In this study, we systematically investigated the characteristics of germline mutations and relevant phenotypes in five types of genitourinary cancers, and found a series of features, including highly cancer type-dependent top mutated genes, predominant mutation types with large fragment alterations, male-dominant patient distribution and age-irrelevant cancer onset. We also revealed significant correlation between pathogenic/likely pathogenic mutations and patient prognosis and the risk of genitourinary cancers in population, suggesting them as prognostic and risk factors. Our study established the clinical relevance of these mutations and highlighted the importance of early detection and intervention in population with pathogenic and likely pathogenic germline mutations.
Cancer patients with pathogenic or likely pathogenic germline mutations are a special group of patients with characteristic phenotypes, including early onset cancers, familial aggregation, multiple organ involvement, high level of malignancy, poor therapeutic response and poor prognosis [13,14,15,16]. The most commonly seen cancers with definite causes of germline mutations include Lynch syndrome and hereditary breast and ovarian cancer (HBOC) [17, 18], while recent evidence suggested that a subset patients with pathogenic germline mutations were also predisposed to higher lung cancer risk and familial aggregation [19,20,21]. It was reported that genes responsible for DNA damage repair (DDR) were mainly involved in germline mutations in hereditary cancers [8, 22]. This includes a series of genes, such as MLH1, MSH2, MSH6, PMS2, ATM, BLM, BRCA1, BRCA2, POLE and POLD1 [8, 22, 23]. Germline mutations of these genes may greatly enhance the risk of cancer, and certain group of mutations may correspond to certain cancer types. For example, genes in mismatch repair (MMR) (MLH1, MSH2, MSH6, PMS2, etc.) are predominantly linked to Lynch syndrome, and genes in homologous recombination repair (HRR) (BRCA1 and BRCA2) are mainly involved in HBOC. Other less-frequent germline mutations are less cancer type-specific and may be found in any cancer.
Although hereditary cancers such as Lynch syndrome and HBOC have been widely studied, the germline mutational status in genitourinary cancers and their correlation with prognosis and cancer risk have been largely uninvestigated. This is possibly due to the low incidence of germline mutation-induced genitourinary cancer and the fact that there have been few definite links between certain germline mutations and certain type of genitourinary cancer . We therefore performed a database research and revealed interesting characteristics of germline mutations in genitourinary cancer and established their correlation with patient prognosis. It was not surprising to find that ATM, BRCA1, PMS2 and BRCA2 were among genes with highest number of mutations. As mentioned above, these genes belong to DDR and are sensitive to DNA damage. DNA damage is a common process happened during carcinogenesis, and factors including chronic inflammation, virus infection, carcinogen or toxin can all lead to DNA damage which initiate repair [25,26,27]. In normal tissues of subjects without germline mutations, repetitive damage and repair alter the microenvironment and the normal cellular cycle controlled by a series of epigenetic and genetic mechanisms. Abnormal gene regulation under repetitive damage and repair ultimately leads to accumulation of somatic mutations, and key mutations at driver genes result in malignant transformation of cells [28,29,30]. In contrast, for subjects with germline mutations at DDR genes, the DNA repair mechanism is impaired congenitally, cellular malignant transformation may therefore happen at early stage of life and lead to tumor growth. This is the reason for high cancer incidence and low onset age for people with Lynch syndrome or HBOC-related germline mutations [25,26,27].
ATM gene mutations were also reported in recent studies on germline mutations in non-small cell lung cancer (NSCLC) [21, 31, 32]. It was reported to be the gene with highest germline mutation frequency in western population . Similarly, BRCA1, BRCA2 and PMS2 were also reported as germline mutations in cancers other than Lynch syndrome and HBOC [21, 31, 32]. BLM and VHL genes also contained germline mutations leading to Bloom syndrome  and Von Hippel-Lindau (VHL) disease , respectively. However, they were also found in other cancers with germline mutations. It is possible that DDR gene germline mutations can cause various types of cancers, with MMR genes preferentially found in Lynch syndrome and HRR genes preferentially found in HBOC. Functional subgroups of DDR genes may differentially affect carcinogenesis of different tissues.
It appeared from our study that bladder cancer and prostate cancer shared some common top mutated genes, while the top mutated genes were quite different in kidney cancer. In kidney cancer, two KIRC and one KIRP patients carried GJB2, three KIRP patients carried MET, one KIRC and two KRIP patients carried MUTYH and three KIRC patients carried VHL germline mutations. GJB2 germline mutations have been previously reported in congenital hearing loss  and rarely been reported in cancer . Our study revealed GJB2 germline pathogenic mutations in KIRC and KIRP for the first time, providing new evidence for the pathogenicity of the gene in kidney cancers. In contrast, MET germline mutations have been implicated in many cancers, including KRIP , and it was not surprising that we also found MET germline mutations in this study. Similarly, MUTYH germline mutations have also been reported in many cancers, including kidney cancer [38, 39]. The germline VHL mutations have been linked to VHL disease, which is an inheritable condition leading to retinal and central nervous system hemangioblastomas, clear cell renal cell carcinomas, pheochromocytomas, pancreatic neuroendocrine tumors and endolymphatic sac tumors . From our observations,.these top mutated genes were kidney-specific and distinguish themselves from those in bladder and prostate cancers, although they all belong to genitourinary cancer. Therefore, the mechanism of aberrancies in kidney cancers with germline mutations may be largely different from that of bladder and prostate cancer.
Determination of pathogenicity of germline mutations is crucial for establishing the link between mutations and phenotypes. Here in this study we interpreted the pathogenicity of all reported mutations based on ACMG guidelines. Frameshift and stop gained mutations were highly possible pathogenic or likely pathogenic mutations, indicating the inherit property of mutations. The interpretation would be more meaningful if the pathogenic mutations happened to key DDR genes related to known phenotypes. In contrast, missense mutations are more difficult to interpret, unless sufficient evidence is available to link single amino acid change with phenotypes. This is more likely to occur in single-gene related hereditary diseases, such as VHL disease [34, 41, 42]. In our study, pathogenic mutations of VHL gene were all missense mutations, reflecting the intrinsic properties of mutations in this disease. In contrast, the ratio of missense mutations was low in other highly mutated genes, such as ATM, BRCA1 and PMS2. It was interesting to find that nearly half of the mutations found in BLCA were stop gained mutations. These mutations spread many genes including both high and low frequency genes. This observation demonstrated characteristic mutational change in BLCA, suggesting high ratio of truncated DDR related proteins in the specific cancer.
It is widely known that male overweight female in patient number with a rough ratio of 2:1 in sporadic kidney and urothelial cancer . We found similar trend in genitourinary cancer with germline mutations, suggesting that male is possibly more susceptible to genitourinary cancer if the chance of mutation heredity is similar for both sexes. It is also possible that male may have higher penetrance than female. KIRC is the most common type of kidney cancer, and it was interesting that female overweight male in the number of KIRC patients with germline mutations, although male overweight female in sporadic KIRC . The reason for this discrepancy may include the manner of heredity, mutation penetrance and environmental factors. Previous studies on Lynch syndrome and HBOC revealed significantly lower onset age compared with sporadic colorectal, breast and ovarian cancer patients [44, 45]. However, we did not find age difference between those with pathogenic or likely pathogenic mutations and those with non-pathogenic mutations. Since non-pathogenic mutations were comprehensive found in sporadic cancer patients and normal subjects, our observation suggested that germline mutations of genitourinary cancers did not affect the onset age.
Previous studies reported that untreated patients with Lynch syndrome or HBOC exhibited significantly worse prognosis than sporadic patients [13,14,15,16]. Our study with genitourinary cancer patients also showed identical trend, in which patients with pathogenic or likely pathogenic mutations exhibited much worse overall survival rate than those with non-pathogenic mutations, suggesting that pathogenic and likely pathogenic germline mutations were risk factors for the prognosis of genitourinary cancer patients. Furthermore, the OR values we calculated strongly supported the notion that those with pathogenic or likely pathogenic germline mutations were in much higher risk for developing all types of genitourinary cancers than those without the germline mutations (general population). Our observation of higher overall OR in likely pathogenic mutations than pathogenic mutations suggested that some likely pathogenic mutation may be essentially pathogenic, although limited available evidence does not support the pathogenic interpretation currently. These observations provided strong evidence for the necessity of early detection of germline mutations for those with strong family history and continuous surveillance and early intervention for those with confirmed pathogenic or likely pathogenic germline mutations. On the other hand, it appeared from our study that no difference was found in overall survival rate between patients with pathogenic mutations and those with likely pathogenic mutations. This suggested that some likely pathogenic mutations may actually be pathogenic, although clinical evidence may be absent for interpretation of pathogenic for many likely pathogenic mutations, especially for frameshift and stop gained mutations.
Genitourinary cancer patients with pathogenic or likely pathogenic mutations may be treated with corresponding targeted drugs based on the availability of matched drugs for certain germline mutations. For example, locally advanced or metastatic genitourinary cancer patients with BRCA1/2 mutations may be treated with poly ADP-ribose polymerase (PARP) inhibitors. Although it has long been known that PARP inhibitors were effective for prostate cancer with pathogenic or likely pathogenic BRCA1/2 mutations [46, 47], it was not until recently that evidence started to emerge that PARP inhibitors were also effective in other genitourinary malignancies [47,48,49,50,51], except in renal cell carcinoma, since no evidence on the presence of BRCA1/2 mutations has been available in the cancer . Similarly, locally advanced or metastatic cancer patients with germline mutations of MMR genes may be treated with immune checkpoint inhibitors such as PD-l inhibitors, as these cancers generally exhibit high tumor mutational burden and/or high microsatellite instability . Future development of targeted drugs for DDR pathway may open the door for new treatment strategies for genitourinary cancer patients with germline mutations.
This study had some limitations. First, the number of genitourinary cancer patients was limited since data was available from only 206 patients, which led to the limited number of patients in each individual cancer. Secondly, due to the lack of ethnic diversity and predominant male population, the current findings could be non-generalizable to non-White and female patients. Thirdly, the prognosis of patients may be influenced by therapeutic strategies, however, the information of therapy is not available in the TCGA database.