A single-center retrospective comparison of pT1 substaging methods in bladder cancer.

Substaging of T1 urothelial cancer is associated with tumor progression and its reporting is recommended by international guidelines. However, it has not been integrated in risk stratification tools and there is no agreement on the best method to use for its reporting. We aimed to investigate the applicability, interobserver variability, and prognostic value of histological landmark based and micrometric (aggregate linear length of invasive carcinoma (ALLICA), microscopic vs. extensive system, Rete Oncologica Lombarda (ROL) system) substaging methods. A total of 79 patients with the primary diagnosis of T1 urothelial cancer treated with conventional transurethral resection and adjuvant BCG therapy between 2000 and 2020 at the Medical University of Vienna were included. The anatomical and metrical substaging systems were evaluated using agreement rate, Cohen's kappa, Kendall's tau, and Spearman rank correlation. Prognostic value for high-grade recurrence or T2 progression was evaluated in uni- and multivariable analysis. Applicability and reproducibility were good to moderate and varied between substaging methods. Obstacles are mainly due to fragmentation of samples. Anatomical substaging was associated with progression in univariable and multivariable analysis. In our cohort, we could only identify anatomical landmark-based substaging to be prognostic for T2 progression. A major obstacle for proper pathological assessment is fragmentation of samples due to operational procedure. Avoiding such fragmentation might improve reproducibility and significance of pathological T1 substaging of urothelial cancer.

Plasmin Generation Analysis in Patients with Bleeding Disorder of Unknown Cause.

Bleeding disorder of unknown cause (BDUC) is a diagnosis of exclusion after evaluation of plasma coagulation and platelet function. The underlying mechanisms are unclear, but increased fibrinolysis and abnormal clot formation may play a role. All BDUC patients (n=375) from the Vienna bleeding biobank were analyzed in comparison to healthy controls (HC, n=100) in this case-control study. Plasmin generation (PG) parameters were analyzed using calibrated fluorescence detection in citrated-plasma samples. Turbidimetric plasma clot formation/ lysis of 293 (78%) BDUC patients and confocal microscopy of clots from representative BDUC patients (n=6) and HC (n=9) were assessed. Fibrinolytic factors were measured using commercially available ELISAs. In PG analysis, BDUC patients exhibited lower velocity and peak plasmin, but a higher endogenous plasmin potential compared to HC. Peak plasmin correlated with maximum clot absorbance, but not with clot lysis time. Clot absorbance is an indicator of clot fiber density. Confocal microscopy analysis revealed that BDUC patients' clots had a tendency towards thicker fibers, which negatively correlated with peak plasmin (r=-0.561, p=0.030). Peak plasmin correlated weakly with FXIII, but not with the other fibrinolytic factors (alpha2-antiplasmin, thrombin activatable fibrinolysis inhibitor or plasminogen activator inhibitor 1) or bleeding severity. A model comprising fibrinogen and parameters of PG yielded high predictive power in discriminating BDUC patients from HC during 5-fold stratified cross validation (80% of data, mean AUC: 0.847). The same model generalized well to unseen data (20% of data, AUC: 0.856). Overall, BDUC patients exhibited counterintuitively reduced peak plasmin, potentially related to altered clot structure.

Circulating Tumor DNA as a Marker for Treatment Response in Metastatic Melanoma Patients Using Next-Generation Sequencing-A Prospective Feasibility Study.

We prospectively performed a longitudinal analysis of circulating tumor DNA (ctDNA) from 149 plasma samples and CT scans in Stage III and IV metastatic melanoma patients (n = 20) treated with targeted agents or immunotherapy using two custom next-generation sequencing (NGS) Ion AmpliSeq™ HD panels including 60 and 81 amplicons in 18 genes, respectively. Concordance of matching cancer-associated mutations in tissue and plasma was 73.3%. Mutant allele frequency (MAF) levels showed a range from 0.04% to 28.7%, well detectable with NGS technologies utilizing single molecule tagging like the AmpliSeq™ HD workflow. Median followup time of the tissue and/or plasma positive cohort (n = 15) was 24.6 months and median progression-free survival (PFS) was 7.8 months. Higher MAF ≥ 1% at baseline was not significantly associated with a risk of progression (Odds Ratio = 0.15; p = 0.155). Although a trend could be seen, MAF levels did not differ significantly over time between patients with and without a PFS event (p = 0.745). Depending on the cell-free DNA amount, NGS achieved a sensitivity down to 0.1% MAF and allowed for parallel analysis of multiple mutations and previously unknown mutations. Our study indicates that NGS gene panels could be useful for monitoring disease burden during therapy with ctDNA in melanoma patients.