scale functional assessment of several VUS and low-penetrance gene variants to elucidate
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