Targeted deep sequencing of urothelial bladder cancers and associated urinary DNA: a 23-gene panel with utility for non-invasive diagnosis and risk stratification.

OBJECTIVES: To develop a focused panel of somatic mutations (SMs) present in the majority of urothelial bladder cancers (UBCs), to investigate the diagnostic and prognostic utility of this panel, and to compare the identification of SMs in urinary cell-pellet (cp)DNA and cell-free (cf)DNA as part of the development of a non-invasive clinical assay. PATIENTS AND METHODS: A panel of SMs was validated by targeted deep-sequencing of tumour DNA from 956 patients with UBC. In addition, amplicon and capture-based targeted sequencing measured mutant allele frequencies (MAFs) of SMs in 314 urine cpDNAs and 153 urine cfDNAs. The association of SMs with grade, stage, and clinical outcomes was investigated by univariate and multivariate Cox models. Concordance between SMs detected in tumour tissue and cpDNA and cfDNA was assessed. RESULTS: The panel comprised SMs in 23 genes: TERT (promoter), FGFR3, PIK3CA, TP53, ERCC2, RHOB, ERBB2, HRAS, RXRA, ELF3, CDKN1A, KRAS, KDM6A, AKT1, FBXW7, ERBB3, SF3B1, CTNNB1, BRAF, C3orf70, CREBBP, CDKN2A, and NRAS; 93.5-98.3% of UBCs of all grades and stages harboured ≥1 SM (mean: 2.5 SMs/tumour). RAS mutations were associated with better overall survival (P = 0.04). Mutations in RXRA, RHOB and TERT (promoter) were associated with shorter time to recurrence (P < 0.05). MAFs in urinary cfDNA and cpDNA were highly correlated; using a capture-based approach, >94% of tumour SMs were detected in both cpDNA and cfDNA. CONCLUSIONS: SMs are reliably detected in urinary cpDNA and cfDNA. The technical capability to identify very low MAFs is essential to reliably detect UBC, regardless of the use of cpDNA or cfDNA. This 23-gene panel shows promise for the non-invasive diagnosis and risk stratification of UBC.

Low-grade Urothelial Carcinoma Recurs at a Tempo that Naturally Accelerates Over Time.

OBJECTIVE: To investigate the cadence of recurrence in patients with low grade intermediate-risk non-muscle invasive bladder cancer (LG IR-NMIBC) based on clinical determinants. We aim to describe patterns in rates of recurrence to better inform surveillance regimens for this chronic, burdensome, and costly disease. METHODS: Using baseline and follow-up data from participants in the West Midlands' (United Kingdom, UK) Bladder Cancer Prognosis Programme (BCPP), we assessed overall recurrence rate and recurrence-free intervals throughout the follow-up period for IR-NMIBC participants. Recurrence-free intervals were calculated using the Kaplan-Meier method. RESULTS: We identified 379 patients with G1/G2 pTa tumors classified as intermediate risk. Median age was 70 and 284/379 (75%) were male. The median follow-up time was 4.2 years (95% CI: 3.9-4.8). After 5 years of follow-up, 53% of patients had at least one recurrence. One-year recurrence-free survival (RFS) was 75% and 4-year RFS was 50%. The median time to or between 1st, 2nd, 3rd, 4th, and 5th sequential recurrences was 49, 19, 12, 14, and 10 months, respectively. CONCLUSION: Over half of patients with IR-NMIBC are destined to recur. Our data suggest that a subset of patients experience acceleration of recurrence over time and that this acceleration may serve as a potential kinetic biomarker for these individuals that could inform surveillance intervals and future treatment strategies.

Tamoxifen for the treatment of myeloproliferative neoplasms: A Phase II clinical trial and exploratory analysis.

Current therapies for myeloproliferative neoplasms (MPNs) improve symptoms but have limited effect on tumor size. In preclinical studies, tamoxifen restored normal apoptosis in mutated hematopoietic stem/progenitor cells (HSPCs). TAMARIN Phase-II, multicenter, single-arm clinical trial assessed tamoxifen's safety and activity in patients with stable MPNs, no prior thrombotic events and mutated JAK2V617F, CALRins5 or CALRdel52 peripheral blood allele burden ≥20% (EudraCT 2015-005497-38). 38 patients were recruited over 112w and 32 completed 24w-treatment. The study's A'herns success criteria were met as the primary outcome ( ≥ 50% reduction in mutant allele burden at 24w) was observed in 3/38 patients. Secondary outcomes included ≥25% reduction at 24w (5/38), ≥50% reduction at 12w (0/38), thrombotic events (2/38), toxicities, hematological response, proportion of patients in each IWG-MRT response category and ELN response criteria. As exploratory outcomes, baseline analysis of HSPC transcriptome segregates responders and non-responders, suggesting a predictive signature. In responder HSPCs, longitudinal analysis shows high baseline expression of JAK-STAT signaling and oxidative phosphorylation genes, which are downregulated by tamoxifen. We further demonstrate in preclinical studies that in JAK2V617F+ cells, 4-hydroxytamoxifen inhibits mitochondrial complex-I, activates integrated stress response and decreases pathogenic JAK2-signaling. These results warrant further investigation of tamoxifen in MPN, with careful consideration of thrombotic risk.

In meta-analyses of proportion studies, funnel plots were found to be an inaccurate method of assessing publication bias.

OBJECTIVE: To assess the utility of funnel plots in assessing publication bias (PB) in meta-analyses of proportion studies. STUDY DESIGN AND SETTING: Meta-analysis simulation study and meta-analysis of published literature reporting peri-operative mortality after abdominal aortic aneurysm (AAA) repair. Data for the simulation study were stochastically generated. A literature search of Medline and Embase was performed to identify studies for inclusion in the published literature meta-analyses. RESULTS: The simulation study demonstrated that conventionally constructed funnel plots (log odds vs. 1/standard error [1/SE]) for extreme proportional outcomes were asymmetric despite no PB. Alternative funnel plots constructed using study size rather than 1/SE showed no asymmetry for extreme proportional outcomes. When used in meta-analyses of the mortality of AAA repair, these alternative funnel plots highlighted the possibility for conventional funnel plots to demonstrate asymmetry when there was no evidence of PB. CONCLUSION: Conventional funnel plots used to assess for potential PB in meta-analyses are inaccurate for meta-analyses of proportion studies with low proportion outcomes. Funnel plots of study size against log odds may be a more accurate way of assessing for PB in these studies.