Postoperative recurrence detection using individualized circulating tumor DNA in upper tract urothelial carcinoma

Biomarkers that could detect the postoperative recurrence of upper tract urothelial carcinoma (UTUC) have not been established. In this prospective study, we aim to evaluate the utility of individualized circulating tumor DNA (ctDNA) monitoring using digital PCR (dPCR) as a tumor recurrence biomarker for UTUC in the perioperative period. Twenty-three patients who underwent radical nephroureterectomy (RNU) were included. In each patient, whole exome sequencing by next-generation sequencing and TERT promoter sequencing of tumor DNA were carried out. Case-specific gene mutations were selected from sequencing analysis to examine ctDNA by dPCR analysis. We also prospectively collected plasma and urine ctDNA from each patient. The longitudinal variant allele frequencies of ctDNA during the perioperative period were plotted. Case-specific gene mutations were detected in 22 cases (96%) from ctDNA in the preoperative samples. Frequently detected genes were TERT (39%), FGFR3 (26%), TP53 (22%), and HRAS (13%). In all cases, we obtained plasma and urine samples for 241 time points and undertook individualized ctDNA monitoring for 2 years after RNU. Ten patients with intravesical recurrence had case-specific ctDNA detected in urine at the time of recurrence. The mean lead time of urinary ctDNA in intravesical recurrence was 60 days (range, 0–202 days). Two patients with distal metastasis had case-specific ctDNA in plasma at the time of metastasis. In UTUC, tumor-specific gene mutations can be monitored postoperatively as ctDNA in plasma and urine. Individualized ctDNA might be a minimally invasive biomarker for the early detection of postoperative recurrence.     

Highly predictive survival nomogram after upper urinary tract urothelial carcinoma

BACKGROUND:

Nephroureterectomy is the surgical standard of care for patients with upper urinary‐tract urothelial carcinoma. The objectives of the current study were to identify the most informative predictors of cancer‐specific mortality after nephroureterectomy, to devise an algorithm capable of predicting the individual probability of cancer‐specific mortality, and to compare its prognostic accuracy to that of the International Union Against Cancer (UICC) staging system.

METHODS:

Within the Surveillance, Epidemiology, and End Results database, the authors identified 5918 patients who had been treated with nephroureterectomy. Within the development cohort (n = 2959), multivariate Cox regression models predicting cancer‐specific mortality were fitted by using age, stage, nodal status, sex, grade, race, type of surgery (nephroureterectomy with or without bladder‐cuff removal), and tumor location (renal pelvis vs ureter). Backward variable elimination according to the Akaike information criterion identified the most accurate and parsimonious model. Model validation and calibration were performed within the external validation cohort (n = 2959). External validation was also applied to the UICC staging system.

RESULTS:

The 5‐year freedom from cancer‐specific mortality rates in both the development and external validation cohorts was 77.3%. The most informative and parsimonious nomogram for cancer‐specific‐mortality–free survival relied on age, pT and pN stages, and tumor grade. In external validation, nomogram prediction of 5‐year cancer‐specific‐mortality–free rate was 75.4% accurate and was significantly better (P < .001) than the UICC staging system (64.8%).

CONCLUSIONS:

The current nomogram is capable of predicting the prognosis in patients with upper urinary‐tract urothelial carcinoma treated by nephroureterectomy with better accuracy than the UICC staging system. The authors recommend the application of this nomogram to routine clinical practice when counseling or making clinical decisions. Cancer 2010. © 2010 American Cancer Society.     

Management of upper urinary tract urothelial carcinoma

Upper urinary tract urothelial carcinoma (UTUC) is relatively uncommon. In this article, we review prognostic factors, criteria and indications for treatment with the available modalities using contemporary data. A systematic search on PubMed was performed using the keywords ‘upper tract urothelial carcinoma’, ’upper tract transitional cell carcinoma’, ’nephroureterectomy’, ‘laparoscopic’, ‘endoscopic’ and ‘prognostic factor’. The literature on UTUC is scarce. No prognostic factors have been formally validated in either the diagnosis or treatment of UTUC. The gold-standard management for invasive UTUC is radical nephroureterectomy with a bladder-cuff excision. Laparoscopic and endoscopic approaches represent alternatives in properly selected individuals. Segmental ureterectomy may also be considered. The extent and role of lymph node dissection remains to be validated. Chemotherapy may also be considered in select patients. Additional multi-institutional studies are needed to identify and validate prognostic factors that can predict the outcomes of patients diagnosed with UTUC. Randomized controlled trials are needed to assess the efficacy of the treatment modalities.     

腔内激光手术治疗肾盂输尿管肿瘤

目的:通过对无法行根治性手术的上尿路上皮肿瘤患者进行腔内红激光治疗情况进行总结,评价该术式的有效性与安全性.方法:对于我院2013年1月到2016年6月期间收治的肾盂、输尿管肿瘤,其中13例行腔内激光手术治疗,分别就术中情况、术后并发症及疗效进行总结.结果:13例患者中,肾盂肿瘤行经皮肾镜激光手术9例,输尿管肿瘤行输尿管镜激光手术4例,平均手术时间分别为(47.22±6.25)min、(25.0±4.84)min;平均出血量分别约为(133.33±24.94)ml、(40.0±7.07)ml;血尿及肾积水情况得到控制,5/13例呈现局部进展,1/13例术后10个月死亡;未出现严重并发症.结论:对于特殊情况下的肾盂、输尿管肿瘤患者,腔内激光手术具有创伤小,止血效果好,可以姑息性切除肿瘤,降低肿瘤负荷,保护肾脏功能,也能减缓肿瘤进展,可以作为这一类患者的首选治疗方案.     

Diagnostic and prognostic role of cell‐free DNA testing for colorectal cancer patients

Circulating cell‐free DNA (cfDNA) was found in increased amounts in cancer patients and tumor‐associated molecular alteration can be detected in cancer patient's samples. For this reason, the cfDNA analysis is actually considered as a new concept of liquid biopsy. We evaluated the presence and integrity of plasma cfDNA by ALU‐based qPCR and the methylation profile of OSMR and SFRP1 genes promoter in a large cohort of colorectal cancer (CRC) patients (n = 114) in comparison to healthy subjects (n = 56) and patients with adenomatous lesions (n = 22). Moreover, we studied the prognosis value focusing on histopathological staging and survival. The cfDNA concentration and the integrity index were increased in CRC patients. The ALU83 and ALU244 fragment dosage showed a moderate discriminant capacity between CRC patients and controls and CRC and adenoma patients. Especially, cfDNA was significantly higher in CRC patients at advanced histopathological stage. In addition, the increased cfDNA level was associated with poor prognosis. A comparison of methylation profile in matched tissue and plasma on 25 CRC patients was performed and only three mismatched cases were observed. A lower methylation quantification was observed in cfDNA than tissue DNA. The cfDNA methylation frequency was statistically different in controls, adenoma and CRC patients and this frequency increased with the histopathological stage of tumor. The adenoma and CRC patients methylated cfDNA showed a higher quantity of ALU83 and ALU244. An integrated approach, combining the detection of ALU fragments and cancer type‐specific epigenetic alteration, can improve diagnostic efficiency and better define the prognostic value for CRC disease.     

上尿路尿路上皮癌淋巴结清扫的研究进展

上尿路尿路上皮癌(UTUC)指发生于肾盂及输尿管的尿路上皮恶性肿瘤，约占全部尿路上皮癌的 5%～10%。目前，UTUC淋巴引流的解剖范围暂不明确，一些研究表明淋巴结清扫范围对患者生存的影响可能大于清扫淋巴结数量的影响，但这一观点尚未得到证实。近年来，由于微创手术的快速发展，虽然在技术操作方面和标准化方面取得了重大进展，但对于淋巴结清扫的意义和范围仍缺乏共识。本文就UTUC淋巴结清扫的意义、范围、适应证、并发症及微创手术地位的研究进展作一综述。     

DNA copy number aberrations associated with lymphovascular invasion in upper urinary tract urothelial carcinoma

Recent studies have reported that lymphovascular invasion (LVI) is a predictor of patient prognosis in upper urinary tract urothelial carcinoma (UUTUC). DNA copy number aberrations (DCNAs) identified by array-based comparative genomic hybridization (aCGH) had not previously been examined in UUTUC. We therefore examined DCNAs in UUTUC and compared them with DCNAs in LVI. We applied aCGH technology using DNA chips spotted with 4,030 BAC clones to 32 UUTUC patients. Frequent copy number gains were detected on chromosomal regions 8p23.1 and 20q13.12, whereas frequent copy number losses were detected on chromosomal regions 13q21.1, 17p13.1, 6q16.3, and 17p11.2. DCNAs occurred more frequently in tumors with LVI than in those without it (P = 0.0002), and this parameter was more closely associated with LVI than with the tumor grade or pT stage. Disease-specific survival rate was higher in tumors without LVI than in those with it (P = 0.0120); however, tumor grade and stage were not significant prognostic factors of patient outcome. These data support our hypothesis that tumors with LVI have more genetic alterations in terms of total numbers of DCNAs than those without, and provide proof that aggressive adjuvant therapy should be considered for UUTUC patients with LVI.     

Prognostic role of PIK3CA mutations of cell‐free DNA in early‐stage triple negative breast cancer

PIK3CA is an oncogene that encodes the p110α component of phosphatidylinositol 3‐kinase (PI3K); it is the second most frequently mutated gene following the TP53 gene. In the clinical setting, PIK3CA mutations may have favorable prognostic value for hormone receptor‐positive breast cancer patients and, during the past few years, PIK3CA mutations of cell‐free DNA (cfDNA) have attracted attention as a potential noninvasive biomarker of cancer. However, there are few reports on the clinical implications of PIK3CA mutations for TNBC patients. We investigated the PIK3CA major mutation status of cfDNA as a noninvasive biomarker of cancer using droplet digital polymerase chain reaction (ddPCR), which has high level sensitivity and specificity for cancer mutation, in early‐stage 49 triple negative breast cancer (TNBC) patients. A total of 12 (24.4%) of 49 patients had PIK3CA mutations of cfDNA. In a median follow up of 54.4 months, the presence of PIK3CA mutations of cfDNA had significant impacts on relapse‐free survival (RFS; P = 0.0072) and breast cancer‐specific survival (BCSS; P = 0.016), according to the log‐lank test. In a Cox proportional hazards model, the presence of PIK3CA mutations of cfDNA had significant prognostic value in the univariate and multivariate analysis. Additionally, the presence of PIK3CA mutations of cfDNA was significantly correlated with positive androgen receptor phosphorylated form depending on PI3K signaling pathway (pAR) which is independent favorable prognostic factors of TNBC. We demonstrated that the presence of PIK3CA major mutations of cfDNA could be a discriminatory predictor of RFS and BCSS in early‐stage TNBC patients and it was associated with PI3K pathway‐dependent AR phosphorylation.     

Molecular classification and diagnostics of upper urinary tract urothelial carcinoma

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Analysis of colorectal cancer‐related mutations by liquid biopsy: Utility of circulating cell‐free DNA and circulating tumor cells

We recruited 56 colorectal cancer patients and compared the mutational spectrum of tumor tissue DNA, circulating cell‐free DNA (ccfDNA) and circulating tumor cell (CTC) DNA (ctcDNA) to evaluate the potential of liquid biopsy to detect heterogeneity of cancer. Tumor tissue DNA, ccfDNA, and ctcDNA were extracted from each patient and analyzed using next‐generation sequencing (NGS) and digital PCR. To maximize yields of CTC, three antibodies were used in the capture process. From 34 untreated patients, 53 mutations were detected in tumor tissue DNA using NGS. Forty‐seven mutations were detected in ccfDNA, including 20 not detected in tissues. Sixteen mutations were detected in ctcDNA, including five not detected in tissues. In 12 patients (35.3%), mutations not found in tumor tissues were detected by liquid biopsy: nine (26.5%) in ccfDNA only and three (8.8%) in ctcDNA only. Combination analysis of the two liquid biopsy samples increased the sensitivity to detect heterogeneity. From 22 stage IV patients with RAS mutations in their primary tumors, RAS mutations were detected in 14 (63.6%) ccfDNA and in eight (36.4%) ctcDNA using digital PCR. Mutations not detected in primary tumors can be identified in ccfDNA and in ctcDNA, indicating the potential of liquid biopsy in complementing gene analysis. Combination analysis improves sensitivity. Sensitivity to detect cancer‐specific mutations is higher in ccfDNA compared with ctcDNA.     

External validation of an online nomogram in patients undergoing radical nephroureterectomy for upper urinary tract urothelial carcinoma

Background:

The objective was to validate an online nomogram developed based on the French collaborative national database on upper urinary tract urothelial carcinoma (UUT-UC) using a different cohort.

Methods:

The study comprised 328 patients with UUT-UC who underwent radical nephroureterectomy. The discrimination of models was quantified using Harrell''s concordance index. The relationship between the model-derived and actuarial cancer-specific mortality was graphically explored within calibration plots. Calibration was also assessed using the quartiles of the predicted survival at 3 and 5 years and calculation of the corresponding observed Kaplan–Meier estimates. Clinical net benefit was evaluated constructing decision curve analysis.

Results:

The discrimination accuracy of the nomograms at 3 and 5 years was 71.6% and 71.8%, respectively. Although nomograms discriminated well by Kaplan–Meier curves, and log-rank tests were all highly significant, the calibration plots tended to exaggerate the overestimation of mortality between predicted and observed probabilities at 3 and 5 years for survival. When compared with the AJCC/UICC staging system, the nomograms performed well across a wide range of threshold probabilities using decision curve analysis.

Conclusion:

The online nomogram is a highly accurate prognostic tool for patients with UUT-UC treated with radical nephroureterectomy. The model can provide an accurate estimate of the individual risk of cancer-specific mortality. Further improvement and implementation of novel molecular marker is needed.     

Fragmentation of cell‐free DNA is induced by upper‐tract urothelial carcinoma–associated systemic inflammation

Reliable biomarkers for upper‐tract urothelial carcinoma (UTUC) have yet to be found. Plasma cell‐free DNA (cfDNA) has been clinically applied as a minimally invasive blood biomarker for various types of cancer. We investigated the utility of plasma cfDNA as a blood biomarker in UTUC patients. The fragment size of plasma cfDNA was shorter and the concentration of plasma cfDNA was higher in UTUC patients than in healthy controls. The fragment size of plasma cfDNA had a moderate accuracy of diagnosing UTUC (area under the curve [AUC] = 0.72), and multivariate analysis indicated that the fragment size of plasma cfDNA was significantly associated with the presence of UTUC (odds ratio = 0.807, 95% confidence interval [CI] 0.653‐0.955, P = .024). Furthermore, we found that the size of plasma cfDNA shortens alongside disease progression (P < .001). The fragment size of plasma cfDNA in UTUC patients may be an auxiliary tool for the diagnosis of UTUC patients. We also found a high correlation between the fragmentation of plasma cfDNA and serum levels of three inflammatory cytokines (TNFα [r = −.837], interleukin‐6 [IL‐6] [r = −.964], interleukin‐1 receptor antagonist [IL‐1ra] [r = −.911]), which were reported to associate with poor prognosis. Also, we found that the proportion of short fragments of cfDNA was significantly increased in the supernatant of peripheral blood mononuclear cells (PBMCs) from healthy controls cultured in media containing TNFα. These results supposed that cancer‐associated systemic inflammation, especially tumor necrosis factor‐α (TNFα), may contribute to the fragmentation of plasma cfDNA in UTUC patients.     

Expression of steroid hormone receptors and its prognostic significance in urothelial carcinoma of the upper urinary tract

To assess the expression status of steroid hormone receptors in upper urinary tract urothelial carcinoma (UUTUC), we immunohistochemically stained for androgen receptor (AR), estrogen receptor-α (ERα), ERβ, glucocorticoid receptor (GR), and progesterone receptor (PR) in 99 UUTUC specimens and paired non-neoplastic urothelial tissues. AR/ERα/ERβ/GR/PR was positive in 20%/18%/62%/63%/16% of tumors, which was significantly lower (except PR) than in benign urothelial tissues [57% (P < 0.001)/40% (P = 0.001)/85% (P = 0.001)/84% (P = 0.002)/13% (P = 0.489)]. There were no significant associations between each receptor expression pattern and histopathological characteristic of the tumors including tumor grade/stage. Kaplan-Meier and log-rank tests revealed no significant prognostic value of each receptor expression in these 99 patients. However, patients with UUTUC positive for either ERα or PR had a significantly higher risk of disease-specific mortality (P = 0.025), compared with those with UUTUC negative for both. PR positivity alone in pT3 or pT4 tumors was also strongly associated with the risk of disease-specific mortality (P = 0.040). Multivariate analysis further identified the expression of ERα and/or PR as a strong predictor for disease-specific mortality in the entire cohort of the patients (hazard ratio, 2.434; P = 0.037). Thus, in accordance with previous observations in bladder specimens, significant decreases in the expression of AR/ERα/ERβ/GR in UUTUC, compared with that in non-neoplastic urothelium, were observed. Meanwhile, the negativity of both ERα and PR in UUTUC as well as the negativity of PR alone in deeply invasive tumor was suggested to serve as a prognosticator.     

The clinicopathological characteristics of muscle-invasive bladder recurrence in upper tract urothelial carcinoma

This study aimed to clarify the clinical characteristics and oncological outcomes of patients with upper tract urothelial carcinoma (UTUC) who developed muscle-invasive bladder cancer (MIBC) after radical nephroureterectomy (RNU). We identified 966 pTa-4N0-2M0 patients with UTUC who underwent RNU and clarified the risk factors for MIBC progression after initial intravesical recurrence (IVR). We also identified 318 patients with primary pT2-4N0-2M0 MIBC to compare the oncological outcomes with those of patients with UTUC who developed or progressed to MIBC. Furthermore, immunohistochemical examination of p53 and FGFR3 expression in tumor specimens was performed to compare UTUC of MIBC origin with primary MIBC. In total, 392 (40.6%) patients developed IVR after RNU and 46 (4.8%) developed MIBC at initial IVR or thereafter. As a result, pT1 stage on the initial IVR specimen, concomitant carcinoma in situ on the initial IVR specimen, and no intravesical adjuvant therapy after IVR were independent factors for MIBC progression. After propensity score matching adjustment, primary UTUC was a favorable indicator for cancer-specific death compared with primary MIBC. Subgroup molecular analysis revealed high FGFR3 expression in non-MIBC and MIBC specimens from primary UTUC, whereas low FGFR3 but high p53 expression was observed in specimens from primary MIBC tissue. In conclusion, our study demonstrated that patients with UTUC who develop MIBC recurrence after RNU exhibited the clinical characteristics of subsequent IVR more than those of primary UTUC. Of note, MIBC subsequent to UTUC may have favorable outcomes, probably due to the different molecular biological background compared with primary MIBC.     

High visfatin expression predicts poor prognosis of upper tract urothelial carcinoma patients

Visfatin, a newly discovered adipocytokine, is a pro-inflammatory cytokine. This study aimed to evaluate the predictive value of visfatin on prognosis of patients with upper tract urothelial carcinoma. One-hundred and five patients (median age=64, range=24-84 years) were included in this study. Visfatin expression in upper tract urothelial carcinoma tissues was analyzed by immunohistochemistry. Visfatin expression was correlated with clinicopathologic variables using the χ² test. The prognostic value of visfatin for recurrence-free and cancer-specific survival was evaluated by Kaplan-Meier estimates, and the significance of differences between curves was evaluated by the log-rank test. Cox regression model was also used to evaluate the hazard ratios of visfatin on survival. High visfatin expression in upper tract urothelial carcinoma tissues was significantly correlated with tumor stage (P=0.001), grade (P=0.007) and p53 expression (P=0.07). High visfatin expression was associated with poor recurrence-free and cancer-specific survival. Cox regression analysis also revealed that visfatin is an independent predictor of recurrence-free (HR=3.22, P=0.009) and cancer-specific survival (HR=5.74, P=0.023). Our findings indicated that higher visfatin expression is a potential biomarker to predict patient survival. Further study is necessary to investigate the role of visfatin in the carcinogenesis of upper tract urothelial carcinoma.     

Personalized peptide vaccination as second‐line treatment for metastatic upper tract urothelial carcinoma

This study investigated the applicability of personalized peptide vaccination (PPV) for patients with metastatic upper tract urothelial cancer (mUTUC) after failure of platinum‐based chemotherapy. In this single arm, open‐label, phase II clinical trial, patients with mUTUC received PPV at a single institution. Personalized peptide vaccination treatment used a maximum of four peptides chosen from 27 candidate peptides according to human leukocyte antigen types and peptide‐reactive IgG titers, for six s.c. injections weekly as one cycle. The safety of PPV, as well as its influence on host immunity and effect on overall survival were assessed. Forty‐eight patients were enrolled in this study. Personalized peptide vaccinations were well tolerated without severe adverse events. Median survival time was 7.3 months (95% confidence interval [CI], 5.3–13.1) with 13.0 months for patients receiving combined salvage chemotherapy (95% CI, 5.7–17.5) and 4.5 months for patients receiving PPV alone (95% CI, 1.7–10.1) (P = 0.080). Patients with positive CTL responses showed a significantly longer survival than patients with negative CTL responses (hazard ratio, 0.37; 95% CI, 0.16–0.85; P = 0.019). Multivariate Cox regression analysis showed that lower numbers of Bellmunt risk factors and lower levels of B‐cell activating factor were significantly associated with favorable overall survival for patients under PPV treatment. This study indicated that PPV for patients with mUTUC after failure of platinum‐based chemotherapy induced substantial peptide‐specific CTL responses without severe adverse events and has the potential to prolong survival when combined with salvage chemotherapy. UMIN Clinical Trials Registry ID: 000001854.     

Nested quantitative PCR approach for urinary cell‐free EZH2 mRNA and its potential clinical application in bladder cancer

EZH2 is overexpressed in bladder cancer (BC) and plays important roles in tumor development and progression. Recent studies show cell free (cf) RNAs released from cancer cells can reflect tissues changes and are stable and detectable in urine. Although conventional quantitative real‐time PCR (qPCR) is highly sensitive, low abundances of urinary cf‐RNAs usually result in false‐negatives. Thus, this study develops a nested qPCR (nqPCR) approach to quantify cf‐EZH2 mRNA in urine and further assess its clinical significance for BC. Forty urine samples were first selected to evaluate feasibility of nqPCR. Then, levels of urinary cf‐EZH2 mRNA were detected using developed method in an independent cohort of subjects with 91 healthy, 81 cystitis, 169 nonmuscle invasive BC (NMIBC) and 103 muscle‐invasive BC (MIBC). In cf‐EZH2 mRNA detection, nqPCR method was significantly associated with qPCR, but it could detect more urine samples and increase detection limit three orders of magnitude. Based on nqPCR method, cf‐EZH2 mRNA levels have been found to be increased in urine of NMIBC and MIBC patients (p < 0.001). Compared with cytology, cf‐EZH2 mRNA showed higher diagnostic ability for MIBC (p < 0.001) while not for NMIBC (p > 0.05). Moreover, it also could distinguish MIBC from NMIBC, with AUC of 0.787. For MIBC patients, high expression of cf‐EZH2 mRNA associated with advanced stage and was an independent predictor of reduced disease free survival or overall survival. In conclusion, detection of cf‐EZH2 mRNA in urine by nqPCR is a sensitive and noninvasive approach and may be used for diagnosis and prognosis prediction of MIBC.