DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation.

Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array "waves", and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance

An increased body mass index is associated with a worse prognosis in patients administered BCG immunotherapy for T1 bladder cancer

PURPOSE: The body mass index (BMI) may be associated with an increased incidence and aggressiveness of urological cancers. In this study, we aimed to evaluate the impact of the BMI on survival in patients with T1G3 non-muscle-invasive bladder cancer (NMIBC). METHODS: A total of 1155 T1G3 NMIBC patients from 13 academic institutions were retrospectively reviewed and patients administered adjuvant intravesical Bacillus Calmette-Guérin (BCG) immunotherapy with maintenance were included. Multivariable Cox regression analysis was performed to identify factors predictive of recurrence and progression. RESULTS: After re-TURBT, 288 patients (27.53%) showed residual high-grade NMIBC, while 867 (82.89%) were negative. During follow-up, 678 (64.82%) suffered recurrence, and 303 (30%) progression, 150 (14.34%) died of all causes, and 77 (7.36%) died of bladder cancer. At multivariate analysis, tumor size (hazard ratio [HR]:1.3; p = 0.001), and multifocality (HR:1.24; p = 0.004) were significantly associated with recurrence (c-index for the model:55.98). Overweight (HR: 4; p < 0.001) and obesity (HR:5.33 p < 0.001) were significantly associated with an increased risk of recurrence. Addition of the BMI to a model that included standard clinicopathological factors increased the C-index by 9.9. For progression, we found that tumor size (HR:1.63; p < 0.001), multifocality (HR:1.31; p = 0.01) and concomitant CIS (HR: 2.07; p < 0.001) were significant prognostic factors at multivariate analysis (C-index 63.8). Overweight (HR: 2.52; p < 0.001) and obesity (HR: 2.521 p < 0.001) were significantly associated with an increased risk of progression. Addition of the BMI to a model that included standard clinicopathological factors increased the C-index by 1.9. CONCLUSIONS: The BMI could have a relevant role in the clinical management of T1G3 NMIBC, if associated with bladder cancer recurrence and progression. In particular, this anthropometric factor should be taken into account at initial diagnosis and in therapeutic strategy decision making.M.D.V is supported by the Scholarship Foundation of the Republic of Austria—OeAD and by the EUSP Scholarship— European Association of Urologyinfo:eu-repo/semantics/publishedVersio