Outcomes of open versus robotic partial nephrectomy: a 20-year single institution experience.

Robotic assisted partial nephrectomy (RPN) has emerged in urologic practice for the management of appropriately sized renal masses. We provide a 20-year comparison of the outcomes of open partial nephrectomy (OPN) versus RPN for renal cell carcinoma (RCC) at our institution. An IRB-approved retrospective review was conducted of RCC patients at a single institution from 2000 to 2022 who underwent RPN or OPN. In addition to demographics, procedural details including ischemia and operative time were collected. Oncologic outcomes were evaluated through Kaplan-Meier statistical analysis to determine recurrence-free survival (RFS), cancer-specific survival (CSS), and overall survival (OS) analysis. 849 patients underwent RPN while 385 underwent OPN. 61% were male with average age of 58.8 ± 12.8 years. Operative time was shorter in the open group (184 vs 200 min, p = 0.002), as was ischemia time (16 vs 19 min, p = 0.047). However, after 2012, RPN became more common than OPN with improving ischemia time. RPN patients had significantly improved RFS (HR 0.45, p = 0.0004) and OS (HR 0.51, p = 0.0016) when controlled for T-stage and margin status. More > pT1 masses were managed with OPN than RPN (11.2 vs 5.4%, p < 0.0001). At our institution, RPN had an increasing incidence with reduced ischemia time compared to OPN over the last 10 years. While higher stage renal masses were more often managed with OPN, selective use of RPN does offer improved oncologic outcomes. Further investigation is needed to evaluate optimization of the selection of RPN versus OPN in the nephron-sparing management of renal masses.

Does larger prostate size provide protection for cancer specific outcomes in localized prostate cancer.

OBJECTIVE: Benign prostatic hyperplasia is common in the aging population and frequently comorbid with localized prostate cancer. Large prostate volume places significant challenges in robotic prostatectomy including reduced mobility and visualization. The goal of this study is to evaluate the effect of prostate volume as a continuous variable on cancer specific outcomes. METHODS: Three thousand four hundred and twenty five patients with localized prostate cancer at a single institution who underwent robotic prostatectomy were retrospectively reviewed. A number of preoperative, operative, and postoperative variables were collected to evaluate cancer specific outcomes including pathologic stage, tissue margins, and biochemical recurrence (BCR). Logistic regression models and univariate and multivariate analyses were implemented for pathologic stage T3 and BCR respectively. RESULTS: The median follow up time was 52 months (IQR 18-95). 37.4% of the patients had a final pathologic stage of T3 or higher, 21.2% experienced positive surgical margins, and 24.7% of patients experienced BCR. Prostate size was a significant predictor of all three outcomes of interest. Increasing prostate size was protective against both higher pathologic stage and positive surgical margins (odds ratio = 0.989, 0.990 respectively, p < 0.001). There was a modest increase in the risk of BCR with increasing gland size (hazard ratio = 1.006, p < 0.001). These results were most significant for patients with Gleason Grade Groups 1 and 2 prostate cancer. CONCLUSION: Prostate size is a commonly determined clinical factor that effects both surgical planning and cancer specific outcomes. Increasing prostate size may offer protection against higher stage disease and positive surgical margins. While surgically challenging, favorable oncologic outcomes can be consistently achieved for patients with low-intermediate risk disease.

Comparing Magnetic Resonance Imaging and Prostate-Specific Membrane Antigen-Positron Emission Tomography for Prediction of Extraprostatic Extension of Prostate Cancer and Surgical Guidance: A Prospective Nonrandomized Clinical Trial.

PURPOSE: Survivors of surgically managed prostate cancer may experience urinary incontinence and erectile dysfunction. Our aim was to determine if 68Ga-prostate-specific membrane antigen-11 positron emission tomography CT (PSMA-PET) in addition to multiparametric (mp) MRI scans improved surgical decision-making for nonnerve-sparing or nerve-sparing approach. MATERIALS AND METHODS: We prospectively enrolled 50 patients at risk for extraprostatic extension (EPE) who were scheduled for prostatectomy. After mpMRI and PSMA-PET images were read for EPE prediction, surgeons prospectively answered questionnaires based on mpMRI and PSMA-PET scans on the decision for nerve-sparing or nonnerve-sparing approach. Final whole-mount pathology was the reference standard. Sensitivity, specificity, positive predictive value, negative predictive value, and receiver operating characteristic curves were calculated and McNemar's test was used to compare imaging modalities. RESULTS: The median age and PSA were 61.5 years and 7.0 ng/dL. The sensitivity for EPE along the posterior neurovascular bundle was higher for PSMA-PET than mpMRI (86% vs 57%, P = .03). For MRI, the specificity, positive predictive value, negative predictive value, and area under the curve for the receiver operating characteristic curves were 77%, 40%, 87%, and 0.67, and for PSMA-PET were 73%, 46%, 95%, and 0.80. PSMA-PET and mpMRI reads differed on 27 nerve bundles, with PSMA-PET being correct in 20 cases and MRI being correct in 7 cases. Surgeons predicted correct nerve-sparing approach 74% of the time with PSMA-PET scan in addition to mpMRI compared to 65% with mpMRI alone (P = .01). CONCLUSIONS: PSMA-PET scan was more sensitive than mpMRI for EPE along the neurovascular bundles and improved surgical decisions for nerve-sparing approach. Further study of PSMA-PET for surgical guidance is warranted in the unfavorable intermediate-risk or worse populations. CLINICALTRIALS.GOV IDENTIFIER: NCT04936334.

Preoperative aspirin and anticoagulants do not affect partial nephrectomy bleeding.

INTRODUCTION: Studies have reached mixed conclusions on the role of antiplatelet and anticoagulant agents on postoperative complications of partial nephrectomies. This study examines whether preoperative anticoagulation use affected the risk of hemorrhagic complications after partial nephrectomy. MATERIALS AND METHODS: This is a retrospective chart review of all partial nephrectomies performed between 2017 and 2022 at a single institution. For each operation, preoperative data was gathered on whether the patient was on anticoagulation, the type and dose of anticoagulation, and how many days the anticoagulation was held preoperatively. Bivariate analyses for continuous measures were performed using Student's t-tests when there were two comparison groups and ANOVA models when there were more than two comparison groups and Chi-Square tests were used for categorical variables, with Fisher's Exact being used when expected cell counts were small. RESULTS: In this study, warfarin was held for an average of 5.43 days, clopidogrel was held for an average of 6.60 days, aspirin was held for an average of 7.65 days, and direct oral anticoagulants (DOACs) were held for an average of 4.00 days. There was no significant difference in hemoglobin (Hb) change, rate of intraoperative transfusion, postoperative transfusion, bleeding complication, pseudoaneurysm rate, or additional bleeding processes between patients on prior anticoagulation therapy and those not on therapy. There was no significant difference in intraoperative or postoperative outcomes based on history of aspirin use and continuation of aspirin through the surgery. While estimated blood loss appeared statistically significant initially, this difference was accounted for by the covariates of comorbidities, RENAL score, surgical approach, and type of renorrhaphy. Overall, there was no difference in complication rate based solely on aspirin use or continuation of aspirin through surgery. CONCLUSIONS: No difference in complication rate of partial nephrectomy was determined to be solely due to prior use of anticoagulation or aspirin use alone with appropriate cessation of anticoagulation preoperatively. Overall, patients on anticoagulation are not at a higher risk of intraoperative or postoperative bleeding complications when undergoing partial nephrectomy.

Data-Independent Acquisition Phosphoproteomics of Urinary Extracellular Vesicles Enables Renal Cell Carcinoma Grade Differentiation.

Translating the research capability and knowledge in cancer signaling into clinical settings has been slow and ineffective. Recently, extracellular vesicles (EVs) have emerged as a promising source for developing disease phosphoprotein markers to monitor disease status. This study focuses on the development of a robust data-independent acquisition (DIA) using mass spectrometry to profile urinary EV phosphoproteomics for renal cell cancer (RCC) grades differentiation. We examined gas-phase fractionated library, direct DIA (library-free), forbidden zones, and several different windowing schemes. After the development of a DIA mass spectrometry method for EV phosphoproteomics, we applied the strategy to identify and quantify urinary EV phosphoproteomes from 57 individuals representing low-grade clear cell RCC, high-grade clear cell RCC, chronic kidney disease, and healthy control individuals. Urinary EVs were efficiently isolated by functional magnetic beads, and EV phosphopeptides were subsequently enriched by PolyMAC. We quantified 2584 unique phosphosites and observed that multiple prominent cancer-related pathways, such as ErbB signaling, renal cell carcinoma, and regulation of actin cytoskeleton, were only upregulated in high-grade clear cell RCC. These results show that EV phosphoproteome analysis utilizing our optimized procedure of EV isolation, phosphopeptide enrichment, and DIA method provides a powerful tool for future clinical applications.

Outcomes of Robot-Assisted Laparoscopic Pyeloplasty Based on Degree of Obstruction on Preoperative Tc-99 MAG-3 Renal Scintigraphy.

Objective: Management of symptomatic ureteropelvic junction (UPJ) obstruction with hydronephrosis and discordant Tc-99 mercaptoacetyltriglycine (MAG-3) renal scintigraphy is challenging. In this study we describe long-term outcomes of patients who underwent robot-assisted laparoscopic pyeloplasty for the correction of symptomatic UPJ obstruction with discordant preoperative Tc-99m MAG-3 renal scintigraphy. Methods: Patients undergoing robot-assisted laparoscopic pyeloplasty for symptomatic UPJ obstruction at a single academic center from 2009 to 2021 were retrospectively reviewed. Patients were categorized into three groups with varying degrees of obstruction based on preoperative MAG-3 imaging: Group 1: no obstruction (Lasix T1/2 clearance <10 minutes), Group 2: equivocal obstruction (Lasix T1/2 clearance 10-20 minutes), and Group 3: obstruction (Lasix T1/2 clearance >20 minutes. Pyeloplasty success was defined as resolution of symptoms and improvement/stable computed tomography (CT) imaging or MAG-3 scintigraphy. Failure was defined as persistence of symptoms with either obstruction on functional imaging, worsening hydronephrosis, or subsequent intervention. Results: A total of 125 cases were identified, with a median patient age of 35 years. Dismembered pyeloplasty technique was performed in 98.4% of cases. Median preoperative split renal function on MAG-3 scintigraphy was the only statistically significant (p = 0.003) difference in preoperative characteristics between the three groups. There were 15 postoperative complications, with a rate of Clavien-Dindo grade 3 or higher complications of 4.8%. Overall pyeloplasty success was 92.8%, with success rates of 100% (15/15) and 97% (32/33) in the no obstruction and equivocal obstruction groups, respectively. Median time to pyeloplasty failure was 20.4 months. Conclusion: Robot-assisted laparoscopic pyeloplasty is a safe and effective surgical intervention for correcting UPJ obstruction. Patients with symptoms of UPJ obstruction and discordant functional imaging studies demonstrate similar or improved success rates after pyeloplasty compared with patients with documented high-grade obstruction. Based on these findings preoperative renal scan may not be reliable in appropriate selection of candidacy for pyeloplasty.

Can Retroperitoneal Lymph Node Dissection (RPLND) be feasibly performed to prolong survival in Renal Cell Carcinoma (RCC) with limited lymph node involvement? An Analysis of Recurrence Patterns.

INTRODUCTION: The therapeutic benefit of performing a lymph node dissection (LND) in patients with renal cell carcinoma (RCC) has been controversial. In prior studies, it was thought that a low event rate for nodal metastases affected the ability to draw any conclusions. Here, we opted to select patients that had low burden 1 or 2 nodes positive to study survival outcomes and recurrence patterns based on limited LND or extended LND with a template retroperitoneal lymph node dissection (RPLND). METHODS: We used our single institutional database from 2000 and 2019 and identified 45 patients that had only 1 or 2 nodes positive on final pathology without any other systemic disease. These patients all underwent nephrectomy with limited LND or a template RPLND on the ipsilateral side. RESULTS: We identified 23 patients in the limited LND and 22 in the template RPLND group. Thirty-one patients included in the study had 1 positive lymph node and 14 patients had 2 positive lymph nodes. For patients undergoing a limited LND, a median 4 (IQR 1-11) lymph nodes were resected and for those undergoing template RPLND, 18 (IQR: 13-23) lymph nodes were resected. On Kaplan-Meier analysis, a difference was noted in overall survival (P = 0.04) when comparing limited LND to template RPLND. We also mapped out patterns of recurrence and found that 6 patients had retroperitoneal lymph node recurrences after a limited LND in the ipsilateral node packet. On univariate analysis, pathologic stage was a major factor for survival, but did not remain as significant with the inclusion of template RPLND status and Charlson Comorbidity Index in multivariate analysis. CONCLUSION: We identified specific patients that had RCC with limited lymph node involvement. We found that a select number of patients had durable improvement in survival outcomes with template RPLND. In examining the recurrence patterns, a greater number of patients may have derived benefit for an initial template RPLND.

Identifying Preoperative Predictors of Operative Time and Their Impact on Outcomes in Robot-Assisted Partial Nephrectomy.

Objective: To identify preoperative characteristics in patients with renal masses that influence operative time during robot-assisted partial nephrectomy (RAPN) and evaluate the relationship between operative time and length of stay (LOS), complication rates, and overall outcome. Materials and Methods: We queried our institutional database to identify a cohort of patients who underwent RAPN by two experienced robotic surgeons between 2012 and 2019. A multivariable regression model was developed to analyze operative time, LOS, and any grade complication within 30 days postoperatively using the bootstrap resampling technique. Results: A total of 392 patients were included. On multivariable analyses, prior abdominal surgery (p = 0.001) was associated with 22 minutes of increase in operating room time, as well as adhesive perirenal fat (22 minutes, p = 0.001). For each one unit increase in nephrometry score, there was a 4-minute increase in operating room time (p = 0.028), and for each one-cm increase in tumor size, there was an associated 12-minute increase in operating room time (p < 0.001). For each 1 year increase in age, there was an associated 0.024-day increase in LOS [odds ratio (OR) (0.013-0.035)]; in addition, for every one-cm increase in tumor size there was a 0.18-day associated increase in LOS [OR (0.070-0.28)]. Each 1-hour increase in operating room time was associated with a 0.25-day increased LOS [OR (0.092-0.41)]. Only tumor size was found to be associated with any grade complication. Conclusions: Patients with a history of abdominal surgery, larger complex tumors, and significant Gerota's fat undergoing robotic partial nephrectomy should anticipate longer operative times. Older patients with larger tumors and longer operative times can anticipate a longer LOS. Tumor size appears to be the common determinant of all three outcomes: operative time, LOS, and any grade Clavien complication.

Human jackstone arms show a protein-rich, X-ray lucent core, suggesting that proteins drive their rapid and linear growth.

Jackstone calculi, having arms that extend out from the body of the stone, were first described over a century ago, but this morphology of stones has been little studied. We examined 98 jackstones from 50 different patient specimens using micro-computed tomography (micro CT) and infrared (IR) spectroscopy. Micro CT showed that jackstone arms consisted of an X-ray lucent core within each arm. This X-ray lucent core frequently showed sporadic, thin layers of apatite arranged transversely to the axis of the arm. The shells of the jackstones were always composed of calcium oxalate (CaOx), and with the monohydrate form the majority or sole mineral. Study of layering in the shell regions by micro CT showed that growth lines extended from the body of the stone out onto jack arms and that the thickness of the shell covering of jack arms often thinned with distance from the stone body, suggesting that the arms grew at a faster radial rate than did the stone body. Histological cross-sections of decalcified jackstone arms showed the core to be more highly autofluorescent than was the CaOx shell, and immunohistochemistry showed the core to be enriched in Tamm-Horsfall protein. We hypothesize that the protein-rich core of a jack arm might preferentially bind more protein from the urine and resist deposition of CaOx, such that the arm grows in a linear manner and at a faster rate than the bulk of the stone. This hypothesis thus predicts an enrichment of certain urine proteins in the core of the jack arm, a theory that is testable by appropriate analysis.

Genetic risk assessment for hereditary renal cell carcinoma: Clinical consensus statement.

BACKGROUND: Although renal cell carcinoma (RCC) is believed to have a strong hereditary component, there is a paucity of published guidelines for genetic risk assessment. A panel of experts was convened to gauge current opinions. METHODS: A North American multidisciplinary panel with expertise in hereditary RCC, including urologists, medical oncologists, clinical geneticists, genetic counselors, and patient advocates, was convened. Before the summit, a modified Delphi methodology was used to generate, review, and curate a set of consensus questions regarding RCC genetic risk assessment. Uniform consensus was defined as ≥85% agreement on particular questions. RESULTS: Thirty-three panelists, including urologists (n = 13), medical oncologists (n = 12), genetic counselors and clinical geneticists (n = 6), and patient advocates (n = 2), reviewed 53 curated consensus questions. Uniform consensus was achieved on 30 statements in specific areas that addressed for whom, what, when, and how genetic testing should be performed. Topics of consensus included the family history criteria, which should trigger further assessment, the need for risk assessment in those with bilateral or multifocal disease and/or specific histology, the utility of multigene panel testing, and acceptance of clinician-based counseling and testing by those who have experience with hereditary RCC. CONCLUSIONS: In the first ever consensus panel on RCC genetic risk assessment, 30 consensus statements were reached. Areas that require further research and discussion were also identified, with a second future meeting planned. This consensus statement may provide further guidance for clinicians when considering RCC genetic risk assessment. LAY SUMMARY: The contribution of germline genetics to the development of renal cell carcinoma (RCC) has long been recognized. However, there is a paucity of guidelines to define how and when genetic risk assessment should be performed for patients with known or suspected hereditary RCC. Without guidelines, clinicians struggle to define who requires further evaluation, when risk assessment or testing should be done, which genes should be considered, and how counseling and/or testing should be performed. To this end, a multidisciplinary panel of national experts was convened to gauge current opinion on genetic risk assessment in RCC and to enumerate a set of recommendations to guide clinicians when evaluating individuals with suspected hereditary kidney cancer.

Definitive surgical therapy for refractory radiation cystitis: Evaluating effectiveness, tolerability, and extent of surgical approach.

INTRODUCTION AND OBJECTIVE: The management of severe symptoms secondary to radiation changes to the bladder can be difficult. Many patients often endure costly procedures, hospitalizations, transfusions, and physician visits for intractable symptoms. Our aim was to evaluate the short-term efficacy and feasibility of urinary diversion in patients with severe, debilitating symptoms related to radiation cystitis by focusing on perioperative data examining surgical feasibility and assess for any improvement in the number of procedures, transfusions, hospitalizations, and office visits required. METHODS: With IRB approval, we queried our institutional database for patients with a diagnosis code of radiation cystitis who underwent urinary diversion with or without bladder removal from 2011 to 2018. We reviewed institutional and regional record to assess pre, peri and postoperative outcomes, including rates of surgical procedures, hospitalizations, transfusions and clinic visits, in the year before and after treatment. Non-parametric statistics and linear regression were used. RESULTS: Of the 286 patients with radiation cystitis, 45 patients underwent definitive urinary diversion - 31 with concomitant cystectomy and 14 with diversion alone. Analysis of perioperative variables such as estimated blood loss, surgical time, post-operative hospital stay or complication rates were similar to our experience with cystectomy in non-radiated patients. With a mean follow up of 14.6 months, we found that the number of procedures, hospitalizations and transfusions objectively improved following radical surgery. Office visits, however, did not seem to be impacted by performing urinary diversion. There were no significant differences in post-operative benefits between patients that received a concomitant cystectomy and those that only underwent diversion. CONCLUSION: Patients suffering from severe refractory symptomatic radiation cystitis may be best treated with a radical surgical approach. Definitive urinary diversion with or without cystectomy can lower burden of disease by reducing the need for additional procedures, hospitalizations, and blood transfusions on short term follow-up.

Clear cell papillary renal cell carcinoma: Characteristics and survival outcomes from a large single institutional series.

OBJECTIVES: To investigate the clinical characteristics and survival outcomes of a large clear cell papillary renal cell carcinoma cohort. METHODS AND MATERIALS: A retrospective review of patients with clear cell papillary renal cell carcinoma at a single academic center was performed after Institutional Review Board approval. Patients underwent either partial or radical nephrectomy from September 2009 to July 2019. Demographic and clinical characteristics, recurrence, and cancer specific and overall survival were reported. RESULTS: A total of 90 patients were included in the study. Median follow up was 26.5 months. Median age was 61 (range 27 to 87). 47.8% of patients were African American. 26.7% of patients had end stage renal disease. 37.8% had multifocal renal tumors. 48.9% underwent partial nephrectomy, while the remainder underwent radical nephrectomy. 43.3% underwent an open surgical approach, 40.0% a robotic approach, and 16.7% a laparoscopic approach. Pathologic stage included T1a (90.0%), T1b (1.1%), and T2b (8.9%). Fuhrman grades 1-3 were present in 18.9%, 77.8%, and 3.3% of patients, respectively. There were no cancer specific deaths. There was one local recurrence and no metastases. The overall survival at a median follow up of 26.5 months was 92.1% (95% confidence interval 83.1%-96.4%). CONCLUSIONS: Clear cell papillary renal cell carcinoma typically presents at a low stage and grade and has favorable survival outcomes. A nephron-sparing approach to treatment should be considered when feasible due to the tumor's indolent nature and propensity towards multifocality.

Evaluation, diagnosis and surveillance of renal masses in the setting of VHL disease.

This brief report focuses on the evaluation and diagnosis of clinically localized renal masses in children and adults with Von Hippel-Lindau (VHL) disease. Counseling considerations pertinent to the urologists, medical oncologists, and multidisciplinary teams involved in the care of these patients are addressed. As practice patterns regarding the evaluation and management of VHL tumors can vary considerably, this report aims to provide guidance on some of the controversies associated with the diagnostic evaluation and initial management of localized renal masses in VHL patients.

Xanthogranulomatous pyelonephritis: a comparison of open and minimally-invasive surgical approaches.

To compare perioperative outcomes between patients undergoing minimally-invasive (MIS) and open surgical approaches for the treatment of Xanthogranulomatous Pyelonephritis (XGP). Between 2007 and 2017 we retrospectively identified 40 patients undergoing nephrectomy at our institution for pathologically confirmed XGP. Patients whose operations were ultimately completed with open technique were analyzed with the open cohort, whereas patients whose operations were completed in entirety using any laparoscopic approach were analyzed with the MIS group. Twenty-three patients were analyzed in the open cohort, compared to seventeen in the MIS group. Three patients in the open cohort were converted intraoperatively from MIS to open approach. Compared to the open group, the MIS group less often had an abscess on preoperative CT (11.8% vs 54.5%; p = 0.006). The MIS group also had lower intraoperative blood loss (100 vs 400 mL; p < 0.001), lower rate of blood transfusion (0% vs 45.5%; p = 0.002), lower postoperative intensive care admission (0% vs 34.8%; p = 0.013), and shorter hospital stay (4 vs 7 days; p = 0.013). However, there was no significant difference in high-grade complications between these groups (5.9% vs 34.8%; p = 0.054). Preoperative CT scan may be an important factor when considering operative approach for treatment of XGP. Patients who are able to undergo MIS approach have less blood loss, shorter hospitalization, and are less likely to require intensive care admission, which may be related to the disease process, the surgical technique, or both.

Understanding the role of salvage lymphadenectomy in node only recurrences after nephrectomy for renal cell carcinoma.

PURPOSE: To investigate oncological outcomes and relapse patterns in retroperitoneal lymph node (LN)-only recurrences with salvage retroperitoneal lymph node dissection (S-RPLND). MATERIALS AND METHODS: We reviewed records of 19 patients undergoing RPLND for RCC recurrences between 2011 and 2018. All patients initially had primary non-metastatic RCC, with subsequent recurrence restricted to the retroperitoneal lymph nodes (LN). LN recurrence sites after nephrectomy and relapses after S-RPLND were assessed. The primary outcomes were post-RPLND Relapse-Free Survival (RFS), and Cancer-Specific Survival (CSS). RESULTS: The median age of our cohort was 60 years at RPLND. Right and left nephrectomies were performed in 14 (73.7%) and 5 (26.3%), respectively. Clear cell carcinoma was found in 10 (52.6%) patients, followed by papillary in 4(21.1%), chromophobe in 2(10.5%), and 'other' in 3 (15.8%). The extent of lymphadenectomy during nephrectomy and S-RPLND varied based on surgical approach. The median follow-up time after S-RPLND of the entire cohort was 31.53 months, and the median RFS was 9.63 months. Overall, 4 patients died of cancer, of which 3 (75%) were N1 at time of nephrectomy. The CSS after RPLND at 3 and 5 years was 81.5% and 61.1%, respectively. The RFS after RPLND at 2 and 5 years was 44.4% and 29.6%, respectively. CONCLUSIONS: Our results suggest that aggressive surgical management provides satisfactory CSS with acceptable complication rates. Moreover, we believe this subset of patients with node-only recurrence showed an unpredictable pattern of lymphatic spread, with predilection for regional dissemination warranting surgical resection of LN recurrences in a bilateral template fashion when feasible.

Plasma-Derived Extracellular Vesicle Phosphoproteomics through Chemical Affinity Purification.

The invasive nature and the pain caused to patients inhibit the routine use of tissue biopsy-based procedures for cancer diagnosis and surveillance. The analysis of extracellular vesicles (EVs) from biofluids has recently gained significant traction in the liquid biopsy field. EVs offer an essential "snapshot" of their precursor cells in real time and contain an information-rich collection of nucleic acids, proteins, lipids, and so on. The analysis of protein phosphorylation, as a direct marker of cellular signaling and disease progression could be an important stepping stone to successful liquid biopsy applications. Here we introduce a rapid EV isolation method based on chemical affinity called EVtrap (extracellular vesicle total recovery and purification) for the EV phosphoproteomics analysis of human plasma. By incorporating EVtrap with high-performance mass spectrometry (MS), we were able to identify over 16 000 unique peptides representing 2238 unique EV proteins from just 5 µL of plasma sample, including most known EV markers, with substantially higher recovery levels compared with ultracentrifugation. Most importantly, more than 5500 unique phosphopeptides representing almost 1600 phosphoproteins in EVs were identified using only 1 mL of plasma. Finally, we carried out a quantitative EV phosphoproteomics analysis of plasma samples from patients diagnosed with chronic kidney disease or kidney cancer, identifying dozens of phosphoproteins capable of distinguishing disease states from healthy controls. The study demonstrates the potential feasibility of our robust analytical pipeline for cancer signaling monitoring by tracking plasma EV phosphorylation.