- Full list of author information is available at the end of the article. - Interestingly, it is observed that following NAC, approxi- mately 20 to 40% of patients have no residual urothelial cancer cells on histological examination of the RC speci- men and on ePLND [9 – 11]. - The 10 – 15% of patients who develop distant metastases early after NAC might also not benefit from removal of the bladder as these patients had chemotherapy-refractory subclinical metastases at the time of RC. - Especially those with pCR after NAC may be suitables candidates for close surveillance by frequent diagnostic investigation of the bladder and pelvic lymph nodes. - computed tomography (PET/CT), albeit a single node in the surgical template of the ePLND (cN0 – 1 M0) is allowed. - Patients with concomitant tumours of the upper urinary tract, tumours of the urachus or an additional malignancy that is progressing or has required active treatment within the past three years are excluded. - Exceptions to these include patients with basal cell carcinoma of the skin, squamous cell carcinoma of the skin that has undergone potentially curative therapy, or carcinoma in situ (e.g., breast carcinoma in situ, cervical carcinoma in situ), who have undergone potentially curative therapy.. - During NAC, a standard clinical response evaluation (CRE) with a diagnostic CT scan of the thorax and abdomen is performed. - Patients who complete less than three cycles of NAC will be excluded for the per- protocol analyses but will continue in the PRE- PREVENCYS trial for the intention-to-treat analyses.. - Second liquid biopsy collection of blood and urine will be scheduled at hospital admission for surgery.. - As explained above, the aim of the liquid biopsies before and after NAC and the re- staging TUR prior to RC is to investigate whether response to NAC can be predicted using clinical, radiological, and histological variables as well as a wide set of predictive molecular biomarkers in tissue, blood and urine.. - Blood samples and urine specimens will be collected and processed to allow assessment of liquid biopsy-based biomarkers including circulating tumour DNA (ctDNA), extracel- lular vesicle (EV)-based biomarkers (e.g., microRNAs) and platelet-based biomarkers (e.g., mRNA) (Protocols available upon request). - liquid biopsy collections, blood haematology and bio- chemistry, and urine cytology will be collected.. - Patients will be re-evaluated at least once, optionally twice, by standard CRE(s) during NAC and before undergoing surgical resection. - The aim of these CRE(s) will be to identify those patients in whom progression of local disease, absence of a local re- sponse and/or disseminated disease is present. - All visits within CREs will be part of standard clinical care and will follow local hospital protocols. - In case only one CRE is performed (CRE1), a CT scan with intravenous contrast of thorax and abdomen will be performed approximately 1–2 weeks after the third cycle of NAC. - In case the patient will be scheduled for two CREs (CRE1 and CRE2), a CT scan is per- formed approximately 1–2 weeks after the second cycle of NAC and approximately 1–2 weeks after the completion of all NAC courses. - Eligibility of the pa- tients for continuation in the PRE-PREVENCYS trial will be assessed based on the findings of the evalu- ation CT scan after three or four cycles of NAC, using the RECIST 1.1 criteria [21]. - patients will be scheduled for RC, ePLND and urinary diversion approximately four to six weeks after the last course of NAC.. - On the day of RC, a cystoscopy of the bladder will be performed with the patients under general anaesthesia.. - During this proced- ure, bimanual examination and a TUR of any visible le- sions suspected for residual tumour within the bladder and of the scar tissue from the previous location of the bladder tumour will be performed. - This re-staging TUR should include at least one biopsy including detrusor muscle but does not have to be a radical resection of the lesion. - In case of absence of tumour lesions and scar tis- sue, random biopsies will be taken of different locations of the bladder including one of the location of the previ- ous bladder tumour. - The diameter of the biggest visible lesions will be reported, as well as the estimated tumour-stage by the urologist and the duration of the re-staging TUR. - The aim of the TUR biopsies is to de- termine the accuracy of a re-staging TUR in the detec- tion of residual disease after NAC. - Subsequently, open or robot-assisted RC will be performed with ePLND and urinary diversion as per local protocol.. - Table 1 Overview of study visits for participants of the PRE-PREVENCYS trial. - Approximately three months after RC, the first follow- up visit will be planned in concordance with local follow-up protocols. - A diagnostic CT scan of thorax and abdomen will be performed to stage for recurrence of disease (CRE3). - abdomen scans during CRE(s) will be revised centrally by a specialized nuclear physicist and radiologist, re- spectively. - 1 Study algorithm of the PRE-PREVENCYS trial with expected distribution of patients. - The CT scans during CRE(s) will be revised according to the RECIST 1.1 criteria [21].. - The following formalin-fixed, paraffin-embedded (FFPE) tissue specimens will be examined in the current study:. - 1) the diagnostic TUR, 2) the re-staging TUR of residual bladder tumour or scar tissue after NAC, collected in the scope of the trial protocol, and 3) the diagnostic RC and ePLND specimens. - All tissue specimens will be re- vised centrally by an expert uropathologist. - Response to chemotherapy in both the RC and PLND specimens will be assessed ac- cording to the Tumour Regression Grade (TRG) ranging from 1 (pathological complete response) to 3 (weak/ no response) [23]. - The pathologist will be blinded to the outcome of radiological imaging such as those of CRE1 and CRE2. - For revision and histopathological evaluation of the study re-staging TUR biopsies, the pathologist will be blinded to the histopathological outcome of the RC resection specimen as well. - FFPE tissue samples of all three time points will be collected for genomic, gene ex- pression, and immunohistochemical studies for bio- marker analyses.. - DNA and RNA will be isolated form cancerous regions of the TUR and RC specimens containing at least more than 70% tumour cells. - In addition, DNA will be isolated from normal non-malignant adjacent tissue for germline DNA. - After quality control, whole exome sequencing (WES) and RNA sequencing will be done and analysed.. - In addition, unsupervised hierarchical clustering will be done to identify differentially expressed genes between responders and non-responders. - Furthermore, immuno- histochemical staining studies with antibodies will be performed.. - Based on results from tissue biomarker studies, candi- date prognostic and predictive DNA and RNA bio- markers will be defined and subsequently assessed at. - Extracellular vesicle next generation microRNA (EV-microRNAs) se- quencing will be performed. - For these 20 patients EV-RNA sequencing will be performed using both urine and plasma specimens collected at baseline and post chemotherapy. - Candidate biomarkers will be established for treatment outcome prediction. - Validation by quantitative reverse transcrip- tion PCR (RT-qPCR) of the most promising candidate miRNA panels will be performed with Taqman assays.. - Such small panels can reach high levels of specificity and sen- sitivity, although validation in independent collected co- horts will be of importance.. - Additionally, isolated EV fractions will be used as an alternative bio-specimen source for qPCR of other po- tential DNA and RNA biomarkers based on tissue-based biomarker studies. - Perioperative complications of the re-staging TUR, such as perforation of the bladder wall, major. - Any adverse events will be reported to the principal investigator (AV) within 1 week of occurrence. - With an inclusion of 180 patients with MIBC, cT2-T4a N0-N1 M0, at baseline, approximately 150 patients will eventually undergo all procedures of the PRE- PREVENCYS trial including liquid biopsy collection as well as a re-staging TUR during RC. - Descriptive analyses will be performed for all pre- operative clinical, radiological, histopathological vari- ables and on the assessment of mean expression of tis- sue and liquid biopsy biomarkers. - Results of each diagnostic modality will be correlated to the (categorical) pathological ypTN stage in the resection specimen using a Chi-square-based test (categorical-categorical) or a 1- way ANOVA test (continuous-categorical) with post-hoc testing. - Multivariate logistic regression analysis will be used to construct a prognostic model for pCR. - Suitable candidate biomarkers will be first selected based on established evidence reported in literature. - The selected variables will be entered into a ridge logistic regression model, which will be fit using the glmnet R package [27, 28]. - Ridge generally leads to a sparse model, where many of the coefficients will be set to values close to zero. - Ridge logistic regression requires tuning of one parameter, which will be carried out by 10-fold cross validation using the cv.glmnet() function from the glmnet package. - A receiver operating curve (ROC) will be constructed for the final model and the area under the curve (AUC) will be calculated as a measure for dis- criminative ability. - To include all patients in the regres- sion analyses, an imputation procedure of missing values will be performed.. - This will be evaluated in the LASSO logistic regression model with threshold selected by Youden’s J statistic.. - The study has been approved by the Medical Ethical Committee of the Amsterdam University Medical Centre (MEC 2019.594) on January 30, 2020 and has been regis- tered in the Netherlands Trial Register (NL8678). - The study will be conducted according to the principles of the Declaration of Helsinki (10th version, Fortaleza, 2013) and will be in accordance with the Dutch Medical Research Involving Human Subjects Act (WMO). - In each participating centre, the principal investigator will be responsible for recruitment, adherence to the study protocol and follow-up of the included patients. - Any other physician of the multidisciplinary team will inform. - The project leader (AV) is responsible for the study design, conduct of the trial, preparation of the protocol and re- visions and for preparation of case report forms. - Revi- sions of the study protocol will be communicated to all local chief investigators. - The project leader will be re- sponsible for data collection and the data master file.. - Data of each participant will be pseudo anonymized using a subject identification code and put into a com- prehensive database using a standardized Web-based Case Record Form (data management system CASTOR EDC) [29]. - An independent data monitoring committee from the Clinical Research Bureau of the Amsterdam UMC will assess the data, according to the Good clinical practice (GCP) requirements. - Source data verification will be im- plemented during the onsite monitoring in observance of original documents. - Furthermore, serious complica- tions during re-staging TUR, as described in “Interim analysis and (serious) adverse events”, will be carefully monitored at 5 and 25 inclusions. - The final dataset will be available to all study investigators. - Given authorships will follow guidelines of the International Committee of Medical Journal Editors [30]. - Results will be communi- cated via international conferences, via publications and via the NTR.. - In case of more than 10% falsely pre- dicted pCR patients, the PREVENCYS trial will be reconsidered.. - If this PRE-PREVENCYS trial shows that residual dis- ease after NAC can be determined reliably prior to RC, a randomized controlled trial comparing NAC plus RC versus NAC followed by a close active surveillance protocol will be conducted (the PREVENCYS trial).. - For the present study, only patients with a predomin- antly muscle-invasive urothelial cell carcinoma of the bladder will be included (cT2–4a). - Therefore, patients with a CrCl between 40 and 60 ml/min are thus candidates for this split-dose regimen and will be included in this study. - This finding underlines the importance of the need for and extensive compari- son between pre- and post NAC samples in experimen- tal studies. - Members of the PRE-PREVENCYS study group:. - JV, JB and AV initiated the trial, participated in the study design and critically revised and supervised the drafting of the manuscript. - All authors read and approved the final manuscript and agree to be accountable for all aspects of the work.. - The funding body has no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. - The study has been approved by the Medical Ethical Committee of the Amsterdam University Medical Center (MEC 2019.594) on 30th January 2020.. - Written, voluntary, informed consent to participate in the study will be obtained from all participants. - European Association of Urology guidelines on muscle-invasive and metastatic bladder Cancer: summary of the 2020 guidelines. - https://doi.org/10.1007/s . - Radical cystectomy for urothelial carcinoma of the bladder without neoadjuvant or adjuvant therapy: long-term results in 1100 patients. - Neoadjuvant chemotherapy for transitional cell carcinoma of the bladder: a systematic review and meta-analysis. - https://doi.org ju . - https://doi.org/10.1016/S . - https://doi.org . - Updates in the eighth edition of the tumor-node-metastasis staging classification for urologic cancers. - https://doi.org/10.1016/j.. - Bladder Cancer
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