Research progress in genomics associated with prognosis of upper tract urothelial carcinoma

LIU You-sheng, WANGFei

Department of Urology, the Affiliated Hainan Hospital of Hainan Medical University, Hainan General Hospital, Haikou 570311, China

Keywords:

ABSTRACT Upper tract urothelial carcinoma and bladder urothelial carcinoma both belong to urothelial carcinoma, which is a malignant tumor occurring in the renal pelvis and ureteral urothelium.The incidence rate of UTUC is higher among Asians and it shows various pathogenic factors.Patients of UTUC have a short lifespan, and most of them have shown invasive malignant tumors at the time of initial diagnosis.The treatment of most UTUC patients is limited to surgical resection, radiotherapy and chemotherapy in clinical.Due to its rarity, the studies on targeted therapy are rare.With the development of the targeted therapy and immunotherapy,genomics exploration that affects the prognosis of UTUC becomes particularly important.In this paper, we intend to review the differential expression, clinical significance and some special types of UTUC genomes through the UTUC genome.

Upper tract urothelial carcinoma (UTUC) and bladder urothelial carcinoma (BUC) are belong to urothelial carcinomas, including carcinoma of the renal pelvis and ureter.The incidence of carcinoma of the renal pelvis is approximately twice that of carcinoma of the ureter.According to the related literatures, UTUC accounts for 5%～10% of all urothelial carcinoma, and the incidence in China are much higher than that in Western countries[1].UTUC is more common in male patients, the ratio of male to female is about 2:1.The incidence of UTUC is highest between 70 and 90 years old,and there are many factors causing UTUC, such as Hereditary nonpolyposis colorectal cancer (Lynch syndrome)[2],smoking,occupational exposure to carcinogenic aromatic amines (benzidine and β-naphthalene) , and aristolochic acid-induced P53 genespecific mutations[3,1].UTUC is less common than BUC, but because 60% of its tumors exhibit aggressive features, patients have shorter overall survival (OS),and the majority of patients present with a poor prognosis postoperatively[4,1].A large number of clinical studies have proved that early detection, early diagnosis and early treatment can prolong the OS of patients.In recent years,with the development of targeted drugs and immunologic agents, there are more and more drugs that can benefit the patients with UTUC.Relatively speaking, its basic research (gene correlation research)needs more and more urgently.Therefore,we need to screen more UTUC prognostic genes to guide clinical treatment and benefit patients.

1.Research progress of prognostic factors of UTUC

1.1 Preoperative factors

Previous studies have shown that age, sex, race, smoking, the location of the primary tumor, and whether the surgery is delayed are associated with the prognosis and overall survival of patients with UTUC[4,2].Behfae et al reviewed the clinical data of 520 patients with UTUC, body mass index (BMI) , tumor recurrence rate, cancer specificity, and whether radical nephroureterectomy(RNU) was performed were found to be associated with overall survival, improving patient outcomes by influencing body mass index is partially instructive[5].Dalpiaz et al reviewed the data of 171 patients with non-metastatic UTUC, patients with a higher preoperative neutrophil-to-lymphocyte ratio (DNLR),which was assessed preoperatively, showed higher cancer-specific and overall mortality after UTUC[6], suggesting that the DNLR ratio serves as a potential predictor for the future; There is some research value.Of note, a recent study found that a combined score of the systemic immune inflammatory index (SII) with fibrinogen (FIB) is an adjunct to the prognosis of patients with UTUC[7],but because of its small study sample size, the reliability of the conclusion is still to be confirmed.Because of its safety and novelty, the preoperative detection method has become a hot topic recently.In conclusion, we believe that BMI, DNLR ratio, SII-FIB joint score, etc.can also be incorporated into the study criteria to construct the prognostic model in the study of UTUC clinical predictive model.

1.2 Postoperative factors

Tumor stage and grade are recognized as the major prognostic factors of UTUC.Lymph node metastasis, tumor invasion to lymphatic vessels and positive resection margin were the important factors of tumor metastasis or recurrence.Among them, pathological grade of high grade, clinical stage of T2-T4 and presence of vascular lymph node invasion (LVI) are independent risk factors affecting the prognosis of patients with UTUC[8].

2.Advances in genomics of UTUC

With the development of clinical therapy for UTUC, more and more researches are concentrated on the molecular level of UTUC.Therefore, exploring the genomic characteristics of UTUC can help guide risk assessment and future clinical decision-making.Currently,the most common genomic alterations seen in UTUC whole genome sequencing include FGFR3(Fibroblast growth factor receptor), TP53MDM2 tumor suppressor genes, and KMT2D/KDM6A(chromatin-modifying gene) and other typical tumor suppressor or proto-oncogenes (CDKN2A/RAS).Of course, not all of the genomic alterations that affect UTUC are of prognostic and clinical significance.According to the influence of the pathogenic factors of UTUC tumor, we can divide its genome into two major categories: 1.Common type of UTUC genome; 2.Special type of UTUC genome.

2.1 the common UTUC genome

2.1.1 FGFR3-FIBROBLAST growth factor receptor 3

2.1.1.1 differences in FGFR3 expression in UTUC

The FGFR3 gene encodes a protein that is a member of the Fibroblast growth factor receptor family, a tyrosine kinase receptor,it is mainly involved in regulating cell growth and differentiation,bone development, angiogenesis and so on.Besides its expression in UTUC, it has also been found in breast cancer, colorectal cancer and skeletal dysplasia.

In UTUC, FGFR3 mutations are predominantly associated with low-grade tumors and T1, T2(early stage).In patients with this mutation, the risk of tumor progression is low, the invasiveness is weak, and the prognosis is generally good.In 2012, Lyle et al identified FGFR3 mutations in 40% of tumor tissues by isolating and purifying tumor DNA and assessing FGFR3 mutation rates using RT-PCR (reverse transcription polymerase chain reaction); It was found that the mutation was mainly associated with low-grade and non-invasive tumors[9].To validate the mutation rate of FGFR3 in UTUC, Sfakianos et al, by analyzing 83 patients comprising both high-grade and low-grade UTUC, found that the mutation rate of FGFR3 was approximately 54%, and that the mutation rate was approximately 35.6% in high-grade tumors[10].Whereas Audenet et al, in a tumor association analysis of UTUC and UCB, found that FGFR3 was mutated in approximately 40% of patients with UTUC,P < 0.001[11].Finally, Robinson et al also demonstrated a high prevalence of FGFR3 mutations by using WES[12].Recently, studies have shown an upward trend in the mutation rate of FGFR3 in highgrade tumors, and in combination with the high mutation rate in lowgrade tumors, targeted drug studies targeting FGFR3 mutations are imminent.

2.1.1.2 FGFR3 point mutation and gene fusion

In FGFR3 gene mutation, the mutation frequency of mutation site is also different, some patients may even appear gene fusion phenomenon, the most common tyrosine kinase of the FGFR3 gene is a fusion of the TACC3 coding domain.Genome-wide analysis of 67 tumor tissues by Pal et al,using immunohistochemistry, found the point mutation rate for FGFR3 R248C is about 50%, and FGFR3 S249C is for 37.5%, and the fusion rate for FGFR3-TACC3 is 12.5%[13].In 2018, Columbia University Medical Center (CUMC)conducted a mechanistic study of FGFR3-TACC3 fusion, tumors with this fusion phenomenon were found to be predominantly in transcriptional subsets characterized by activation of mitochondrial function, inducing sensitivity to inhibitors of oxidative metabolism by activating oxidative phosphorylation and mitochondrial synthesis[14].The researchers found that this gene fusion is an oncogene that is sensitive to FGFR inhibitors and depends on activation of mitochondrial function.Therefore, the use of FGFR inhibitors to treat FGFR3-TACC3 fusion tumors may become a future landmark.In light of this mechanistic study, we hypothesized that the presence of such gene fusions in tumors may be associated with ferroptosis.Regarding the gene fusion phenomenon, Moss’s team discovered a novel gene fusion SH3KBP1-CNTNAP5 in their research.SH3KBP1, also known as CIN85, promotes the progression of several cancers, including breast, colon, and head and neck cancers[15].The researchers found that this new type of gene fusion may play a role in signal receptor recycling or degradation.

2.1.1.3 Clinical significance of FGFR3 mutation

Similar to bladder and breast cancer, a rich uropapillary subtype has also been found in tumors of patients with high-grade UTUC FGFR3 mutations, which have been shown to be associated with deletion of luminal gene expression and t-cell characteristics, activated FGFR signaling may cause cancer cells to deplete immune cells in an autonomous manner[12,15].Of course, the mechanism of interaction between activated FGFR3 signaling and the immune system leading to the expression subtype of T cell deletion has not been specifically described; Whether inhibition of FGFR3 signaling can activate the immune system in vivo remains to be explored[16].With successive iterations of sequencing techniques, there is increasing evidence that patients with UTUC with FGFR3 mutation types have a relatively good prognosis.Gene testing, IHC (immunohistochemistry), and FISH (Fluorescence in situ hybridization) have shown that different subtypes of expression can be used to guide clinical treatment and may benefit patients.

2.1.2 TP53

2.1.2.1 TP53 expression differences in UTUC

TP53 is a tumor suppressor gene.The tumor suppressor protein encoded by TP53 contains DNA binding, transcriptional activation and other functions.This protein can regulate the expression of target genes, it can induce cell cycle arrest, senescence,apoptosis and DNA repair.Genetic mutations in TP53 are associated with a variety of cancers and can occur in more than 50% of all malignancies[17].Of course, TP53 mutations are also seen in UTUC.In contrast to FGFR3, mutations in TP53 are more common in high-grade tumors and have higher genomic instability[15].Moss found by sequencing that the mutation rate of TP53 was approximately 22.2%, and in high-grade tumors it was approximately 33.4%[15].Lee whole genome sequencing 31 tumor samples and found that the TP53 mutation rate was as high as 70.9%, which may be related to highgrade tumors in most of his samples[18].

2.1.2.2 Clinical significance of TP53 mutations

In response to the prognostic impact of TP53 mutations in patients with UTUC, as early as 2013, a Meta-analysis combining the results of seven studies suggested that TP53 over expression might be an important factor affecting the prognosis of patients with UTUC[19].Lee et al also analyzed P53 protein expression in 12 patients with UTUC and found a significant correlation between P53 expression and serum creatinine levels (P=0.005), and p53 was an independent predictor of progression-free malignancy (hazard ratio = 3.74,P=0.025) and tumor-specific survival (hazard ratio=5.87, P=0.030)[20].These results suggest that TP53 may be used as a predictor of tumor prognosis.Significantly, Chen found that TP53 mutation type was significantly associated with p53 IHC staining by comparing TP53 mutation type with IHC.Moreover, he found that TP53 mutations simultaneously increase the risk of bladder recurrence and metastasis in UTUC, suggesting that TP53 may be a potential biomarker for predicting recurrence and metastasis in patients with UTUC[21].In follow-up published articles, we found that patients with TP53 mutations may benefit more from chemotherapy or immunotherapy[16].

2.1.3 Chromatin modification genes

2.1.3.1 Differential expression of chromatin modification genes in UTUC

KMT2D, KDM6A and CREBBP all belong to chromatin modification genes.Among them, KMT2D encoded protein belongs to Histone methyltransferase, and KDM6A encoded protein belongs to histone demethylase, the protein encoded by CREBBP belongs to histone acetyltransferase.Most studies have shown that mutations in chromatin modifiers are common between low-grade and high-grade UTUC and do not appear to differ[10].In their study, Sfakianos found mutation rates of approximately 35% for KMT2D, approximately 34% for KDM6A, lower mutation rates for CREBBP, approximately 16%, and more common in low-grade tumors[10].Audenet found a mutation rate of approximately 37% for KMT2D and approximately 32% for KDM6A in his experiments[11], which is similar to the results of the former experiment.Unfortunately, in the study of chromatin modifiers, the mutational pathways and mechanisms leading to UTUC still have some defects.

2.1.3.2 The clinical significance of chromatin modification gene mutation

The changes of KMT2D and its protein expression may be related to the location of UTUC and tumor multifocus[22].KDM6A expression level in UTUC patients was significantly lower than that in normal subjects, and the decrease of KDM6A expression level was significantly correlated with UTUC grades and survival time of patients, this suggests that KDM6A may be a useful prognostic factor for patients with poor prognosis[23].Unfortunately, the research on CREBBP has made no progress at home and abroad.

2.1.4 HRAS

HRAS gene encodes HRAS protein, which is owned by a small GTP.The main signal pathways are related to cell proliferation,angiogenesis and immunity.Sfakianos found in his experiments that the mutation rate of HRAS is about 10%[10].Yang sequenced tumor samples from 31 patients with UTUC and 81 patients with UCB and found that HRAS had a mutation rate of approximately 22.0% in UTUC tumors, significantly higher than 3.7% in UCB[24].Audenet also found in its experiments that HRAS changes in UTUC were significantly more frequent in patients with clinical stage < PT2,approximately 17%[11].These studies do not address the impact of HRAS gene mutations on the prognosis of patients with UTUC, nor do they generalize the differences in HRAS between high-grade and low-grade tumors; However, considering its high mutation rate in UTUC tumors, we can carry out follow-up study on this gene.

2.2 Specific types of UTUC genomes

2.2.1 The Hereditary nonpolyposis colorectal cancer UTUC genome

2.2.1.1 Clinical characteristics of 1.1 LS-UTUC

Lynch Syndrome is an autosomal dominant cancer predisposition Syndrome characterized by Hereditary nonpolyposis colorectal cancer mutations in the mismatch repair (MMR) genes MLH1,MSH2, MSH6, and PMS2.Lynch Syndrome is a genetic disorder cancer predisposition Syndrome, can lead to an increased risk of colorectal cancer, endometrial cancer, UTUC and other cancers[25].In LS-Patients, UTUC is the third most common malignancy after colon cancer and endometrial cancer cancer[26].LS-Patients have a 22-fold higher risk of developing UTUC than the general population,with a median age of onset approximately 10-15 years earlier than patients with sporadic UTUC[27].Most of the patients are female,the ratio of male to female is about 0.95/1, and the average age is between 55-60 years old.Among them, the incidence of ureteral tumor is high, accounting for about 51%, and most patients have a family history of malignant tumor[28].

2.2.1.2 Molecular mechanism of LS-UTUC

The molecular mechanism of LS-UTUC is characterized by germline mutations in MMR genes in LS-Patients.Because MMR gene is involved in DNA mismatch repair, its mutation or inactivation will lead to DNA base pairing errors.Whereas cancers with defects in DNA mismatch repair function (dMMR) contain numerous mutations defined as having Microsatellite instability(MSI) and, therefore, MSI is a hallmark of dMMR[29].We can detect and recognize MSI/dMMR by detecting MMR protein expression[29].Based on the fullness of microsatellites in the samples, tumors can be classified into three types: high MSI, low MSI, and stable MSI.UTUC patients have an increased risk of LS when they have germline mutations in their MMR genes.

2.2.1.3 LS-UTUC expression difference at gene level

Is there any difference in gene mutation and prognosis in patients with LS-UTUC? Donahu performed targeted sequencing on 17 patients with confirmed LS-UTUC and compared the results with those of 82 patients with sporadic UTUC,it was found that the median mutation number of LS-UTUC was significantly higher than that of Sporadic Type UTUC (58:6; P <.001),and the mutation frequency of KMT2D, CREBBP, ARID1A or DNA damage and repair genes was significantly higher in LS-UTUC than that in Sporadic Type UTUC (58:6; P <.001),genetic alterations in Notch1,NOTCH3, RB1, and CDKN1B are almost exclusively present in LSUTUC[30].Interestingly, FGFR3 R248C point mutations are highly enriched in LS-UTUC, which may serve as a potential biomarker for screening patients with LS-UTUC.

2.2.1.4 LS-UTUC prognosis

Therkildsen’s analysis of LS-UTUC patients’ prognosis provides the answer in his study.In LS-UTUC patients,the 10-year survival rate is approximately 50%, whereas in patients with MMRmutational features, MSI has no significant effect on overall survival[31].In terms of treatment, Hollande believes that adjuvant chemotherapy may be more effective in LS-UTUC patients who have undergone radical nephroureterectomy[32].Moreover, in 2015,Le found that patients with LS-UTUC might benefit more from anti-PD-1 therapy, which has direct clinical implications for personalized treatment of patients with LS-UTUC[33].However, most study data suggest that there is no single marker that can identify all UTUC tumors with MMR-deficient features.Therefore, we need to screen out patients with UTUC with MMR-deficient features through a comprehensive assessment of genetic testing, the MSIsensor score,and tumor mutational signature analysis in order to implement individualized precision medicine.Assessment of MSI and detection of dMMR by whole-exome sequencing (WES) technology may be the future for screening LS-UTUC[34].

2.2.2 The aristolochic acids (AA)-associated UTUC genome

2.2.2.1 Clinical characteristics of AA-UTUC

Aristolochic acids (AA) , a chemical extracted from Aristolochic,has hepatotoxicity and nephrotoxicity.Long-term intake of AAcontaining herbs or plants is one of the major risk factors for AA nephropathy (AAN) and UTUC).Since 1992, Vanherweghem has carried out an epidemiological survey at the kidney disease research centre in Brussels and found that some women from Belgium who took weight-loss drugs containing Chinese herbal medicines experienced rapid deterioration in their kidney function,the researchers subsequently found a link between this particular type of fibrotic interstitial nephritis and the Chinese herb[35].In follow-up studies, Balkan endemic nephropathy (BEN) was also found to be associated with AA.Although most countries prohibit the use of products containing AA because of their nephrotoxicity and carcinogenicity, Chinese herbal medicines and related products containing AA are still used in our country and in some Asian countries, there is an increased risk of AAN and UTUC in the Asian population.

2.2.2.2 AA-UTUC differential expression at gene level

In recent years, with the further study of AA and BEN, the study of AA-utuc’s pathogenesis and genome is becoming more and more intensive.Compared with Sporadic Type UTUC genome, AAUTUC gene expression was significantly different.Hoang found that the AA-UTUC genome had a higher mutational load, with approximately 72% of the mutations in its tumor samples being a: T to T: a translocations and expressed predominantly on nontranscribed strands.Mutations in the splicing site also play an important role in the pathogenesis of AA-UTUC[36].At the same time, AA-derived DNA compounds can lead to typical genomewide mutations, and this DNA compound is an important specific biomarker for the diagnosis of AA-associated nephropathy[37].Whereas Poon’s epidemiological observations found that AA-UTUC patients appeared to be younger than non-AA-UTUC patients and that AA-UTUC patients had a higher contralateral tumor recurrence rate[38].In a follow-up study of AA-UTUC mutations,Castells designed a novel sequencing method-LC-WES (lowcoverage whole-exome sequencing)-for AA mutation detection.He found that TP53 was the most commonly mutated gene compared with the non-AA-UTUC mutant genome, with a mutation rate of approximately 46% (tumor sample: 17/37) , and all of its mutations were a > T translocations.KMT2D mutations were found in 15 of these tumor samples and with different mutation types, which should be associated with a high mutation burden and increased genomic instability[39].

2.2.2.3 Prognosis of AA-UTUC

As for prognostic impact of AA-UTUC,Lu presented some new viewpoints.By whole genome sequencing 90 UTUC samples and using hierarchical clustering based on differences in the number of snvs, the researchers identified two major subtypes: AA Sig and NO-AA Sig.The number of AA Sig Subtype was significantly higher than that of no-AA Sig Subtype, and AA Sig subtypes were significantly associated with AA-containing herbal intake, female patients, poor renal function, multifocal tumors, and lower T staging[40].They suggested that the AA Sig subtype demonstrated better outcomes in both cancer-specific survival (CSS) and metastasisfree survival (MFS) compared with the No-AA Sig Subtype.For patients with advanced disease, Cisplatin chemotherapy remains the preferred treatment in the clinic, but may also be influenced from AA nephropathy[41].It is also because immune checkpoint inhibitors have a stronger antitumor effect on tumors with a high mutation burden that immune checkpoint inhibitors have become a target of choice for patients with advanced stages of AA-UTUC[42].

3.Conclusion

In conclusion, UTUC is a kind of multi-gene-induced malignant tumor.Early screening, early diagnosis, early prevention and even early treatment have important clinical significance for its prognosis.At now, although the stage and grade of the tumor is still the most important factor, but for patients who have undergone genetic testing, the type of gene mutation has more accurate clinical guiding significance for the follow-up treatment and prognosis.

Conflict of interest and author contribution

Conflict of interest: the content of this article does not involve related conflict of interest, the study does not involve any manufacturers or other economic organizations directly or indirectly economic and interest sponsorship.

Authors’ contribution

Liu Yousheng: mainly for literature search, reading, recording,writing and other work.Wang Fei: the overall control of the article,the article content review and so on.

Project support: Construction Project of Hainan Provincial Clinical Medical Center.